1 00:00:05,200 --> 00:00:07,760 WELCOME TO TODAY'S WEDNESDAY 2 00:00:07,760 --> 00:00:11,200 AFTERNOON LECTURE SERIES 3 00:00:11,200 --> 00:00:13,680 PRESENTATION. 4 00:00:13,680 --> 00:00:15,400 I'M THE CHIEF OF THE SECTION 5 00:00:15,400 --> 00:00:16,920 ON MICROBIAL PATHOGENESIS OF 6 00:00:16,920 --> 00:00:17,120 NICHD. 7 00:00:17,120 --> 00:00:21,080 ON BEHALF OF THE 8 00:00:21,080 --> 00:00:21,680 PROTEIN TRAFFICKING DYNAMICS 9 00:00:21,680 --> 00:00:24,160 INTEREST GROUP I WOULD LIKE TO 10 00:00:24,160 --> 00:00:27,080 WELCOME BACK TO THE NIH TODAY'S 11 00:00:27,080 --> 00:00:29,960 WALS SPEAKER, FRIEND AND FORMER 12 00:00:29,960 --> 00:00:33,200 COLLEAGUE DR. MANU HEGDE. 13 00:00:33,200 --> 00:00:35,080 MANY OF YOU REMEMBER MANU FROM 14 00:00:35,080 --> 00:00:37,240 HIS TIME AT NIH, AS A MEMBER OF 15 00:00:37,240 --> 00:00:39,560 THE NCI SCHOLARS PROGRAM, LATER 16 00:00:39,560 --> 00:00:42,080 STARTING IN 2002 AS INVESTIGATOR 17 00:00:42,080 --> 00:00:44,680 IN WHAT WAS FORMERLY KNOWN AS 18 00:00:44,680 --> 00:00:48,880 THE CELL BIOLOGY METABOLISM 19 00:00:48,880 --> 00:00:50,400 BRANCH OF NICHD. 20 00:00:50,400 --> 00:00:53,040 SAME PROGRAM I JOINED A FEW 21 00:00:53,040 --> 00:00:54,320 YEARS LATER. 22 00:00:54,320 --> 00:00:57,800 MANU RECEIVED TENURE IN 2008, 23 00:00:57,800 --> 00:00:59,360 2011 SADLY MOVED HIS LABORATORY 24 00:00:59,360 --> 00:01:00,560 TO THE UNIVERSITY OF CAMBRIDGE 25 00:01:00,560 --> 00:01:04,520 IN ENGLAND TO JOIN THE MEDICAL 26 00:01:04,520 --> 00:01:05,600 RESEARCH COUNCIL LABORATORY OF 27 00:01:05,600 --> 00:01:07,400 MOLECULAR BIOLOGY WHERE HE'S 28 00:01:07,400 --> 00:01:09,880 BEEN SINCE THEN. 29 00:01:09,880 --> 00:01:14,040 MANU IS AN ABSOLUTE LEADER IN 30 00:01:14,040 --> 00:01:16,760 QUALITY PROTEIN CONTROL, AT THE 31 00:01:16,760 --> 00:01:18,400 FOREFRONT OF STUDYING PROTEINS, 32 00:01:18,400 --> 00:01:19,480 MEMBRANE PROTEINS IN PARTICULAR, 33 00:01:19,480 --> 00:01:21,280 FROM THE MOMENT THEY ARE 34 00:01:21,280 --> 00:01:22,160 SYNTHESIZED OR BORN AT THE 35 00:01:22,160 --> 00:01:24,880 RIBOSOME TO DELIVERED TO THE 36 00:01:24,880 --> 00:01:25,640 FINAL DESTINATION COMPARTMENT 37 00:01:25,640 --> 00:01:28,040 WITHIN CELLS AND HOW CELLS 38 00:01:28,040 --> 00:01:30,560 RECOGNIZE ERRORS THAT IF NOT 39 00:01:30,560 --> 00:01:32,200 CORRECTED WOULD SIGNIFICANTLY 40 00:01:32,200 --> 00:01:34,480 IMPACT HUMAN HEALTH. 41 00:01:34,480 --> 00:01:35,800 MANU'S CONTRIBUTIONS TO THE 42 00:01:35,800 --> 00:01:37,640 FIELD OF PROTEIN BIOGENESIS AND 43 00:01:37,640 --> 00:01:39,800 QUALITY CONTROL ARE EXCEPTIONAL 44 00:01:39,800 --> 00:01:43,120 IN ORIGINALITY, AND MORE THAN 45 00:01:43,120 --> 00:01:44,040 130 PUBLICATIONS TODAY EVIDENCE 46 00:01:44,040 --> 00:01:46,840 HE'S IN A CLASS OF HIS OWN, 47 00:01:46,840 --> 00:01:48,400 REALLY. 48 00:01:48,400 --> 00:01:53,080 SO FUN FACT, MANU SENT HIS HIS 49 00:01:53,080 --> 00:01:54,280 CV, 13 PAGES LONG, PUBLICATION 50 00:01:54,280 --> 00:01:56,560 RECORD COVERED 11 OF THE 13 51 00:01:56,560 --> 00:01:57,040 PAGES. 52 00:01:57,040 --> 00:02:00,120 WHAT MAKES HIM AN OUTSTANDING 53 00:02:00,120 --> 00:02:02,600 SCIENTIST IS INSTINCT FOR 54 00:02:02,600 --> 00:02:03,680 IMPORTANT BIOLOGICAL QUESTIONS, 55 00:02:03,680 --> 00:02:05,880 AND HIS CLEVERNESS IN DEVELOPING 56 00:02:05,880 --> 00:02:07,080 ELEGANT EXPERIMENTAL APPROACHES 57 00:02:07,080 --> 00:02:10,160 TO FIND THE CORRECT ANSWER. 58 00:02:10,160 --> 00:02:12,200 MANY DISCOVERIES MANU HAS MADE 59 00:02:12,200 --> 00:02:22,640 OVER THE YEARS ONE MIGHT 60 00:07:18,760 --> 00:07:19,960 DRAG 61 00:07:19,960 --> 00:07:25,680 ADJACENT PART OF HYDROPHILIC POP 62 00:07:25,680 --> 00:07:26,680 POLLY PEPTIDE ACROSS, THIS 63 00:07:26,680 --> 00:07:31,800 DIDN'T MAKE SENSE TO SOME 64 00:07:31,800 --> 00:07:35,320 PEOPLE, AND FELT STRONGLY THE 65 00:07:35,320 --> 00:07:36,280 MOVEMENT ACROSS THE MEMBRANE 66 00:07:36,280 --> 00:07:39,200 MUST GO THROUGH A CHANNEL. 67 00:07:39,200 --> 00:07:42,120 AND SO AROUND 1980 OR SO IT WAS 68 00:07:42,120 --> 00:07:43,600 PROPOSED THE WAY A MEMBRANE 69 00:07:43,600 --> 00:07:46,800 PROTEIN GETS MADE IS YOU HAVE 70 00:07:46,800 --> 00:07:49,200 THESE HYDROPHOBIC SEGMENTS OF 71 00:07:49,200 --> 00:07:54,640 POLYPEPTIDE OUT OF THE RIBOSOME 72 00:07:54,640 --> 00:07:55,640 INSERTED THROUGH A CHANNEL. 73 00:07:55,640 --> 00:07:57,680 WHEN I WAS A GRADUATE STUDENT, I 74 00:07:57,680 --> 00:08:00,680 WROTE A REVIEW ON HOW MEMBRANE 75 00:08:00,680 --> 00:08:02,520 PROTEINS GET MADE, OF COURSE THE 76 00:08:02,520 --> 00:08:04,840 PICTURE HADN'T CHANGED AT ALL IN 77 00:08:04,840 --> 00:08:07,560 THE 17 YEARS SINCE IT WAS 78 00:08:07,560 --> 00:08:09,360 PROPOSED, AND WHEN I WROTE THIS 79 00:08:09,360 --> 00:08:12,160 REVIEW, THE IDEA IS THAT A 80 00:08:12,160 --> 00:08:15,880 RIBOSOME IS DOCKED AT 81 00:08:15,880 --> 00:08:17,360 TRANSLOCATION CHANNEL OR 82 00:08:17,360 --> 00:08:18,880 TRANSLOCON, AS THESE BLACK BARS 83 00:08:18,880 --> 00:08:29,680 COME OUT OF THE RIBOSOME THEY RD 84 00:08:35,440 --> 00:08:38,360 THINGS FORWARD WAS THE IDENTITY 85 00:08:38,360 --> 00:08:41,280 OF THIS, AND THE CENTRAL 86 00:08:41,280 --> 00:08:43,920 COMPONENT AROSE FROM A 87 00:08:43,920 --> 00:08:45,560 COMBINATION OF YEAST AND 88 00:08:45,560 --> 00:08:46,560 BACTERIAL GENETICS AND 89 00:08:46,560 --> 00:08:48,400 CHEMISTRY, AND A REAL ADVANCE 90 00:08:48,400 --> 00:08:55,000 WAS TO GET THE STRUCTURE OF AN 91 00:08:55,000 --> 00:08:58,920 ARC HEEL VERSION IN 2004, AND 92 00:08:58,920 --> 00:09:01,200 THE PROTEIN WASN'T ACTUALLY IN 93 00:09:01,200 --> 00:09:11,640 THE PROCESS OPEN INTO THE 94 00:09:17,280 --> 00:09:18,160 LIPID BILAYER THROUGH THE 95 00:09:18,160 --> 00:09:22,760 LATERAL GATE FOR INSERTION OF 96 00:09:22,760 --> 00:09:23,520 HYDROPHOBIC ELEMENTS, THE IDEA 97 00:09:23,520 --> 00:09:25,160 TURNS OUT TO BE TRUE, OUR 98 00:09:25,160 --> 00:09:26,600 CONTRIBUTION WAS TO LEVERAGE 99 00:09:26,600 --> 00:09:29,000 ADVANCES IN CryoEM TO STAGE 100 00:09:29,000 --> 00:09:31,640 THE PROCESS OF INSERTION OF 101 00:09:31,640 --> 00:09:34,680 HYDROPHOBIC SEGMENT BEFORE AND 102 00:09:34,680 --> 00:09:37,200 AFTER IT ENGAGES THIS SEC61 103 00:09:37,200 --> 00:09:38,600 TRANSLOCATION CHANNEL AND ONE 104 00:09:38,600 --> 00:09:40,680 CAN SEE HOW THIS MIGHT WORK. 105 00:09:40,680 --> 00:09:43,120 SO, THIS IS JUST A MORPH BETWEEN 106 00:09:43,120 --> 00:09:47,040 AN INACTIVE AND ACTIVE VERSION 107 00:09:47,040 --> 00:09:49,880 OF SEC61, ACTIVE MEANING ENGAGED 108 00:09:49,880 --> 00:09:51,760 BY HYDROPHOBIC SEGMENT, IN THIS 109 00:09:51,760 --> 00:09:54,360 CASE A SIGNAL PEPTIDE, AS 110 00:09:54,360 --> 00:09:55,160 PREDICTED ORIGINALLY FROM 111 00:09:55,160 --> 00:09:56,800 BIOCHEMICAL AND STRUCTURAL WORK 112 00:09:56,800 --> 00:09:58,320 IS SITTING AT THIS LATERAL GATE. 113 00:09:58,320 --> 00:10:00,960 IN ORDER TO SIT THERE, HALF OF 114 00:10:00,960 --> 00:10:02,600 THE MOLECULE MOVES APART FROM 115 00:10:02,600 --> 00:10:06,080 THE OTHER HALF, AND IN THAT 116 00:10:06,080 --> 00:10:11,760 PROCESS THE CENTRAL OROIFICE 117 00:10:11,760 --> 00:10:12,080 OPENS. 118 00:10:12,080 --> 00:10:14,720 IN THIS TYPE OF PICTURE HOW THIS 119 00:10:14,720 --> 00:10:16,360 TRANSLOCATION CHANNEL WORKS, YOU 120 00:10:16,360 --> 00:10:19,000 HAVE A HYDROPHOBIC SEGMENT OF 121 00:10:19,000 --> 00:10:21,200 POLYPEPTIDE THAT ENGAGES LATERAL 122 00:10:21,200 --> 00:10:23,600 PART OF THIS CHANNEL, DRAGGING 123 00:10:23,600 --> 00:10:29,600 THE HYDROPHILIC SEGMENT 124 00:10:29,600 --> 00:10:32,480 DOWNSTREAM AND AT A LATER STEP 125 00:10:32,480 --> 00:10:33,640 MOVED INTO THE BILAYER AND 126 00:10:33,640 --> 00:10:35,080 THROUGH THE MEMBRANE THROUGH 127 00:10:35,080 --> 00:10:36,920 THIS CHANNEL. 128 00:10:36,920 --> 00:10:38,880 YOU CAN IMAGINE THEN, AGAIN I 129 00:10:38,880 --> 00:10:40,480 EMPHASIZE THE FACT THAT IT 130 00:10:40,480 --> 00:10:43,640 REALLY IS A WORKING MODEL, OF 131 00:10:43,640 --> 00:10:47,600 HOW YOU WOULD THEN HAVE AS MORE 132 00:10:47,600 --> 00:10:48,480 AND MORE HYDROPHOBIC SEGMENTS 133 00:10:48,480 --> 00:10:49,880 COME OUT OF THE RIBOSOME THEY DO 134 00:10:49,880 --> 00:10:51,320 THE SAME THING. 135 00:10:51,320 --> 00:10:54,480 YOU INSERT THEM ALL ONE BY ONE 136 00:10:54,480 --> 00:10:57,120 THROUGH THE LATERAL GATE OF THE 137 00:10:57,120 --> 00:10:59,080 SEC61 CHANNEL, THE WORKING MODEL 138 00:10:59,080 --> 00:11:00,720 IN REVIEWER TEXT BOOK THAT 139 00:11:00,720 --> 00:11:01,720 YOU'LL SEE. 140 00:11:01,720 --> 00:11:02,880 NOW, THAT MAKES A LOT OF SENSE. 141 00:11:02,880 --> 00:11:04,560 THERE'S A GOOD REASON WHY THIS 142 00:11:04,560 --> 00:11:06,480 MODEL HAS BEEN EXTREMELY 143 00:11:06,480 --> 00:11:07,400 DURABLE. 144 00:11:07,400 --> 00:11:10,320 BUT WHEN YOU LOOK AT A REAL 145 00:11:10,320 --> 00:11:12,040 MEMBRANE PROTEIN AS OPPOSED TO 146 00:11:12,040 --> 00:11:13,840 MODELS, YOU START TO SEE SOME 147 00:11:13,840 --> 00:11:14,320 PROBLEMS. 148 00:11:14,320 --> 00:11:19,880 SO, WHAT I'VE DEPICTED HERE IS A 149 00:11:19,880 --> 00:11:21,400 G-PROTEIN COUPLED RECEPTOR, BETA 150 00:11:21,400 --> 00:11:22,400 ADRENERGIC RECEPTOR. 151 00:11:22,400 --> 00:11:23,800 WHEN THE PROTEIN IS MADE, AND 152 00:11:23,800 --> 00:11:27,080 THIS IS A STRUCTURE OF IT, THE 153 00:11:27,080 --> 00:11:28,520 HYDROPHOBIC PARTS OF THE 154 00:11:28,520 --> 00:11:30,680 POLYPEPTIDE ARE IN THE MEMBRANE 155 00:11:30,680 --> 00:11:33,760 AND ALL THE HYDROPHOBIC SURFACES 156 00:11:33,760 --> 00:11:35,360 FACE THE LIPID BILAYER, HERE 157 00:11:35,360 --> 00:11:36,520 HYDROPHOBIC IS RED. 158 00:11:36,520 --> 00:11:40,400 THE INTERIOR OF THAT IS FILLED 159 00:11:40,400 --> 00:11:42,080 WITH LOTS OF HYDROPHILIC AMINO 160 00:11:42,080 --> 00:11:44,440 ACIDS, AND THOSE ARE 161 00:11:44,440 --> 00:11:45,120 FUNCTIONALLY IMPORTANT FOR 162 00:11:45,120 --> 00:11:47,320 THINGS LIKE BINDING TO LIGANDS, 163 00:11:47,320 --> 00:11:49,080 IN THIS CASE ADRENALINE. 164 00:11:49,080 --> 00:11:51,480 SO IF YOU LOOK AT KIND OF A 165 00:11:51,480 --> 00:11:53,120 DECONSTRUCTED VERSION OF THIS, 166 00:11:53,120 --> 00:11:55,280 YOU CAN SEE THE INDIVIDUAL 167 00:11:55,280 --> 00:11:56,920 SEGMENTS OF POLYPEPTIDE THAT 168 00:11:56,920 --> 00:11:58,920 SPAN THE MEMBRANE ARE HIGHLY 169 00:11:58,920 --> 00:11:59,720 VARIABLE. 170 00:11:59,720 --> 00:12:01,000 SO SOME ARE REMARKABLE HIGH 171 00:12:01,000 --> 00:12:06,280 DOUGH FILL -- HYDROPHILIC. 172 00:12:06,280 --> 00:12:09,440 IF YOU TEST WHETHER THEY CAN 173 00:12:09,440 --> 00:12:12,960 ENGAGE SEC61 AND INSERT INTO THE 174 00:12:12,960 --> 00:12:13,880 LIPID BILAYER, SEVERAL DON'T. 175 00:12:13,880 --> 00:12:17,000 THREE OUT OF SEVEN DON'T. 176 00:12:17,000 --> 00:12:18,640 IN MANY, MANY MULTI-PASS 177 00:12:18,640 --> 00:12:21,120 MEMBRANE PROTEINS AROUND HALF OF 178 00:12:21,120 --> 00:12:23,800 THE MEMBRANE PROTEIN SEGMENTS 179 00:12:23,800 --> 00:12:25,360 CAN'T ENGAGE SEC61 WHEN TESTED 180 00:12:25,360 --> 00:12:27,680 IN A MODEL SYSTEM RAISING THE 181 00:12:27,680 --> 00:12:30,400 PROBLEM OF HOW THESE SEGMENTS 182 00:12:30,400 --> 00:12:32,280 GET INSERTED. 183 00:12:32,280 --> 00:12:33,520 THE SECOND PROBLEM OF 184 00:12:33,520 --> 00:12:34,840 HYDROPHILIC PARTS OF A PROTEIN 185 00:12:34,840 --> 00:12:36,960 LIKE THIS IN THE MEMBRANE IS 186 00:12:36,960 --> 00:12:40,440 THESE PROTEINS ARE SYNTHESIZED 187 00:12:40,440 --> 00:12:43,320 CO-TRANSLATIONALLY, INSERTED 188 00:12:43,320 --> 00:12:43,760 CO-TRANSLATIONALLY. 189 00:12:43,760 --> 00:12:44,720 TRANSLATION IS RELATIVELY SLOW, 190 00:12:44,720 --> 00:12:50,960 YOU MAKE FIVE OR SIX AMINO ACIDS 191 00:12:50,960 --> 00:12:51,240 A SECOND. 192 00:12:51,240 --> 00:12:56,080 A 350 AMINO ACID PASSED PROTEIN 193 00:12:56,080 --> 00:12:59,600 WILL TAKE OFF SECONDS TO GET 194 00:12:59,600 --> 00:13:00,880 MADE, THEY ARE WAITING FOR QUITE 195 00:13:00,880 --> 00:13:03,640 A LONG TIME, ON THE SCALE OF 196 00:13:03,640 --> 00:13:06,080 MOLECULAR DYNAMICS TENS OF 197 00:13:06,080 --> 00:13:08,680 SECONDS IS AN ENORMITY, A VERY 198 00:13:08,680 --> 00:13:10,520 LONG TIME. 199 00:13:10,520 --> 00:13:12,520 HOW HAD HYDROPHILIC SEGMENTS 200 00:13:12,520 --> 00:13:23,040 STABILIZED UNTIL YOU MAKE THE -- 201 00:13:28,600 --> 00:13:30,320 WE TRIED TO UNDERSTAND THIS, ONE 202 00:13:30,320 --> 00:13:32,440 CLASSIC APPROACH TO TAKE A 203 00:13:32,440 --> 00:13:33,760 BIOCHEMICAL STRATEGY, AND THE 204 00:13:33,760 --> 00:13:36,080 APPROACH HERE IS TO TAKE A CRUDE 205 00:13:36,080 --> 00:13:38,320 EXTRACT THAT CAN SYNTHESIZE 206 00:13:38,320 --> 00:13:42,880 PROTEINS, IN THIS CASE AN 207 00:13:42,880 --> 00:13:44,360 EXTRACT MADE FROM LYSATE, ADD 208 00:13:44,360 --> 00:13:47,360 AN mRNA THAT CODES FOR PROTEIN 209 00:13:47,360 --> 00:13:52,160 YOU'RE INTERESTED IN, THE IDEA 210 00:13:52,160 --> 00:14:02,440 IS THAT WOULD GET 211 00:14:04,920 --> 00:14:05,200 GPCR. 212 00:14:05,200 --> 00:14:09,040 SO IF YOU TRANSLATE THE PROTEIN, 213 00:14:09,040 --> 00:14:11,400 IN THIS IN VITRO REACTION, YOU 214 00:14:11,400 --> 00:14:15,360 PRODUCE A PRODUCT THAT CODES FOR 215 00:14:15,360 --> 00:14:21,360 THE GPCR, IN THIS WAYS BETA 1 216 00:14:21,360 --> 00:14:23,640 RECEPTOR, IN PRESENCE YOU CAN 217 00:14:23,640 --> 00:14:26,400 SEE POLYPEPTIDEA FAIR AMOUNT, 218 00:14:26,400 --> 00:14:27,480 SHIFTS TO HIGHER MOLECULAR 219 00:14:27,480 --> 00:14:29,000 WEIGHT, THAT TURNS OUT IS 220 00:14:29,000 --> 00:14:29,800 GLYCOSYLATION, THAT TELLS YOU 221 00:14:29,800 --> 00:14:31,320 THAT AT LEAST THE BEGINNING PART 222 00:14:31,320 --> 00:14:34,960 OF THE PROTEIN GOT INSERTED INTO 223 00:14:34,960 --> 00:14:35,600 THE LIPID BILAYER. 224 00:14:35,600 --> 00:14:46,280 WE SHOWED THE OTHER PARTS WERE S 225 00:14:51,160 --> 00:14:54,760 NOT ONLY BEEN INSERTED BUT 226 00:14:54,760 --> 00:14:55,160 FOLDED CORRECTLY. 227 00:14:55,160 --> 00:14:56,720 IF YOU IMMOBILIZE A LIGAND ON 228 00:14:56,720 --> 00:14:59,000 THIS CLASS FOR SOME BEADS AND DO 229 00:14:59,000 --> 00:15:00,760 A PULLDOWN, YOU CAN APPRECIATE 230 00:15:00,760 --> 00:15:03,200 THAT ONLY THE INSERTED AND 231 00:15:03,200 --> 00:15:05,120 GLYCOSYLATED POPULATION GETS 232 00:15:05,120 --> 00:15:05,440 PULLED OUT. 233 00:15:05,440 --> 00:15:06,760 SO, WHAT THIS TELLS YOU IS THAT 234 00:15:06,760 --> 00:15:09,240 IN THIS IN VITRO REACTION, EVEN 235 00:15:09,240 --> 00:15:12,160 THOUGH IT DOESN'T NECESSARILY 236 00:15:12,160 --> 00:15:14,920 WORK TO 100% EFFICIENCY, ALL THE 237 00:15:14,920 --> 00:15:16,560 COMPONENTS THAT ARE NEEDED TO 238 00:15:16,560 --> 00:15:19,480 SYNTHESIZE THE PROTEIN TAKE THE 239 00:15:19,480 --> 00:15:25,160 RIBOSOME TO THE ER, INSERT 240 00:15:25,160 --> 00:15:26,320 TRANSMEMBRANE SEGMENTS, THIS IS 241 00:15:26,320 --> 00:15:28,040 AT LEAST A COMPLETE SYSTEM AND 242 00:15:28,040 --> 00:15:29,200 THE TRICK IS TO FIGURE OUT 243 00:15:29,200 --> 00:15:30,440 WHAT'S INVOLVED. 244 00:15:30,440 --> 00:15:32,200 WE FACE TWO BIG PROBLEMS. 245 00:15:32,200 --> 00:15:34,280 HOW DO YOU ACTUALLY FIND THE 246 00:15:34,280 --> 00:15:36,680 FACTORS OF A PROCESS THAT OCCURS 247 00:15:36,680 --> 00:15:39,840 CO-TRANSLATIONALLY AND IS VERY 248 00:15:39,840 --> 00:15:40,440 DYNAMIC? 249 00:15:40,440 --> 00:15:41,160 THE SECOND AND MORE CHALLENGING 250 00:15:41,160 --> 00:15:43,240 PROBLEM IS HOW DO YOU CONVINCE 251 00:15:43,240 --> 00:15:45,000 SOMEBODY TO LOOK FOR FACTORS 252 00:15:45,000 --> 00:15:47,080 WHEN ALL OF THE TEXT BOOKS TELL 253 00:15:47,080 --> 00:15:51,000 YOU THAT EVERYTHING IS ALREADY 254 00:15:51,000 --> 00:15:52,520 KNOWN, RIGHT? 255 00:15:52,520 --> 00:15:53,840 AND HERE WE BASICALLY WERE 256 00:15:53,840 --> 00:15:55,200 STUMPED, RIGHT? 257 00:15:55,200 --> 00:15:56,720 SO IT WAS DIFFICULT TO KIND OF 258 00:15:56,720 --> 00:15:58,280 CONVINCE PEOPLE TO WORK ON THIS 259 00:15:58,280 --> 00:16:01,760 AND OF COURSE WE DIDN'T HAVE 260 00:16:01,760 --> 00:16:03,840 GREAT IDEAS HOW TO FIGURE THIS 261 00:16:03,840 --> 00:16:05,440 OUT. 262 00:16:05,440 --> 00:16:07,640 AT THIS POINT I'M REMINDED OF A 263 00:16:07,640 --> 00:16:10,720 BOOK GIVEN TO ME BY MY DAD 264 00:16:10,720 --> 00:16:12,880 GROWING UP, "HOW TO SOLVE IT," 265 00:16:12,880 --> 00:16:14,920 IT'S A MATHEMATICS BOOK, HE WAS 266 00:16:14,920 --> 00:16:16,320 A MATHEMATICIAN. 267 00:16:16,320 --> 00:16:18,400 IT'S HOW TO SOLVE MATHEMATICAL 268 00:16:18,400 --> 00:16:18,840 PROBLEMS. 269 00:16:18,840 --> 00:16:21,440 AND ONE OF THE KEY PRINCIPLES IN 270 00:16:21,440 --> 00:16:23,400 THIS BOOK IS IF YOU CAN'T SOLVE 271 00:16:23,400 --> 00:16:25,080 THE PROBLEM YOU'RE INTERESTED 272 00:16:25,080 --> 00:16:27,480 IN, TRY TO SOLVE A SIMPLER 273 00:16:27,480 --> 00:16:28,440 RELATED PROBLEM. 274 00:16:28,440 --> 00:16:31,000 AND THE HOPE IS THAT IF THE 275 00:16:31,000 --> 00:16:32,160 PROBLEM IS RELATED ENOUGH, 276 00:16:32,160 --> 00:16:33,160 INSIGHTS FROM THE SIMPLER 277 00:16:33,160 --> 00:16:35,760 VERSION WILL OF COURSE GIVE YOU 278 00:16:35,760 --> 00:16:37,080 SOME TOE HOLD INTO THE PROBLEM 279 00:16:37,080 --> 00:16:39,040 THAT WILL HELP YOU SOLVE THE 280 00:16:39,040 --> 00:16:39,840 MORE COMPLICATED ONE. 281 00:16:39,840 --> 00:16:41,160 WE OF COURSE USE THIS ALL THE 282 00:16:41,160 --> 00:16:41,720 TIME. 283 00:16:41,720 --> 00:16:43,320 THIS IS THE BASIS OF MODEL 284 00:16:43,320 --> 00:16:45,440 SYSTEMS, WHY WE MAKE SIMPLE 285 00:16:45,440 --> 00:16:48,200 VERSIONS OF CONSTRUCTS AND SO 286 00:16:48,200 --> 00:16:49,160 FORTH BUT WORTH REMINDING 287 00:16:49,160 --> 00:16:57,080 OURSELVES ONCE IN A WHILE. 288 00:16:57,080 --> 00:16:57,920 MEMBRANE PROTEINS COME IN 289 00:16:57,920 --> 00:17:00,720 VARIETIES AND FLAVORS, A COUPLE 290 00:17:00,720 --> 00:17:04,120 LABELED HERE, GPCR IN THE 291 00:17:04,120 --> 00:17:04,360 MIDDLE. 292 00:17:04,360 --> 00:17:05,720 ABC TRANSPORTERS, RECEPTOR 293 00:17:05,720 --> 00:17:07,080 TYROSINE KINASES, AND SO FORTH. 294 00:17:07,080 --> 00:17:14,760 A NUMBER OF YEARS AGO WE GOT 295 00:17:14,760 --> 00:17:15,760 INTERESTED IN UNDERSTANDING HOW 296 00:17:15,760 --> 00:17:20,600 THE CLASS OF PROTEINS GET MADE, 297 00:17:20,600 --> 00:17:21,640 AN IMPORTANT SUBSET ARE SNARE 298 00:17:21,640 --> 00:17:24,200 PROTEIN INVOLVED IN PROTEIN 299 00:17:24,200 --> 00:17:25,520 TRAFFICKING WITH A SINGLE 300 00:17:25,520 --> 00:17:27,360 SEGMENT, IT WASN'T CLEAR HOW 301 00:17:27,360 --> 00:17:29,240 THEY GET TARGETED TO THE 302 00:17:29,240 --> 00:17:30,320 MEMBRANE AND INSERTED. 303 00:17:30,320 --> 00:17:31,760 IT MIGHT SEEM LIKE INVOLVING 304 00:17:31,760 --> 00:17:33,320 THAT PROBLEM WOULD GIVE US 305 00:17:33,320 --> 00:17:37,360 INSIGHT INTO THESE OTHERS WAS A 306 00:17:37,360 --> 00:17:39,200 BIT FANCIFUL, THAT'S THE CASE 307 00:17:39,200 --> 00:17:41,800 AND I'LL EXPLAIN WHY. 308 00:17:41,800 --> 00:17:44,360 SO, THIS GOES BACK QUITE MANY 309 00:17:44,360 --> 00:17:46,400 YEARS NOW, BUT HOW THIS WORKS 310 00:17:46,400 --> 00:17:49,000 WAS REALLY UNCLEAR, AND WHAT WE 311 00:17:49,000 --> 00:17:51,760 DID WAS RESEARCHED FOR FACTORS 312 00:17:51,760 --> 00:17:54,640 THAT RECOGNIZED THIS HYDROPHOBIC 313 00:17:54,640 --> 00:17:55,840 TRANSMEMBRANE SEGMENT AND 314 00:17:55,840 --> 00:17:56,480 DELIVER PROTEIN TO THE MEMBRANE 315 00:17:56,480 --> 00:17:58,720 WHERE IT NEEDS TO BE INSERTED. 316 00:17:58,720 --> 00:18:00,640 USING A BIOCHEMICAL STRATEGY WE 317 00:18:00,640 --> 00:18:04,520 IDENTIFIED A FACTOR IN MAMMALIAN 318 00:18:04,520 --> 00:18:06,240 CELLS WHICH IS ATPase WIDELY 319 00:18:06,240 --> 00:18:08,000 CONSERVED, IT SEEMED OF SEEMS TO 320 00:18:08,000 --> 00:18:09,080 BE TARGETING FACTOR THAT 321 00:18:09,080 --> 00:18:11,960 DELIVERS IT TO THE E.R. FOR 322 00:18:11,960 --> 00:18:12,360 INSERTION. 323 00:18:12,360 --> 00:18:13,480 AND THE REASON THIS WAS AN 324 00:18:13,480 --> 00:18:14,360 IMPORTANT ADVANCE IS BECAUSE 325 00:18:14,360 --> 00:18:15,880 ONCE YOU KNOW THE FIRST 326 00:18:15,880 --> 00:18:17,320 COMPONENT OF ANY PATHWAY, YOU 327 00:18:17,320 --> 00:18:25,720 CAN APPLY LOTS OF OTHER TOOLS, 328 00:18:25,720 --> 00:18:26,360 GENETIC TOOLS, BIOCHEMICALS 329 00:18:26,360 --> 00:18:28,640 TOOLS AND SO FORTH TO WORK OUT 330 00:18:28,640 --> 00:18:29,000 PATHWAY. 331 00:18:29,000 --> 00:18:30,680 WE NOW HAVE A COMPLETE PICTURE 332 00:18:30,680 --> 00:18:34,480 OF HOW THIS WORKS INCLUDING 333 00:18:34,480 --> 00:18:35,920 STRUCTURES, RECONSTITUTED WITH 334 00:18:35,920 --> 00:18:36,600 PURIFIED COMPONENTS AND SO FORTH 335 00:18:36,600 --> 00:18:39,760 BUT ONE OF THE KEY INSIGHTS IS 336 00:18:39,760 --> 00:18:41,520 AT THE MEMBRANE RECEPTOR FOR 337 00:18:41,520 --> 00:18:45,560 THIS TARGETING FACTOR IS A 338 00:18:45,560 --> 00:18:46,960 TWO-PROTEIN COMPLEX THAT 339 00:18:46,960 --> 00:18:48,360 RELEASES THE SUBSTRATE FROM THIS 340 00:18:48,360 --> 00:18:50,680 TARGETING FACTOR AND THEN 341 00:18:50,680 --> 00:18:52,000 FACILITATES ITS INSERTION INTO 342 00:18:52,000 --> 00:18:53,640 THE BILAYER. 343 00:18:53,640 --> 00:18:55,840 THE REASON THIS WAS IMPORTANT TO 344 00:18:55,840 --> 00:18:56,840 UNDERSTAND AND THESE ARE THE 345 00:18:56,840 --> 00:18:59,880 NAMES OF THE YEAST PROTEINS, GET 346 00:18:59,880 --> 00:19:02,000 1, GET 2, GET 3, I SHOULD SAY A 347 00:19:02,000 --> 00:19:03,280 NUMBER OF GROUPS AROUND THE 348 00:19:03,280 --> 00:19:06,320 WORLD CONTRIBUTED TO WORKING OUT 349 00:19:06,320 --> 00:19:10,080 THIS PATHWAY, IS THAT THESE TWO 350 00:19:10,080 --> 00:19:11,880 PROTEINS ALTHOUGH INITIALLY 351 00:19:11,880 --> 00:19:13,920 DIFFICULT TO FIND HOMOLOGS OF 352 00:19:13,920 --> 00:19:17,080 IT, WITH BETTER METHODS OF 353 00:19:17,080 --> 00:19:19,720 SEARCHING SUCH AS STRUCTURE 354 00:19:19,720 --> 00:19:24,800 BASED METHODS LIKE HH PRED COULD 355 00:19:24,800 --> 00:19:25,440 APPRECIATE THERE WERE HOMOLOGS, 356 00:19:25,440 --> 00:19:29,120 IN THE MAMMALIAN TWO OTHER 357 00:19:29,120 --> 00:19:33,720 HOMOLOGS, TWO THE CENTRAL PART 358 00:19:33,720 --> 00:19:37,120 OF A MUCH LARGER COMPLEX, EMC, 359 00:19:37,120 --> 00:19:40,520 AND THERE WAS A VERY POORLY 360 00:19:40,520 --> 00:19:41,840 UNDERSTOOD PAIR OF PROTEINS THAT 361 00:19:41,840 --> 00:19:44,480 LATER WERE SHOWN TO FORM A 362 00:19:44,480 --> 00:19:46,680 COMPLEX, AND THAT WE NAMED THE 363 00:19:46,680 --> 00:19:48,640 GEL COMPLEX, AND I'LL COME BACK 364 00:19:48,640 --> 00:19:49,120 TO THAT. 365 00:19:49,120 --> 00:19:52,000 OF COURSE, IF THIS IS AN 366 00:19:52,000 --> 00:19:53,440 INSERTION FACTOR FOR INSERTING 367 00:19:53,440 --> 00:19:55,280 TRANSMEMBRANE SEGMENT INTO THE 368 00:19:55,280 --> 00:19:56,360 BILAYER, IT STANDS TO REASON 369 00:19:56,360 --> 00:19:58,240 THESE OTHERS MIGHT HAVE A 370 00:19:58,240 --> 00:20:01,160 SIMILAR FUNCTION IF THEY ARE 371 00:20:01,160 --> 00:20:03,360 STRUCTURALLY SIMILAR. 372 00:20:03,360 --> 00:20:06,040 AND SO, MY LAB DECIDED TO 373 00:20:06,040 --> 00:20:08,840 INVESTIGATE THIS FOR EMC, AND 374 00:20:08,840 --> 00:20:10,080 OUR COLLABORATOR BOB KEENAN WENT 375 00:20:10,080 --> 00:20:12,600 TO WORK TO STUDY WHAT THE OTHER 376 00:20:12,600 --> 00:20:14,920 COMPLEX, THE GEL COMPLEX, DOES. 377 00:20:14,920 --> 00:20:16,640 AND THIS WINDS UP CONVERGING A 378 00:20:16,640 --> 00:20:18,600 BIT LATER. 379 00:20:18,600 --> 00:20:20,600 SO, WHAT WE FOUND, THE OTHER 380 00:20:20,600 --> 00:20:23,400 IMPORTANT THING TO SAY, I SHOULD 381 00:20:23,400 --> 00:20:25,800 GO BACK HERE, IS THAT THESE ARE 382 00:20:25,800 --> 00:20:27,360 THE MAMMALIAN PROTEINS AND 383 00:20:27,360 --> 00:20:27,680 NAMES. 384 00:20:27,680 --> 00:20:29,760 BUT IT TURNS OUT A REALLY 385 00:20:29,760 --> 00:20:32,280 IMPORTANT INSIGHT FROM THIS KIND 386 00:20:32,280 --> 00:20:34,440 OF BIOINFORMATIC ANALYSIS IS 387 00:20:34,440 --> 00:20:36,600 THAT IT APPEARED THAT THE GET1 388 00:20:36,600 --> 00:20:39,360 WAS RELATED TO A BACTERIAL 389 00:20:39,360 --> 00:20:44,880 PROTEIN, AND THE BACTERIAL 390 00:20:44,880 --> 00:20:47,040 PROTEIN EMC, LINKED COMPONENTS 391 00:20:47,040 --> 00:20:49,560 IN BACTERIA WITH COMPONENTS IN 392 00:20:49,560 --> 00:20:51,120 EUKARYOTES, AND SEEMED LIKE THIS 393 00:20:51,120 --> 00:20:54,560 WAS A VERY UNIVERSAL FAMILY AND 394 00:20:54,560 --> 00:20:58,880 IT'S NOW KNOWN AS THE OXA 1 395 00:20:58,880 --> 00:20:59,920 FAMILY, NOMENCLATURE IS TERRIBLE 396 00:20:59,920 --> 00:21:01,480 BUT NOT THAT IMPORTANT FOR OUR 397 00:21:01,480 --> 00:21:02,240 DISCUSSION. 398 00:21:02,240 --> 00:21:07,160 WHAT'S KNOWN FROM STUDYING THIS 399 00:21:07,160 --> 00:21:09,440 FAMILY, THE E. COLI PROTEIN 400 00:21:09,440 --> 00:21:11,440 LOOKS LIKE THIS, A BEAUTIFUL 401 00:21:11,440 --> 00:21:15,160 STRUCTURE, THE WAY IT MEDIATES 402 00:21:15,160 --> 00:21:16,080 TRANSMEMBRANE DOMAIN INSERTION 403 00:21:16,080 --> 00:21:17,880 IS HYPOTHESIZED TO BE THE 404 00:21:17,880 --> 00:21:18,280 FOLLOWING. 405 00:21:18,280 --> 00:21:23,920 REMEMBER THAT AT THE BEGINNING I 406 00:21:23,920 --> 00:21:25,320 MENTIONED THAT HYDROPHOBIC 407 00:21:25,320 --> 00:21:27,360 DOMAINS WILL SPONTANEOUSLY 408 00:21:27,360 --> 00:21:29,720 INSERT INTO THE LIPID BILAYER 409 00:21:29,720 --> 00:21:31,880 BUT BARRIER IS THE TRANSLOCATION 410 00:21:31,880 --> 00:21:33,360 OF THE HYDROPHILIC PART ACROSS 411 00:21:33,360 --> 00:21:35,400 THE MEMBRANE SO THE ENERGY 412 00:21:35,400 --> 00:21:46,000 GAINED BY PARTITIONING THIS THS 413 00:21:47,720 --> 00:21:50,480 HYDROPHOBIC, REDUCES THE ENERGY 414 00:21:50,480 --> 00:21:52,080 BARRIER FOR THE HYDROPHOBIC 415 00:21:52,080 --> 00:21:52,480 PART. 416 00:21:52,480 --> 00:21:54,360 THE PROTEIN IS STRUCTURED THAT 417 00:21:54,360 --> 00:21:55,760 IT HAS THIS VESTIBULE THAT GOES 418 00:21:55,760 --> 00:22:00,360 PART OF THE WAY ACROSS THE 419 00:22:00,360 --> 00:22:01,880 MEMBRANE SO YOU CAN IMAGINE THE 420 00:22:01,880 --> 00:22:03,840 LIPID BILAYER WILL BE A BIT 421 00:22:03,840 --> 00:22:05,200 THINNER NEAR THE VESTIBULE. 422 00:22:05,200 --> 00:22:07,800 SO IT REDUCES THE BARRIER TO 423 00:22:07,800 --> 00:22:14,320 TRANSLOCATION WHICH FACILITATES 424 00:22:14,320 --> 00:22:16,600 THIS INSERTION REACTION. 425 00:22:16,600 --> 00:22:18,200 THERE'S REASONABLE SUPPORT BUT 426 00:22:18,200 --> 00:22:26,000 RIGOROUS PROOF STILL AWAITS MORE 427 00:22:26,000 --> 00:22:27,400 STUDIES. 428 00:22:27,400 --> 00:22:30,120 EMC MEDIATES THIS REACTION. 429 00:22:30,120 --> 00:22:35,120 WE COULD SHOW THIS IN DIFFERENT 430 00:22:35,120 --> 00:22:37,800 CONTEXTS BUT THE ONE IMPORTANT 431 00:22:37,800 --> 00:22:44,000 FOR GPCR DOES THE JOB FOR THE 432 00:22:44,000 --> 00:22:46,760 FIRST SEGMENT. 433 00:22:46,760 --> 00:22:48,520 PATRICK CHITWOOD SHOWS EMC IS 434 00:22:48,520 --> 00:22:50,960 REQUIRED TO INSERT INTO THE 435 00:22:50,960 --> 00:22:52,760 LIPID BILAYER AS LONG AS THE 436 00:22:52,760 --> 00:22:54,160 PART THAT GOES ACROSS THE 437 00:22:54,160 --> 00:22:57,120 MEMBRANE IS SHORT, THE CASE FOR 438 00:22:57,120 --> 00:22:58,040 90% OF GPCRs. 439 00:22:58,040 --> 00:22:59,600 THIS DEMONSTRATES THIS WHERE WE 440 00:22:59,600 --> 00:23:03,120 MADE CELL LINES THAT ARE LACKING 441 00:23:03,120 --> 00:23:04,560 EMC BY DELETING A 442 00:23:04,560 --> 00:23:06,880 SUBOPPORTUNITY, AND THIS IS A 443 00:23:06,880 --> 00:23:07,560 FLOW CYTOMETRY ASSAY THAT 444 00:23:07,560 --> 00:23:12,880 MEASURES HOW THE OF THE BETA 1 445 00:23:12,880 --> 00:23:14,000 ADRENERGIC RECEPTOR GETS 446 00:23:14,000 --> 00:23:16,640 PRODUCED, IN THE PRODUCTION IS 447 00:23:16,640 --> 00:23:18,400 REDUCED BY 50%, IN EMC 448 00:23:18,400 --> 00:23:21,000 KNOCKOUTS, THAT CAN BE RESCUED 449 00:23:21,000 --> 00:23:22,840 BY REINTRODUCING COMPLEX BACK 450 00:23:22,840 --> 00:23:25,240 INTO THE CELLS. 451 00:23:25,240 --> 00:23:29,640 WE CAN RECONSTITUTE WITH 452 00:23:29,640 --> 00:23:31,200 PURIFIED COMPONENTS, EMC WAS 453 00:23:31,200 --> 00:23:33,080 SUFFICIENT AND LATER STUDIES 454 00:23:33,080 --> 00:23:40,640 COULD MUTATE CERTAIN RESIDUES TO 455 00:23:40,640 --> 00:23:44,920 SEE HOW THIS WORKS. 456 00:23:44,920 --> 00:23:47,280 EMC MEDIATES THE FIRST SEGMENT 457 00:23:47,280 --> 00:23:51,400 OF THE GPCR, FOR EXAMPLE, AND 458 00:23:51,400 --> 00:23:56,240 THE RIBOSOME DOCKS ON THIS SEC 459 00:23:56,240 --> 00:23:57,560 61 WHERE ADDITIONAL STUFF 460 00:23:57,560 --> 00:23:57,920 HAPPENS. 461 00:23:57,920 --> 00:24:00,880 IT WAS IMPORTANT TO ASSIGN A 462 00:24:00,880 --> 00:24:07,640 FUNCTION TO EMC, IMPORTANT FOR 463 00:24:07,640 --> 00:24:10,160 GETTING INSIGHT 2000 GPCRs, 464 00:24:10,160 --> 00:24:11,800 MORE IMPORTANT FOR ILLUSTRATING 465 00:24:11,800 --> 00:24:13,760 SEC61 IS NOT THE ONLY COMPONENT 466 00:24:13,760 --> 00:24:14,800 THAT DOES EVERYTHING. 467 00:24:14,800 --> 00:24:16,440 I THINK THAT IF YOU WOULD HAVE 468 00:24:16,440 --> 00:24:19,360 ASKED ANYONE, THEY WOULD HAVE 469 00:24:19,360 --> 00:24:21,560 SAID THIS TRANSMEMBRANE DOMAIN 470 00:24:21,560 --> 00:24:26,120 IS INSERTED BY SEC61 BUT 471 00:24:26,120 --> 00:24:27,960 INHIBITORS BLOCK THAT LATERAL 472 00:24:27,960 --> 00:24:29,400 DATE AND SEGMENTS GET INSERTED 473 00:24:29,400 --> 00:24:32,040 FINE IN THE PRESENCE OF 474 00:24:32,040 --> 00:24:32,480 INHIBITORS. 475 00:24:32,480 --> 00:24:33,120 THAT PSYCHOLOGICALLY CHANGED 476 00:24:33,120 --> 00:24:34,640 QUITE A BIT, RIGHT? 477 00:24:34,640 --> 00:24:36,680 IT ALLOWED FOR THE POSSIBILITY 478 00:24:36,680 --> 00:24:38,280 THAT THERE WERE FACTORS DESPITE 479 00:24:38,280 --> 00:24:40,600 THIS FIELD HAVING EXISTED FOR 480 00:24:40,600 --> 00:24:41,680 DECADES, THAT THERE WERE FACTORS 481 00:24:41,680 --> 00:24:42,960 WE DIDN'T KNOW ABOUT. 482 00:24:42,960 --> 00:24:44,840 THAT MADE IT MUCH EASIER FOR US 483 00:24:44,840 --> 00:24:46,960 TO BE MOTIVATED TO FIND 484 00:24:46,960 --> 00:24:48,120 ADDITIONAL COMPONENTS. 485 00:24:48,120 --> 00:24:50,080 AND SO THE NEXT QUESTION WAS 486 00:24:50,080 --> 00:24:52,360 AFTER YOU GET THAT FIRST 487 00:24:52,360 --> 00:24:53,360 TRANSMEMBRANE SEGMENT INSERTED 488 00:24:53,360 --> 00:24:56,040 WHAT THEN HAPPENS? 489 00:24:56,040 --> 00:24:57,920 AND WHAT WE SPECULATED IS THAT 490 00:24:57,920 --> 00:25:01,200 REMEMBER THAT MANY OF THESE 491 00:25:01,200 --> 00:25:03,960 HYDROPHOBIC SEGMENTS ARE 492 00:25:03,960 --> 00:25:04,480 PEPPERED WITH HYDROPHILIC 493 00:25:04,480 --> 00:25:07,560 RESIDUES, THERE MUST BE A 494 00:25:07,560 --> 00:25:11,080 CHAPERONE THAT TEMPORARY 495 00:25:11,080 --> 00:25:12,960 STABILIZED INTERMEDIATES WHILE 496 00:25:12,960 --> 00:25:15,560 YOU SYNTHESIZE THE REST OF THE 497 00:25:15,560 --> 00:25:19,400 PROTEIN UNTIL IT HOLDS TOGETHER, 498 00:25:19,400 --> 00:25:20,560 ANALOGOUS IN THE CYTOSOL, WHERE 499 00:25:20,560 --> 00:25:21,920 THEY BIND UNTIL YOU FOLD THE 500 00:25:21,920 --> 00:25:25,560 WHOLE THING, MAKE THE WHOLE 501 00:25:25,560 --> 00:25:27,080 PROTEIN AND THEN EVENTUALLY FOLD 502 00:25:27,080 --> 00:25:28,200 CHAPERONES IN THE MEMBRANE WOULD 503 00:25:28,200 --> 00:25:29,560 MAKE SENSE BUT WE DIDN'T KNOW 504 00:25:29,560 --> 00:25:32,760 WHAT THEY WERE. 505 00:25:32,760 --> 00:25:35,480 SO, PATRICK MOTIVATED BY THE 506 00:25:35,480 --> 00:25:38,440 POSSIBILITY OF ADDITIONAL 507 00:25:38,440 --> 00:25:39,520 FACTORS USED A CROSS-LINKING 508 00:25:39,520 --> 00:25:40,920 STRATEGY TO IDENTIFY SUCH 509 00:25:40,920 --> 00:25:42,120 CHAPERONES. 510 00:25:42,120 --> 00:25:43,360 THE EXPERIMENT IS A 511 00:25:43,360 --> 00:25:44,480 STRAIGHTFORWARD ONE. 512 00:25:44,480 --> 00:25:47,080 HE USED THIS IN VITRO 513 00:25:47,080 --> 00:25:48,600 TRANSLATION SYSTEM TO SYNTHESIZE 514 00:25:48,600 --> 00:25:51,160 A PROTEIN UP TO THE POINT WHERE 515 00:25:51,160 --> 00:25:52,360 THE FIRST TRANSMEMBRANE SEGMENT 516 00:25:52,360 --> 00:25:53,560 HAD BEEN SYNTHESIZED AND 517 00:25:53,560 --> 00:25:55,320 INHERITED BUT NO FURTHER. 518 00:25:55,320 --> 00:25:57,680 AND THERE'S SOME TRICKS ONE CAN 519 00:25:57,680 --> 00:26:00,080 USE IN IN VITRO TRANSLATION TO 520 00:26:00,080 --> 00:26:04,160 GENERATE SUCH A STALLED 521 00:26:04,160 --> 00:26:04,800 INTERMEDIATE USING TRUNCATED 522 00:26:04,800 --> 00:26:08,560 mRNA WITHOUT A STOP CODE. 523 00:26:08,560 --> 00:26:11,640 WHAT IS ADJACENT TO THE SEGMENT, 524 00:26:11,640 --> 00:26:15,240 USING CYSTEINE REACTIVE 525 00:26:15,240 --> 00:26:15,920 CROSS-LINKING, THE YELLOW 526 00:26:15,920 --> 00:26:19,200 CIRCLE, BUT LATER COULD SHOW 527 00:26:19,200 --> 00:26:23,360 THIS USING SITE-SPECIFIC PHOTO 528 00:26:23,360 --> 00:26:24,560 CROSS-LINKING USING AMINO ACIDS. 529 00:26:24,560 --> 00:26:27,640 THE EXPERIMENT IS BEAUTIFUL IN 530 00:26:27,640 --> 00:26:27,960 SIMPLICITY. 531 00:26:27,960 --> 00:26:29,600 ONE ASSEMBLED WITH OR WITHOUT 532 00:26:29,600 --> 00:26:32,120 THE CYSTEINE IN THIS 533 00:26:32,120 --> 00:26:34,320 TRANSMEMBRANE SEGMENT, ADD A 534 00:26:34,320 --> 00:26:35,840 CYSTEINE REACTIVE CROSS-LINKER, 535 00:26:35,840 --> 00:26:40,160 ONE CAN SEE THERE'S A VERY 536 00:26:40,160 --> 00:26:43,000 PROMINENT UNKNOWN CROSS-LINKED 537 00:26:43,000 --> 00:26:45,160 THAT CROSS-LINKS TO GLYCOSYLATED 538 00:26:45,160 --> 00:26:47,360 INSERTED TRANSMEMBRANE SEGMENT. 539 00:26:47,360 --> 00:26:49,440 YOU SEE SOME CROSS-LINKS TO 540 00:26:49,440 --> 00:26:51,600 SEC61 WHICH WE KNOW IS NEARBY. 541 00:26:51,600 --> 00:26:53,920 IT TOOK A WHILE TO FIGURE OUT 542 00:26:53,920 --> 00:26:56,640 WHAT THIS CROSS-LINK IS, SKILLED 543 00:26:56,640 --> 00:26:58,360 UP, MASS SPEC, DIDN'T INITIALLY 544 00:26:58,360 --> 00:27:00,240 IDENTIFY THE CROSS-LINK WE 545 00:27:00,240 --> 00:27:01,600 IDENTIFIED A PARTNER THAT IT WAS 546 00:27:01,600 --> 00:27:03,000 ASSOCIATED WITH AND THEN USED 547 00:27:03,000 --> 00:27:05,760 THE PARTNER TO FIGURE OUT WHAT 548 00:27:05,760 --> 00:27:07,520 THE COMPLEX WAS. 549 00:27:07,520 --> 00:27:11,200 AND IT TURNS OUT THAT IT 550 00:27:11,200 --> 00:27:15,800 CROSS-LINKS TO A TWO-PROTEIN 551 00:27:15,800 --> 00:27:18,320 COMPLEX VIA SMALL SUBUNIT, 552 00:27:18,320 --> 00:27:21,480 ASTERIX, AND LARGE IS CCD47, THE 553 00:27:21,480 --> 00:27:22,240 FUNCTION OF NEITHER WAS KNOWN AT 554 00:27:22,240 --> 00:27:23,440 THE TIME. 555 00:27:23,440 --> 00:27:25,520 WHAT WE COULD SHOW BY THIS 556 00:27:25,520 --> 00:27:27,160 CROSS-LINKING IS IT'S ADJACENT 557 00:27:27,160 --> 00:27:28,360 TO A TRANSMEMBRANE SEGMENT 558 00:27:28,360 --> 00:27:30,240 THAT'S JUST BEEN INSERTED INTO 559 00:27:30,240 --> 00:27:35,160 THE LIPID BILAYER, AND OBVIOUSLY 560 00:27:35,160 --> 00:27:36,360 ITS FUNCTION WASN'T CLEAR. 561 00:27:36,360 --> 00:27:37,760 WHAT WE COULD FURTHER SHOW IS 562 00:27:37,760 --> 00:27:40,960 THAT IF WE MOVE THAT 563 00:27:40,960 --> 00:27:42,600 CROSS-LINKER, FOR EXAMPLE, 564 00:27:42,600 --> 00:27:44,400 PHOTOCROSS-LINKER TO VARIOUS 565 00:27:44,400 --> 00:27:49,120 POSITIONS ALONG THE POLYPEPTIDE 566 00:27:49,120 --> 00:27:51,040 CROSS-LINKS ARE ONLY SEEN IN THE 567 00:27:51,040 --> 00:27:51,800 TRAN MEMBRANE SEGMENT, 568 00:27:51,800 --> 00:27:53,400 RECOGNIZING IN THE LIPID 569 00:27:53,400 --> 00:27:55,720 BILAYER, OBVIOUSLY THE KIND OF 570 00:27:55,720 --> 00:27:57,840 PROTEIN WE WERE LOOKING FOR, A 571 00:27:57,840 --> 00:27:59,560 CHAPERONE IN THE MEMBRANE. 572 00:27:59,560 --> 00:28:01,640 THE SECOND THING IS THAT FIRST 573 00:28:01,640 --> 00:28:03,440 TRANSMEMBRANE SEGMENT TURNS OUT 574 00:28:03,440 --> 00:28:05,560 HAS A COUPLE HYDROPHILIC 575 00:28:05,560 --> 00:28:08,000 RESIDUES, IF YOU CHANGE THOSE TO 576 00:28:08,000 --> 00:28:10,000 HYDROPHOBIC, THEN YOU NO LONGER 577 00:28:10,000 --> 00:28:11,400 SEE THIS INTERACTION. 578 00:28:11,400 --> 00:28:13,040 AND IF YOU PUT HYDROPHILIC 579 00:28:13,040 --> 00:28:14,680 RESIDUES IN OTHER PARTS OF THE 580 00:28:14,680 --> 00:28:15,560 TRANSMEMBRANE SEGMENT YOU CAN 581 00:28:15,560 --> 00:28:16,520 GET BACK THE INTERACTION. 582 00:28:16,520 --> 00:28:19,160 WHAT WE LEARNED FROM THAT IS 583 00:28:19,160 --> 00:28:21,760 THAT THIS FACTOR IS RECOGNIZING 584 00:28:21,760 --> 00:28:26,120 TRANSMEMBRANE SEGMENTS IN THE 585 00:28:26,120 --> 00:28:29,520 LIPID BILAYER VIA PARTIAL 586 00:28:29,520 --> 00:28:34,680 HYDROPHILICICT, HELPING WITH 587 00:28:34,680 --> 00:28:37,840 UNDERSTANDING HOW THESE 588 00:28:37,840 --> 00:28:39,160 PARTIALLY HYDROPHOBIC SEGMENTS 589 00:28:39,160 --> 00:28:41,320 MAY BE STABILIZED UNTIL YOU MAKE 590 00:28:41,320 --> 00:28:41,640 THE PROTEIN. 591 00:28:41,640 --> 00:28:43,200 THE COMPLEX IS CONSERVED IN 592 00:28:43,200 --> 00:28:43,600 EUKARYOTES. 593 00:28:43,600 --> 00:28:46,360 IF YOU KNOCK IT OUT, THE CELLS 594 00:28:46,360 --> 00:28:48,760 ARE STILL ALIVE, AND AS ARE 595 00:28:48,760 --> 00:28:50,560 YEAST, BUT SEEMS TO CAUSE ER 596 00:28:50,560 --> 00:28:53,000 STRESS, WHAT YOU WOULD EXPECT 597 00:28:53,000 --> 00:28:54,960 FOR PROTEIN FOLDING PROBLEMS, IF 598 00:28:54,960 --> 00:28:59,400 YOU CAN'T ASSEMBLE MEMBRANE 599 00:28:59,400 --> 00:29:00,120 PROTEINS CORRECTLY. 600 00:29:00,120 --> 00:29:03,400 KNOCKOUT OF CCCD47 MOUSE IN 601 00:29:03,400 --> 00:29:05,440 EMBRYONIC LETHAL, AND THERE ARE 602 00:29:05,440 --> 00:29:10,080 POINT MUTANTS THAT HAVE BEEN 603 00:29:10,080 --> 00:29:15,600 DESCRIBED IN HUMANS PRESUMABLY 604 00:29:15,600 --> 00:29:16,960 HYPOMORPHIC ALLELES, APPEARS TO 605 00:29:16,960 --> 00:29:23,920 BE AN IMPORTANT WIDELY 606 00:29:23,920 --> 00:29:24,360 CONSERVES. 607 00:29:24,360 --> 00:29:25,600 THAT'S A GOOD SIGN. 608 00:29:25,600 --> 00:29:27,640 AND THEN IN FUNCTIONAL 609 00:29:27,640 --> 00:29:29,960 EXPERIMENTS, USING AGAIN OUR 610 00:29:29,960 --> 00:29:33,240 FLOW CYTOMETRY ASSAY, IT SEEMS 611 00:29:33,240 --> 00:29:36,640 TO REDUCE THE BIOGENESIS OF 612 00:29:36,640 --> 00:29:38,320 MULTI-PASS MEMBRANE PROTEINS, 613 00:29:38,320 --> 00:29:39,880 DIFFERENT CLASSES SHOWN HERE, 614 00:29:39,880 --> 00:29:41,680 BUT NOT SINGLE PASS MEMBRANE 615 00:29:41,680 --> 00:29:42,040 PROTEINS. 616 00:29:42,040 --> 00:29:43,640 REMEMBER, THIS CLASS IS THE ONE 617 00:29:43,640 --> 00:29:47,720 WE STARTED WITH, IT DEPENDS ON 618 00:29:47,720 --> 00:29:51,160 THE GET PATHWAY, AND SO THE 619 00:29:51,160 --> 00:29:52,040 MULTI-PASS PROTEINS ARE 620 00:29:52,040 --> 00:29:52,720 SELECTIVELY AFFECTED, THAT MAKES 621 00:29:52,720 --> 00:29:54,880 SENSE FOR THE KIND OF FACTORS 622 00:29:54,880 --> 00:29:58,600 WE'RE LOOKING FOR. 623 00:29:58,600 --> 00:30:01,520 SO, WHAT WHAT WE'RE LEFT WITH AT 624 00:30:01,520 --> 00:30:03,320 THIS POINT THERE'S SOME FACTOR 625 00:30:03,320 --> 00:30:05,080 THAT ENGAGES THE EARLY PARTS OF 626 00:30:05,080 --> 00:30:07,840 A MEMBRANE PROTEIN AND WE COULD 627 00:30:07,840 --> 00:30:09,800 SHOW IN EXPERIMENTS IT SEEMS TO 628 00:30:09,800 --> 00:30:11,440 REMAIN ASSOCIATED OR AT LEAST 629 00:30:11,440 --> 00:30:12,840 NEARBY UNTIL YOU MAKE THE REST 630 00:30:12,840 --> 00:30:14,680 OF THE PROTEIN AND THEN ONCE THE 631 00:30:14,680 --> 00:30:17,240 PROTEIN FOLDS IT'S NO LONGER 632 00:30:17,240 --> 00:30:17,520 ASSOCIATED. 633 00:30:17,520 --> 00:30:18,880 SO THAT KIND OF FITS THE IDEA 634 00:30:18,880 --> 00:30:22,280 THAT IT IS SOME TYPE OF 635 00:30:22,280 --> 00:30:23,080 CHAPERONE THAT OPERATES INSIDE 636 00:30:23,080 --> 00:30:23,720 THE MEMBRANE. 637 00:30:23,720 --> 00:30:26,720 IT SEEMS TO DO SO, IT'S NOT AN 638 00:30:26,720 --> 00:30:27,680 ENERGY DEPENDENT FACTORS, SEEMS 639 00:30:27,680 --> 00:30:33,200 TO DO SO BY BINDING EXPOSED 640 00:30:33,200 --> 00:30:34,400 HYDROPHILICITY IN THE MEMORY. 641 00:30:34,400 --> 00:30:36,240 THAT'S THE WORKING MODEL. 642 00:30:36,240 --> 00:30:37,320 HOW DOES IT WORK? 643 00:30:37,320 --> 00:30:43,040 WHAT DOES IT LOOK LIKE AND SO 644 00:30:43,040 --> 00:30:43,480 FORTH? 645 00:30:43,480 --> 00:30:45,880 SO, LUCCA, A Ph.D. STUDENT IN 646 00:30:45,880 --> 00:30:47,440 THE LAB, DECIDED TO INVESTIGATE. 647 00:30:47,440 --> 00:30:49,880 WHAT SHE DECIDED TO DO WAS FIRST 648 00:30:49,880 --> 00:30:58,960 ASK WHEN IS IT STABLY RECRUITED 649 00:30:58,960 --> 00:31:01,600 TO THE RIBOSOME TRANSLOCON 650 00:31:01,600 --> 00:31:02,400 COMPLEX. 651 00:31:02,400 --> 00:31:03,280 TWO LENGTHS ARE SHOWN HERE, IN 652 00:31:03,280 --> 00:31:05,640 THE FIRST ONE THE TRANSMEMBRANE 653 00:31:05,640 --> 00:31:07,000 SEGMENT HAS JUST BEEN INSERTED, 654 00:31:07,000 --> 00:31:12,240 IN THE SECOND IT'S BEEN INSERTED 655 00:31:12,240 --> 00:31:14,040 AND MORE POLYPEPTIDE IS 656 00:31:14,040 --> 00:31:15,120 SYNTHESIZED, NOW COMING OUT OF 657 00:31:15,120 --> 00:31:18,200 THE RIBOSOME. 658 00:31:18,200 --> 00:31:20,880 YOU CAN AFFINITY PURIFY EACH 659 00:31:20,880 --> 00:31:21,440 SEQUENTIAL INTERMEDIATES, 660 00:31:21,440 --> 00:31:22,680 ANALYZE BY MASS SPEC, BLOTTING, 661 00:31:22,680 --> 00:31:23,800 AND SO FORTH. 662 00:31:23,800 --> 00:31:26,320 SHOWN IS AN EXPERIMENT LIKE 663 00:31:26,320 --> 00:31:29,000 THAT, WHERE WE HAVE INCREASING 664 00:31:29,000 --> 00:31:29,520 LENGTHS OF POLYPEPTIDE 665 00:31:29,520 --> 00:31:31,360 DOWNSTREAM OF THE FIRST 666 00:31:31,360 --> 00:31:32,160 TRANSMEMBRANE SEGMENT. 667 00:31:32,160 --> 00:31:34,560 AND I THINK WHAT YOU CAN 668 00:31:34,560 --> 00:31:35,480 APPRECIATE IS THAT ALL OF THEM 669 00:31:35,480 --> 00:31:40,720 ASSOCIATE OF COURSE WITH THE 670 00:31:40,720 --> 00:31:43,440 RIBOSOME, WHAT IS SYNTHESIZING, 671 00:31:43,440 --> 00:31:46,560 AND THE SEC61 PROTEIN TO WHICH 672 00:31:46,560 --> 00:31:48,160 THE RIBOSOME IS DOCKED. 673 00:31:48,160 --> 00:31:49,040 THE PAT COMPLEX IS RECRUITED 674 00:31:49,040 --> 00:31:50,880 AFTER YOU MAKE A CERTAIN AMOUNT 675 00:31:50,880 --> 00:31:53,640 OF POLYPEPTIDE, AND IN THIS 676 00:31:53,640 --> 00:31:55,560 EXPERIMENT ONE HAS SOLUBILIZED 677 00:31:55,560 --> 00:31:58,680 MEMBRANE AND AFFINITY PURIFIED 678 00:31:58,680 --> 00:32:00,640 IT, TELLING YOU WHERE IT 679 00:32:00,640 --> 00:32:02,840 RECRUITS THE PAT COMPLEX THAT'S 680 00:32:02,840 --> 00:32:04,720 A FAIRLY STABLE COMPLEX, THERE'S 681 00:32:04,720 --> 00:32:07,120 NO CROSS-LINKING OR ANYTHING 682 00:32:07,120 --> 00:32:07,320 HERE. 683 00:32:07,320 --> 00:32:14,240 IT'S BEEN STABLY RECRUITED TO 684 00:32:14,240 --> 00:32:16,360 THE TRANSLOCON, ANALYZE BY 685 00:32:16,360 --> 00:32:17,960 SINGLE PARTICLE CryoEM. 686 00:32:17,960 --> 00:32:20,680 ANOTHER MEMBER OF THE LAB, MIN, 687 00:32:20,680 --> 00:32:22,440 DECIDED TO DO THIS. 688 00:32:22,440 --> 00:32:23,640 IT WAS DISAPPOINTING INITIALLY 689 00:32:23,640 --> 00:32:26,560 BECAUSE ALTHOUGH WE COULD GET 690 00:32:26,560 --> 00:32:29,080 GOOD DENSITY FOR THE SEC61 691 00:32:29,080 --> 00:32:39,360 COMPLEX, GREEN HERE, AND SOME 692 00:32:39,360 --> 00:32:41,040 OTHER PARTS, THE PART THAT'S 693 00:32:41,040 --> 00:32:42,240 NEXT TO THE RIBOSOME IS WELL 694 00:32:42,240 --> 00:32:44,320 ORDERED BUT THE PART IN THE 695 00:32:44,320 --> 00:32:45,960 MEMBRANE WAS RATHER BLOBY. 696 00:32:45,960 --> 00:32:52,640 YOU CAN'T REALLY SEE MUCH THERE. 697 00:32:52,640 --> 00:32:54,840 ALPHA FOLD IS PUBLISHED AT THIS 698 00:32:54,840 --> 00:32:56,160 PUBLISHED, TURNS OUT IT'S 699 00:32:56,160 --> 00:32:57,800 PARTICULARLY GOOD AT PREDICTING 700 00:32:57,800 --> 00:33:03,880 STRUCTURES OF MEMBRANE PROTEINS. 701 00:33:03,880 --> 00:33:08,640 AND SO ASTRIX IS 10 KILODALTONS, 702 00:33:08,640 --> 00:33:12,200 AND CCCD47 IS BIGGER, IT PREDICT 703 00:33:12,200 --> 00:33:16,160 AS BEAUTIFUL STRUCTURE, TOGETHER 704 00:33:16,160 --> 00:33:17,880 IT PREDICTS A STRUCTURE OF THE 705 00:33:17,880 --> 00:33:19,640 COMPLEX THAT FITS NICELY INTO 706 00:33:19,640 --> 00:33:20,760 THIS DENSITY. 707 00:33:20,760 --> 00:33:23,920 YOU HAVE A SMALL THREE-HELIX 708 00:33:23,920 --> 00:33:25,440 BUNDLE OF THIS ASTRIX PROTEIN, 709 00:33:25,440 --> 00:33:30,480 ADJACENT TO WHICH IS THE SINGLE 710 00:33:30,480 --> 00:33:32,480 TRANSMEMBRANE DOMAIN OF CCD47, 711 00:33:32,480 --> 00:33:33,960 LARGE GLOBULAR DOMAIN AND OTHER 712 00:33:33,960 --> 00:33:36,480 PARTS THAT BIND THE RIBOSOME. 713 00:33:36,480 --> 00:33:38,320 IT'S IMPORTANT OF COURSE TO KNOW 714 00:33:38,320 --> 00:33:40,880 THAT THIS IS SOME SEMBLANCE OF 715 00:33:40,880 --> 00:33:43,240 REALITY, AS OPPOSED TO COMPLETE 716 00:33:43,240 --> 00:33:44,680 ARTIFACT, BUT OF COURSE FITTING 717 00:33:44,680 --> 00:33:45,960 INTO THIS LOW RESOLUTION DENSITY 718 00:33:45,960 --> 00:33:47,880 WAS A GOOD START. 719 00:33:47,880 --> 00:33:50,480 WHAT LUCCA DID WAS PUT 720 00:33:50,480 --> 00:33:52,560 PHOTOCROSS-LINKERS IN DIFFERENT 721 00:33:52,560 --> 00:33:55,120 PARTS OF ASTRIX, ESSENTIALLY 722 00:33:55,120 --> 00:33:55,840 RECONSTITUTED ASTRIX KNOCKOUT 723 00:33:55,840 --> 00:33:59,440 CELLS WITH VERSIONS IN WHICH ONE 724 00:33:59,440 --> 00:34:01,960 USED AMBER SUPPRESSION TO PUT A 725 00:34:01,960 --> 00:34:03,640 PHOTOCROSS-LINKER IN ANY ONE 726 00:34:03,640 --> 00:34:05,480 LOCATION. 727 00:34:05,480 --> 00:34:06,800 I'M COMPARING A 728 00:34:06,800 --> 00:34:08,480 PHOTOCROSS-LINKER AT POSITION 42 729 00:34:08,480 --> 00:34:10,720 VERSES 62 SHOWN BY THIS RED AND 730 00:34:10,720 --> 00:34:12,040 BLUE STAR. 731 00:34:12,040 --> 00:34:14,000 AND THEN YOU IRRADIATE WITH UV 732 00:34:14,000 --> 00:34:16,320 LIGHT, WHAT ONE CAN SEE IS THIS 733 00:34:16,320 --> 00:34:19,160 IS THE ASTRIX AT THE BOTTOM OF 734 00:34:19,160 --> 00:34:21,680 THE GEL, IN UV DEPENDENT MANNER 735 00:34:21,680 --> 00:34:27,000 YOU SEE ADDITIONAL BAN 736 00:34:27,000 --> 00:34:30,760 IMMUNOPRECIPITATED WITH 737 00:34:30,760 --> 00:34:32,600 ANTIBODIES AGAINST CCD47 738 00:34:32,600 --> 00:34:34,240 REPRESENTING A CROSS-LINK AS 739 00:34:34,240 --> 00:34:37,720 EXPECTED FROM STRUCTURAL MODEL. 740 00:34:37,720 --> 00:34:39,920 THE OTHER DOESN'T CROSS-LINK TO 741 00:34:39,920 --> 00:34:41,800 CCD47, IT'S ON THE OTHER SIDE. 742 00:34:41,800 --> 00:34:44,320 HEE THEE PLACES THE PROBE IN -- 743 00:34:44,320 --> 00:34:48,080 SHE PLACES THE PROBE IN A DOZEN 744 00:34:48,080 --> 00:34:50,760 PLACES, IT'S A SMALL PROTEIN, 745 00:34:50,760 --> 00:34:55,240 AND SO WE CAN, ONE, VERIFY WHICH 746 00:34:55,240 --> 00:34:58,520 PART IS ADJACENT TO CCCD47, 747 00:34:58,520 --> 00:35:00,800 VALIDATING THE ALPHAFOLD MODEL. 748 00:35:00,800 --> 00:35:05,520 TWO, WE CAN THEN FIND THAT THIS 749 00:35:05,520 --> 00:35:09,880 PART OF ASTRIX IS THE PART THAT 750 00:35:09,880 --> 00:35:10,360 RECOGNIZES SUBSTRATE. 751 00:35:10,360 --> 00:35:12,440 SO, KNOWING KIND OF THAT THIS IS 752 00:35:12,440 --> 00:35:14,080 A REASONABLE PLAUSIBLE MODEL, 753 00:35:14,080 --> 00:35:17,560 STRUCTURAL MODEL, WHAT DO WE 754 00:35:17,560 --> 00:35:19,240 LEARN FROM THIS? 755 00:35:19,240 --> 00:35:21,960 HERE'S THE LATERAL GATE OF 756 00:35:21,960 --> 00:35:22,840 SEC61. 757 00:35:22,840 --> 00:35:24,520 IT'S CLOSED. 758 00:35:24,520 --> 00:35:25,920 SO, ALTHOUGH THIS COMPLEX IS IN 759 00:35:25,920 --> 00:35:28,000 THE PROCESS OF MAKING A MEMBRANE 760 00:35:28,000 --> 00:35:29,960 PROTEIN AND YOU MIGHT IMAGINE 761 00:35:29,960 --> 00:35:34,280 THAT THE MEMBRANE PROTEIN WOULD 762 00:35:34,280 --> 00:35:37,720 GET INSERTED BILATERAL GATE, 763 00:35:37,720 --> 00:35:39,480 THAT'S NOT HAPPENING. 764 00:35:39,480 --> 00:35:41,640 THE CHAPERONE PAT COMPLEX IS NOT 765 00:35:41,640 --> 00:35:45,160 LOCATED NEAR THE LATERAL GATE, 766 00:35:45,160 --> 00:35:46,560 IT'S BEHIND SEC61, QUITE FAR 767 00:35:46,560 --> 00:35:46,760 AWAY. 768 00:35:46,760 --> 00:35:49,080 YOU COULD EXPECT A CHAPERONE TO 769 00:35:49,080 --> 00:35:49,960 CAPTURE MEMBRANE PROTEINS AS 770 00:35:49,960 --> 00:35:54,720 THEY COME OUT OF THE SEC61 771 00:35:54,720 --> 00:35:56,440 TRANSLOCON, THIS DOESN'T MAKE 772 00:35:56,440 --> 00:35:57,120 SENSE WITH THAT. 773 00:35:57,120 --> 00:36:00,760 NOW IF YOU HAVE A CLOSER LOOK AT 774 00:36:00,760 --> 00:36:01,800 ASTRIX, SOME THINGS DO MAKE 775 00:36:01,800 --> 00:36:02,280 SENSE. 776 00:36:02,280 --> 00:36:05,680 SO THIS IS THE SURFACE OF ASTRIX 777 00:36:05,680 --> 00:36:08,200 THAT BY CROSS-LINKING ENGAGES 778 00:36:08,200 --> 00:36:08,520 SUBSTRATE. 779 00:36:08,520 --> 00:36:11,360 AND ONE THING YOU SEE IS IT'S 780 00:36:11,360 --> 00:36:14,000 HIGHLY CONSERVED, SO PURPLE IS 781 00:36:14,000 --> 00:36:15,280 CONSERVED HERE, BACK SIDE WHICH 782 00:36:15,280 --> 00:36:17,080 ISN'T SHOWN IS NOT WELL 783 00:36:17,080 --> 00:36:19,640 CONSERVED BUT THE PART THAT 784 00:36:19,640 --> 00:36:21,600 BINDS SUBSTRATE IS CONSERVED. 785 00:36:21,600 --> 00:36:26,680 SECOND, IT'S HYDROPHILIC. 786 00:36:26,680 --> 00:36:30,800 THE JOB BIOCHEMICAL IS TO 787 00:36:30,800 --> 00:36:32,800 RECOGNIZE HYDROPHILIC SEGMENTS, 788 00:36:32,800 --> 00:36:34,360 SO HAVING A HYDROPHILIC SURFACE 789 00:36:34,360 --> 00:36:36,400 SEEMS TO MAKE SENSE. 790 00:36:36,400 --> 00:36:38,600 IF YOU MUTATE THIS SURFACE TO 791 00:36:38,600 --> 00:36:42,200 MAKE IT LESS HYDROPHILIC, IT NO 792 00:36:42,200 --> 00:36:50,200 LONGER ENGAGES SUBSTRATE. 793 00:36:50,200 --> 00:36:52,600 THE HYDROPHILICITY IS IMPORTANT. 794 00:36:52,600 --> 00:36:56,880 MANY SURFACES ARE MADE OF 795 00:36:56,880 --> 00:36:58,200 METHININES, MANY PROTEINS 796 00:36:58,200 --> 00:37:01,440 INVOLVED IN TRANSMEMBRANE DOMAIN 797 00:37:01,440 --> 00:37:02,720 RECOGNITION ARE METHININES RICH, 798 00:37:02,720 --> 00:37:04,040 A FEATURE OF THIS PROTEIN AS 799 00:37:04,040 --> 00:37:06,160 WELL, THOUGHT THAT PART OF THE 800 00:37:06,160 --> 00:37:08,600 REASON IS BECAUSE IT IS THE MOST 801 00:37:08,600 --> 00:37:10,040 FLEXIBLE SIDE CHAIN AND SO MAYBE 802 00:37:10,040 --> 00:37:12,640 IT CAN ACCOMMODATE A WIDE RANGE 803 00:37:12,640 --> 00:37:18,320 OF SEQUENCES THROUGH THAT 804 00:37:18,320 --> 00:37:18,600 PROPERTY. 805 00:37:18,600 --> 00:37:21,080 WHAT WE LEARN FROM THIS 806 00:37:21,080 --> 00:37:23,560 STRUCTURE IS THAT ASTRIX HAS A 807 00:37:23,560 --> 00:37:27,120 SUBSTRATE BINDING REGION, THAT 808 00:37:27,120 --> 00:37:28,800 ENGAGES PARTIALLY HYDROPHILIC 809 00:37:28,800 --> 00:37:29,080 SUBSTRATES. 810 00:37:29,080 --> 00:37:32,120 BUT THE PARTS THAT ARE PUZZLING, 811 00:37:32,120 --> 00:37:34,040 IT DOES SO BEHIND SEC61. 812 00:37:34,040 --> 00:37:37,560 WHAT IT MEANS IS THAT THE 813 00:37:37,560 --> 00:37:38,520 POLYPEPTIDE IS REROUTED OVER 814 00:37:38,520 --> 00:37:41,880 HERE TO THE LEFT IN THIS GREEN, 815 00:37:41,880 --> 00:37:43,680 AWAY FROM THE LATERAL GATE. 816 00:37:43,680 --> 00:37:45,960 IN THE CONVENTIONAL MODEL OF HOW 817 00:37:45,960 --> 00:37:47,720 MEMBRANE INSERTION SHOULD WORK 818 00:37:47,720 --> 00:37:49,800 THIS NEXT TRANSMEMBRANE SEGMENT 819 00:37:49,800 --> 00:37:51,560 NEEDS TO ENGAGE THE LATERAL GATE 820 00:37:51,560 --> 00:37:53,760 BUT IS BEING PULLED AWAY. 821 00:37:53,760 --> 00:37:56,280 SO THAT IS REALLY PUZZLING. 822 00:37:56,280 --> 00:37:59,480 AS IF THAT WASN'T BAD ENOUGH, A 823 00:37:59,480 --> 00:38:01,520 STUDENT IN THE LAB HAPPENED TO 824 00:38:01,520 --> 00:38:04,800 BE LOOKING AT OUR STRUCTURE, HE 825 00:38:04,800 --> 00:38:08,720 SAID THERE'S ANOTHER PROBLEM. 826 00:38:08,720 --> 00:38:12,400 THAT'S BECAUSE THE CCD47, THIS 827 00:38:12,400 --> 00:38:15,120 PINK-COLORED POLY PEPTIDE, SITS 828 00:38:15,120 --> 00:38:17,360 CLOSE TO SEC61 AND WHAT AARON 829 00:38:17,360 --> 00:38:22,600 NOTICED IS THAT THAT PART OF 830 00:38:22,600 --> 00:38:24,720 CCD47 IS WEDGED BETWEEN SEC61 831 00:38:24,720 --> 00:38:25,680 AND RIBOSOME. 832 00:38:25,680 --> 00:38:28,880 YOU CAN APPRECIATE IN THIS 833 00:38:28,880 --> 00:38:30,200 SPACE-FILLING MODEL IT'S A TIGHT 834 00:38:30,200 --> 00:38:31,360 FIT. 835 00:38:31,360 --> 00:38:33,720 THE CONSEQUENCE IS SEC61 CANNOT 836 00:38:33,720 --> 00:38:34,040 OPEN. 837 00:38:34,040 --> 00:38:36,280 I'M SHOWING AGAIN WHAT THE MOVIE 838 00:38:36,280 --> 00:38:38,080 OF SEC61 OPENING LOOKS LIKE. 839 00:38:38,080 --> 00:38:39,520 I THINK YOU CAN APPRECIATE THIS 840 00:38:39,520 --> 00:38:42,080 IS THE SAME VIEW HERE THAT THIS 841 00:38:42,080 --> 00:38:43,200 KIND OF MOVEMENT SIMPLY CANNOT 842 00:38:43,200 --> 00:38:48,440 HAPPEN WHEN THIS WEDGE IS 843 00:38:48,440 --> 00:38:49,240 SITTING HERE. 844 00:38:49,240 --> 00:38:52,600 SO, WHAT HAS HAPPENED WHEN YOU 845 00:38:52,600 --> 00:38:54,920 RECRUITED THE PAT COMPLEX YOU 846 00:38:54,920 --> 00:38:56,840 WEDGE SEC61 TO A CLOSED 847 00:38:56,840 --> 00:38:58,400 CONFIRMATION AND PULL THE 848 00:38:58,400 --> 00:39:00,680 SUBSTRATE AWAY FROM THE LATERAL 849 00:39:00,680 --> 00:39:01,600 GATE. 850 00:39:01,600 --> 00:39:05,080 AND SO THAT IS NOT CONDUCIVE TO 851 00:39:05,080 --> 00:39:05,960 INSERTING THE NEXT TRANSMEMBRANE 852 00:39:05,960 --> 00:39:08,480 SEGMENT INTO THE LATERAL GATE OF 853 00:39:08,480 --> 00:39:12,320 SEC61 AS CONVENTIONAL MOD WOMAN 854 00:39:12,320 --> 00:39:13,920 -- MODEL WOULD HAVE YOU 855 00:39:13,920 --> 00:39:14,960 LEAVE. 856 00:39:14,960 --> 00:39:18,760 THE ONLY PLAUSIBLE MODEL IS THAT 857 00:39:18,760 --> 00:39:22,000 AS YOU ELONGATE MORE, YOU MAKE 858 00:39:22,000 --> 00:39:23,720 THE NEXT TRANSMEMBRANE SEGMENT, 859 00:39:23,720 --> 00:39:25,600 YOU HAVE TWO TRANSMEMBRANE 860 00:39:25,600 --> 00:39:27,080 SEGMENTS THAT WOULD INSERT AS A 861 00:39:27,080 --> 00:39:29,200 PAIR INTO THE SPACE BEHIND SEC 862 00:39:29,200 --> 00:39:31,640 61 BETWEEN THE CHAPERONE AND 863 00:39:31,640 --> 00:39:32,440 BACK OF SEC61. 864 00:39:32,440 --> 00:39:35,640 AND SO THE QUESTION IS, IS THAT 865 00:39:35,640 --> 00:39:36,840 A PLAUSIBLE SCENARIO? 866 00:39:36,840 --> 00:39:39,000 AND IF IT IS, THEN IS THERE 867 00:39:39,000 --> 00:39:41,680 MACHINERY THAT CATALYZEs THAT 868 00:39:41,680 --> 00:39:42,080 REACTION? 869 00:39:42,080 --> 00:39:47,280 SO I WILL TELL YOU THAT IT IS 870 00:39:47,280 --> 00:39:49,120 PLAUSIBLE IN THAT THIS LOOP 871 00:39:49,120 --> 00:39:50,440 BETWEEN THE TWO TRANSMEMBRANE 872 00:39:50,440 --> 00:39:52,840 SEGMENTS, IF YOU PUT A FOLDED 873 00:39:52,840 --> 00:39:54,160 DOMAIN THERE, THIS INSERTION 874 00:39:54,160 --> 00:39:56,320 REACTION IN FACT DOESN'T WORK 875 00:39:56,320 --> 00:39:57,760 VERY EFFICIENTLY, SUGGESTING IN 876 00:39:57,760 --> 00:40:00,000 FACT THAT MIGHT BE WHAT'S 877 00:40:00,000 --> 00:40:00,280 HAPPENING. 878 00:40:00,280 --> 00:40:03,160 BUT TO FIGURE OUT WHAT WAS GOING 879 00:40:03,160 --> 00:40:05,120 ON, WE THEN ISOLATED THESE 880 00:40:05,120 --> 00:40:06,120 INTERMEDIATES, AT THE POINT 881 00:40:06,120 --> 00:40:08,640 WHERE THE NEXT TWO TRANSMEMBRANE 882 00:40:08,640 --> 00:40:11,120 SEGMENTS WOULD BE INSERTING. 883 00:40:11,120 --> 00:40:13,320 AND THEN AFFINITY PURIFIED, DID 884 00:40:13,320 --> 00:40:15,080 MASS SPEC, VARIOUS BLOTTING 885 00:40:15,080 --> 00:40:16,280 EXPERIMENTS AND CryoEM TO SEE 886 00:40:16,280 --> 00:40:17,680 WHAT WAS GOING ON. 887 00:40:17,680 --> 00:40:20,200 AND THIS TURNS OUT TO BE 888 00:40:20,200 --> 00:40:21,040 REVEALING. 889 00:40:21,040 --> 00:40:24,160 THIS IS A COMPLICATED SLIDE WITH 890 00:40:24,160 --> 00:40:25,440 A FAIRLY SIMPLE CONCLUSION. 891 00:40:25,440 --> 00:40:27,120 THIS IS A MASS SPEC EXPERIMENT 892 00:40:27,120 --> 00:40:28,960 WHERE WE'RE COMPARING AN EARLY 893 00:40:28,960 --> 00:40:31,160 INTERMEDIATE, WHERE THE FIRST 894 00:40:31,160 --> 00:40:32,240 TRANSMEMBRANE SEGMENT HAS BEEN 895 00:40:32,240 --> 00:40:34,040 INSERTED, BUT THE PAT COMPLEX 896 00:40:34,040 --> 00:40:35,600 HAS NOT BEEN RECRUITED. 897 00:40:35,600 --> 00:40:38,520 COMPARED TO A LATE INTERMEDIATE, 898 00:40:38,520 --> 00:40:40,880 WHERE THE PAT COMPLEX IS 899 00:40:40,880 --> 00:40:41,840 RECRUITED, NEXT TWO SEGMENTS ARE 900 00:40:41,840 --> 00:40:43,240 RIGHT ABOUT THE POINT WHERE THEY 901 00:40:43,240 --> 00:40:45,720 ARE INSERTING IN THE BILAYER. 902 00:40:45,720 --> 00:40:49,040 AND BY COMPARING THESE TWO 903 00:40:49,040 --> 00:40:49,600 COMPLEXES USING QUANTITATIVE 904 00:40:49,600 --> 00:40:51,480 MASS SPEC, THERE ARE ONLY A 905 00:40:51,480 --> 00:40:53,000 HANDFUL OF PROTEINS THAT ARE 906 00:40:53,000 --> 00:40:55,320 ENRICHED IN THE LATE COMPLEX. 907 00:40:55,320 --> 00:40:56,640 ONE OF THEM -- ALL OF THEM TURN 908 00:40:56,640 --> 00:41:00,040 OUT TO BE PARTS OF LARGER 909 00:41:00,040 --> 00:41:07,200 COMPLEXES THAT OUR LAB AND BOB 910 00:41:07,200 --> 00:41:09,200 KEENAN'S LAB CHARACTERIZED. 911 00:41:09,200 --> 00:41:12,440 THIS IS A BLOT VERIFYING IT'S 912 00:41:12,440 --> 00:41:14,800 RECRUITED TO THE LATE COMPLEX, 913 00:41:14,800 --> 00:41:17,040 ASTRIX IS NOT IDENTIFIED BY MASS 914 00:41:17,040 --> 00:41:21,680 SPEC BECAUSE IT HAS VERY FEW 915 00:41:21,680 --> 00:41:24,320 CRYPTIC PEPTIDES. 916 00:41:24,320 --> 00:41:28,600 THIS PROTEIN NICALIN AND NOMO 917 00:41:28,600 --> 00:41:30,880 ARE PART OF 47, PART OF AN 918 00:41:30,880 --> 00:41:32,880 OBLIGATE COMPLEX, WE CALLED THIS 919 00:41:32,880 --> 00:41:38,880 THE BACK OF SEC61 COMPLEX, OR 920 00:41:38,880 --> 00:41:39,160 BOS 921 00:41:39,160 --> 00:41:40,560 COMPLEX, NO IDEA WHAT IT DOES 922 00:41:40,560 --> 00:41:43,680 BUT WE KNOW WHERE IT IS. 923 00:41:43,680 --> 00:41:46,000 THIRD IS TWO-PROTEIN COMPLEX 924 00:41:46,000 --> 00:41:47,200 TMCL 1 AND INTERACTION PARTNER, 925 00:41:47,200 --> 00:41:49,960 AND THIS IS THE COMPLEX THAT I 926 00:41:49,960 --> 00:41:54,800 MENTIONED BRIEFLY AT THE 927 00:41:54,800 --> 00:41:57,560 BEGINNING, HOMOLOGOUS TO THE GET 928 00:41:57,560 --> 00:42:00,280 COMPLEX, AND EMC, ALSO MEDIATING 929 00:42:00,280 --> 00:42:01,920 TRANSMEMBRANE DOMAIN INSERTION, 930 00:42:01,920 --> 00:42:05,280 CALL IT THE GEL COMPLEX, FOR GET 931 00:42:05,280 --> 00:42:06,440 AND EMC-LIKE. 932 00:42:06,440 --> 00:42:09,200 THIS FACTORS SEEMS LIKE IT WOULD 933 00:42:09,200 --> 00:42:09,800 BE MEDIATING TRANSMEMBRANE 934 00:42:09,800 --> 00:42:11,840 DOMAIN INSERTION. 935 00:42:11,840 --> 00:42:12,800 WE HAVE THREE COMPLEXES HERE, 936 00:42:12,800 --> 00:42:16,240 REALLY NICE WORK FROM THE KEENAN 937 00:42:16,240 --> 00:42:18,520 LAB HAS SHOWN THESE ARE THREE 938 00:42:18,520 --> 00:42:19,840 INDEPENDENT COMPLEXES ASSEMBLED 939 00:42:19,840 --> 00:42:21,360 TOGETHER ON THE RIBOSOME BUT 940 00:42:21,360 --> 00:42:24,120 OTHERWISE DON'T INTERACT WITH 941 00:42:24,120 --> 00:42:24,960 EACH OTHER. 942 00:42:24,960 --> 00:42:27,720 SO, ONE COULD OF COURSE THEN 943 00:42:27,720 --> 00:42:30,080 ANALYZE THIS BY EM, RESOLUTION 944 00:42:30,080 --> 00:42:32,480 AGAIN IS MEAGER. 945 00:42:32,480 --> 00:42:35,920 HOWEVER, THE ABILITY TO DOCK IN 946 00:42:35,920 --> 00:42:38,560 REASONABLY GOOD PREDICTIONS IS 947 00:42:38,560 --> 00:42:38,840 VERY GOOD. 948 00:42:38,840 --> 00:42:40,560 AND SO WHAT YOU'RE LOOKING AT 949 00:42:40,560 --> 00:42:45,120 HERE IS THE VIEW OF THIS 950 00:42:45,120 --> 00:42:47,080 TRANSLOCON FROM THE LUMEN OF THE 951 00:42:47,080 --> 00:42:47,800 E.R. 952 00:42:47,800 --> 00:42:49,600 YOU'RE LOOKING TOWARDS THE 953 00:42:49,600 --> 00:42:51,880 RIBOSOME, AND THE PROTEIN THAT'S 954 00:42:51,880 --> 00:42:54,000 BEING SYNTHESIZED IS COMING OUT 955 00:42:54,000 --> 00:42:55,200 OF THE RIBOSOME TOWARDS YOU. 956 00:42:55,200 --> 00:42:58,480 AND WHAT WE WIND UP SEEING IS 957 00:42:58,480 --> 00:43:00,640 THE THIRD TRANSMEMBRANE DOMAIN 958 00:43:00,640 --> 00:43:01,960 OF THE INSERTING POLYPEPTIDE BUT 959 00:43:01,960 --> 00:43:05,920 CAN'T SEE THE OTHER TWO. 960 00:43:05,920 --> 00:43:07,880 HOWEVER, WE CAN DEDUCE WHICH 961 00:43:07,880 --> 00:43:09,440 PARTS OF THE SUBSTRATE IS 962 00:43:09,440 --> 00:43:19,680 ADJACENT TO THESE OTHER 963 00:43:19,680 --> 00:43:20,240 COMPLEXES USING 964 00:43:20,240 --> 00:43:21,280 PHOTOCROSS-LINKING. 965 00:43:21,280 --> 00:43:24,840 THIS IS SEEN ON THE RIGHT. 966 00:43:24,840 --> 00:43:26,440 AND THEN YOU IRRADIATE WITH UV 967 00:43:26,440 --> 00:43:26,880 LIGHT. 968 00:43:26,880 --> 00:43:29,520 WHAT YOU CAN SEE IS THAT HERE'S 969 00:43:29,520 --> 00:43:32,360 A CROSS-LINK TO THE FIRST 970 00:43:32,360 --> 00:43:33,120 TRANSMEMBRANE SEGMENT, PRIMARY 971 00:43:33,120 --> 00:43:34,240 CROSS-LINK. 972 00:43:34,240 --> 00:43:37,080 AND THAT TURNS OUT TO BE ASTRIX. 973 00:43:37,080 --> 00:43:38,720 I'M NOT SHOWING THE IPS HERE BUT 974 00:43:38,720 --> 00:43:40,760 THAT PLACES THE FIRST 975 00:43:40,760 --> 00:43:42,640 TRANSMEMBRANE SEGMENT HERE. 976 00:43:42,640 --> 00:43:44,600 THE SECOND ONE DOES NOT 977 00:43:44,600 --> 00:43:46,480 CROSS-LINK TO ASTRIX BUT TO 978 00:43:46,480 --> 00:43:47,280 SOMETHING SLIGHTLY BIGGER, WHICH 979 00:43:47,280 --> 00:43:51,640 TURNS OUT TO BE A COMPONENT OF 980 00:43:51,640 --> 00:43:54,680 THE GEL COMPLEX, TMCL1 SUBUNIT. 981 00:43:54,680 --> 00:43:56,640 THE THIRD SEGMENT CROSS-LINKS TO 982 00:43:56,640 --> 00:43:57,880 THE SEC61 COMPLEX AND DEPENDING 983 00:43:57,880 --> 00:44:00,800 ON WHERE YOU PUT THE 984 00:44:00,800 --> 00:44:01,400 PHOTOCROSS-LINKER, IT 985 00:44:01,400 --> 00:44:05,560 CROSS-LINKS TO ALPHA OR BETA 986 00:44:05,560 --> 00:44:08,680 SUBUNIT, BETA IS RIGHT HERE, 987 00:44:08,680 --> 00:44:11,200 THIS NICELY ILLUSTRATES HOW 988 00:44:11,200 --> 00:44:21,680 SPECIFIC PHOTOCROSS-LINKER, 989 00:44:22,240 --> 00:44:25,200 INCREDIBLY SPECIFIC. 990 00:44:25,200 --> 00:44:26,240 WE FEEL REASONABLE COULD HAVE 991 00:44:26,240 --> 00:44:27,760 DENT DRAWING THE SUBSTRATE HERE. 992 00:44:27,760 --> 00:44:29,200 WE THINK WE'RE LOOKING AT 993 00:44:29,200 --> 00:44:32,280 INTERMEDIATE IN WHICH THE SECOND 994 00:44:32,280 --> 00:44:33,560 AND THIRD TRANSMEMBRANE SEGMENTS 995 00:44:33,560 --> 00:44:35,560 HAVE JUST BEEN INSERTED, AND 996 00:44:35,560 --> 00:44:37,800 OBVIOUSLY THEY ARE NOT AT THE 997 00:44:37,800 --> 00:44:39,360 LATERAL GATE OF SEC 61. 998 00:44:39,360 --> 00:44:42,400 THEY ARE BEHIND SEC 61, ADJACENT 999 00:44:42,400 --> 00:44:43,280 TO POORLY CHARACTERIZED 1000 00:44:43,280 --> 00:44:46,760 COMPLEXES THAT FORM THIS 1001 00:44:46,760 --> 00:44:47,760 HORSESHOE SHAPE BEHIND SEC61, 1002 00:44:47,760 --> 00:44:51,240 WHY WE CALL THIS THE MULTI-PASS 1003 00:44:51,240 --> 00:44:53,000 TRANSLOCON, IT SEEMS TO BE 1004 00:44:53,000 --> 00:44:55,120 INVOLVED IN INSERTING MULTI-PASS 1005 00:44:55,120 --> 00:44:55,360 PROTEINS. 1006 00:44:55,360 --> 00:44:57,600 THE IDEA THEN IS THAT AFTER YOU 1007 00:44:57,600 --> 00:45:01,000 INSERT THE FIRST TRANSMEMBRANE 1008 00:45:01,000 --> 00:45:02,240 SEGMENT, THE PARTIAL HYDROPHILIC 1009 00:45:02,240 --> 00:45:05,840 CHARACTER SEEMS TO PLAY A ROLE 1010 00:45:05,840 --> 00:45:08,120 IN RECRUITING PAT COMPLEX, WHICH 1011 00:45:08,120 --> 00:45:10,160 SEEMS TO BE CO-RECRUITED WITH 1012 00:45:10,160 --> 00:45:12,280 THE OTHER TWO COMPLEXES, GEL AND 1013 00:45:12,280 --> 00:45:13,160 BOS. 1014 00:45:13,160 --> 00:45:16,320 WHAT THE PAT COMPLEX DOES, SEEMS 1015 00:45:16,320 --> 00:45:25,960 TO LOCK SEC61 CLOSED BY WEDGING 1016 00:45:25,960 --> 00:45:28,920 WITH THE RIBOSOME PULLING THE 1017 00:45:28,920 --> 00:45:29,720 NASCENT CHAIN. 1018 00:45:29,720 --> 00:45:33,200 AS YOU MAKE MORE SEGMENTS OF 1019 00:45:33,200 --> 00:45:37,200 POLY PEPTIDE WE GET INSERTED 1020 00:45:37,200 --> 00:45:38,640 BEHIND SEC61. 1021 00:45:38,640 --> 00:45:40,520 WE THINK THIS REACTION IS 1022 00:45:40,520 --> 00:45:42,040 MEDIATED BY GEL COMPLEX. 1023 00:45:42,040 --> 00:45:44,040 AND ONCE YOU GET THAT INSERTION 1024 00:45:44,040 --> 00:45:46,480 THEN YOU CAN IMAGINE THAT AS YOU 1025 00:45:46,480 --> 00:45:47,200 SYNTHESIZE MORE POLYPEPTIDE THEY 1026 00:45:47,200 --> 00:45:50,040 CAN THEN CONTINUE TO BE INSERTED 1027 00:45:50,040 --> 00:45:52,480 BEHIND SEC61, UNTIL YOU MAKE THE 1028 00:45:52,480 --> 00:45:54,720 WHOLE PROTEIN, IT FOLDS, AND 1029 00:45:54,720 --> 00:45:58,000 ONCE ALL THE HYDROPHILIC PARTS 1030 00:45:58,000 --> 00:45:59,560 OF THE POLYPEPTIDE ARE BURIED 1031 00:45:59,560 --> 00:46:00,880 THERE'S NOTHING FOR COMPLEXES TO 1032 00:46:00,880 --> 00:46:03,160 BIND TO ANYMORE AND WE IMAGINE 1033 00:46:03,160 --> 00:46:04,720 THE COMPLEX DISASSEMBLES AND 1034 00:46:04,720 --> 00:46:08,000 YOU'VE MADE YOUR PROTEIN. 1035 00:46:08,000 --> 00:46:09,400 SO, THIS OBVIOUSLY IS A 1036 00:46:09,400 --> 00:46:11,160 COMPLETELY DIFFERENT WAY OF 1037 00:46:11,160 --> 00:46:12,080 THINKING ABOUT HOW MEMBRANE 1038 00:46:12,080 --> 00:46:14,320 INSERTION WORKS THAN THE KIND OF 1039 00:46:14,320 --> 00:46:16,080 SEQUENTIAL INSERTION VIA THE 1040 00:46:16,080 --> 00:46:18,040 LATERAL GATE OF SEC61. 1041 00:46:18,040 --> 00:46:19,600 THE FIRST TRANSMEMBRANE SEGMENT 1042 00:46:19,600 --> 00:46:22,040 IS INSERTED BY THIS PROTEIN 1043 00:46:22,040 --> 00:46:24,520 COMPLEX, EMC, WHICH ISN'T SHOWN 1044 00:46:24,520 --> 00:46:25,880 IN THE DIAGRAM. 1045 00:46:25,880 --> 00:46:28,360 AND LATER SEGMENTS OF THIS GPCR 1046 00:46:28,360 --> 00:46:31,320 ARE INSERTED BY OTHER COMPONENTS 1047 00:46:31,320 --> 00:46:33,240 BEHIND SEC61. 1048 00:46:33,240 --> 00:46:35,160 SO NONE OF THEM ARE INSERTED BY 1049 00:46:35,160 --> 00:46:37,480 THE LATERAL GATE OF SEC61. 1050 00:46:37,480 --> 00:46:39,760 SO THAT MAKES A PREDICTION WHICH 1051 00:46:39,760 --> 00:46:42,680 IS THAT IF YOU BLOCK THE LATERAL 1052 00:46:42,680 --> 00:46:45,640 GATE OF SEC61, INSERTION OF THIS 1053 00:46:45,640 --> 00:46:47,640 GPCR SHOULD STILL WORK. 1054 00:46:47,640 --> 00:46:49,760 AND ONE THING THAT'S HAPPENED IN 1055 00:46:49,760 --> 00:46:52,040 THE PAST NUMBER OF YEARS, IS 1056 00:46:52,040 --> 00:46:55,640 THAT THERE HAVE BEEN A NUMBER OF 1057 00:46:55,640 --> 00:46:57,360 GOOD INHIBITORS OF SEC61 LATERAL 1058 00:46:57,360 --> 00:47:01,720 GATE, AND NICE WORK BY PARK AND 1059 00:47:01,720 --> 00:47:03,160 JACK TAUNTON HAVE GOTTEN 1060 00:47:03,160 --> 00:47:04,120 STRUCTURES OF SUCH INHIBITORS 1061 00:47:04,120 --> 00:47:05,600 AND BIND RIGHT AT THE LATERAL 1062 00:47:05,600 --> 00:47:08,160 GATE AND BLOCK IT. 1063 00:47:08,160 --> 00:47:12,400 AND SO WE CAN THEN TEST THIS 1064 00:47:12,400 --> 00:47:12,720 IDEA. 1065 00:47:12,720 --> 00:47:14,040 SO, HERE'S A POSITIVE CONTROL 1066 00:47:14,040 --> 00:47:16,560 WHICH IS A SINGLE PASS PROTEIN 1067 00:47:16,560 --> 00:47:19,280 THAT IS KNOWN TO INSERT VIA 1068 00:47:19,280 --> 00:47:21,360 LATERAL GATE OF SEC61, AND THIS 1069 00:47:21,360 --> 00:47:26,280 IS A FLOW CYTOMETRY ASSAY IN 1070 00:47:26,280 --> 00:47:28,520 WHICH THE GRAY IS CONTROL CELLS, 1071 00:47:28,520 --> 00:47:29,680 NO INHIBITORS, NOTHING KNOCKED 1072 00:47:29,680 --> 00:47:30,120 DOWN. 1073 00:47:30,120 --> 00:47:32,440 OBVIOUSLY IF YOU KNOCK DOWN 1074 00:47:32,440 --> 00:47:33,840 SEC61 INSERT DOESN'T WORK. 1075 00:47:33,840 --> 00:47:35,920 BUT IF YOU ADD THE INHIBITOR OF 1076 00:47:35,920 --> 00:47:36,800 THE LATERAL GATE INSERTION 1077 00:47:36,800 --> 00:47:38,480 DOESN'T WORK AS WELL. 1078 00:47:38,480 --> 00:47:41,480 THAT MAKES COMPLETE SENSE. 1079 00:47:41,480 --> 00:47:42,920 BUT HERE'S RHODOPSIN. 1080 00:47:42,920 --> 00:47:45,640 OBVIOUSLY IF YOU KNOCK DOWN 1081 00:47:45,640 --> 00:47:46,640 SEC61, INSERTION DOESN'T WORK, 1082 00:47:46,640 --> 00:47:48,600 BUT THAT'S THE BINDING SITE FOR 1083 00:47:48,600 --> 00:47:49,400 THE RIBOSOME. 1084 00:47:49,400 --> 00:47:50,800 RIBOSOME HAS NOTHING TO BIND TO 1085 00:47:50,800 --> 00:47:54,040 AT THE MEMBRANE. 1086 00:47:54,040 --> 00:47:56,960 THE INHIBITOR IS COMPLETELY 1087 00:47:56,960 --> 00:47:58,240 INEFFECTIVE AGAINST INHIBITING 1088 00:47:58,240 --> 00:48:00,120 RHODOPSIN INSERTION. 1089 00:48:00,120 --> 00:48:01,840 I HAVE TO SAY, SURPRISINGLY 1090 00:48:01,840 --> 00:48:03,600 CLEAN HOW GOOD THIS RESULT IS. 1091 00:48:03,600 --> 00:48:06,640 WE DID THIS WITH THREE 1092 00:48:06,640 --> 00:48:08,960 COMPLETELY UNRELATED CHEMICALLY 1093 00:48:08,960 --> 00:48:11,640 UNRELATED INHIBITORS, AND WE'VE 1094 00:48:11,640 --> 00:48:15,240 ALSO DONE THIS WITH MULTIPLE 1095 00:48:15,240 --> 00:48:15,880 DIFFERENT GPCRs. 1096 00:48:15,880 --> 00:48:17,840 AND SO THIS CLASS OF PROTEINS 1097 00:48:17,840 --> 00:48:20,000 DOESN'T SEEM TO USE THE LATERAL 1098 00:48:20,000 --> 00:48:21,200 GATE OF SEC61. 1099 00:48:21,200 --> 00:48:23,280 YOU MIGHT ASK WHAT EXACTLY IS 1100 00:48:23,280 --> 00:48:24,480 SEC61 NEEDED FOR? 1101 00:48:24,480 --> 00:48:26,920 OBVIOUSLY IT'S NEEDED HERE IN 1102 00:48:26,920 --> 00:48:30,000 THIS CASE FOR BINDING TO THE 1103 00:48:30,000 --> 00:48:35,320 RIBOSOME AND SERVING AS 1104 00:48:35,320 --> 00:48:35,680 TEMPLATES. 1105 00:48:35,680 --> 00:48:38,160 IF YOU'RE IN THE PROCESS OF 1106 00:48:38,160 --> 00:48:39,160 MAKING A MEMBRANE PROTEIN, 1107 00:48:39,160 --> 00:48:40,720 SUPPOSE THAT THERE'S A LOOP IN 1108 00:48:40,720 --> 00:48:42,880 THE MIDDLE THAT'S REALLY LONG. 1109 00:48:42,880 --> 00:48:45,360 THE COMPONENTS I TOLD YOU ABOUT, 1110 00:48:45,360 --> 00:48:48,360 THEY ARE NOT WELL SUITED FOR 1111 00:48:48,360 --> 00:48:49,360 TRANSLOCATING LONG SEGMENTS OF 1112 00:48:49,360 --> 00:48:50,960 POLYPEPTIDE BECAUSE THEY DON'T 1113 00:48:50,960 --> 00:48:53,400 HAVE A CHANNEL IN THEM LIKE THE 1114 00:48:53,400 --> 00:48:54,600 CHANNEL THAT'S IN SEC61. 1115 00:48:54,600 --> 00:48:56,480 AND IT TURNS OUT THAT IF YOU 1116 00:48:56,480 --> 00:49:02,280 HAVE SUCH A PROTEIN AND THIS IS 1117 00:49:02,280 --> 00:49:05,320 THE C3A COMPLEMENT RECEPTOR, THE 1118 00:49:05,320 --> 00:49:07,960 LOOP IS 150 AMINO ACIDS LONG, 1119 00:49:07,960 --> 00:49:09,360 TRANSLOCATION OF THE LOOP 1120 00:49:09,360 --> 00:49:10,040 REQUIRES SEC61. 1121 00:49:10,040 --> 00:49:12,320 HERE IS THE EXPERIMENT TO SHOW 1122 00:49:12,320 --> 00:49:12,480 IT. 1123 00:49:12,480 --> 00:49:14,480 THIS PROTEIN HAS TWO 1124 00:49:14,480 --> 00:49:15,320 GLYCOSYLATION SITES. 1125 00:49:15,320 --> 00:49:19,160 AND THE SECOND ONE IS IN THIS 1126 00:49:19,160 --> 00:49:19,440 LOOP. 1127 00:49:19,440 --> 00:49:21,600 SO HERE'S AN EXPERIMENT WITHOUT 1128 00:49:21,600 --> 00:49:23,160 SEC61 INHIBITOR. 1129 00:49:23,160 --> 00:49:24,400 THE PROTEIN GETS GLYCOSYLATED 1130 00:49:24,400 --> 00:49:26,000 ONCE AND TWICE, RIGHT HERE. 1131 00:49:26,000 --> 00:49:29,320 BUT IF YOU HAVE SEC61 INHIBITOR, 1132 00:49:29,320 --> 00:49:31,480 THE FIRST GLYCOSYLATION STILL 1133 00:49:31,480 --> 00:49:32,680 OCCURS, AND THAT'S OCCURRING AT 1134 00:49:32,680 --> 00:49:38,280 THE N N-TERMINUS, IT'S MEDIATEY 1135 00:49:38,280 --> 00:49:40,480 EMC, BUT THE SECOND IS 1136 00:49:40,480 --> 00:49:42,280 PREFERENTIALLY GONE. 1137 00:49:42,280 --> 00:49:44,520 AND THAT'S BECAUSE YOU CANNOT 1138 00:49:44,520 --> 00:49:45,440 TRANSLOCATE THAT LOOP. 1139 00:49:45,440 --> 00:49:50,480 WE CAN THEN SHOW THIS USING 1140 00:49:50,480 --> 00:49:53,560 PROTEASE PROTECTION, IF YOU 1141 00:49:53,560 --> 00:49:54,600 DISSOLVE WITH PROTEASE, FIRST 1142 00:49:54,600 --> 00:49:57,160 THREE ARE PROTECTED FROM 1143 00:49:57,160 --> 00:49:59,600 PROTEASE, WHAT THE FRAGMENT HERE 1144 00:49:59,600 --> 00:50:00,000 REPRESENTS. 1145 00:50:00,000 --> 00:50:02,160 AND AMAZINGLY ENOUGH, IF YOU 1146 00:50:02,160 --> 00:50:04,720 SHORTEN THAT LOOP TO BE 20 OR SO 1147 00:50:04,720 --> 00:50:07,040 AMINO ACIDS, NOW WHETHER YOU 1148 00:50:07,040 --> 00:50:10,440 HAVE SEC61 INHIBITOR OR NOT, 1149 00:50:10,440 --> 00:50:11,960 INSERTION WORKS PERFECTLY FINE. 1150 00:50:11,960 --> 00:50:15,480 SO, WHAT ONE THEN IS LEFT WITH 1151 00:50:15,480 --> 00:50:18,280 IS THIS IDEA THAT THE OXA 1152 00:50:18,280 --> 00:50:19,640 FAMILY, WHICH I REMIND YOU IS 1153 00:50:19,640 --> 00:50:23,320 COULD BE SERVED ACROSS ALL 1154 00:50:23,320 --> 00:50:25,320 ORGANISMS, MEDIATES INSERTION OF 1155 00:50:25,320 --> 00:50:27,480 TRANSMEMBRANE SEGMENTS WHEN THE 1156 00:50:27,480 --> 00:50:28,560 ADJACENT FLANKING DOMAIN IS 1157 00:50:28,560 --> 00:50:29,560 RELATIVELY SHORT. 1158 00:50:29,560 --> 00:50:31,520 THAT'S BECAUSE IT SEEMS TO WORK 1159 00:50:31,520 --> 00:50:33,240 BY USING A VESTIBULE THAT IS NOT 1160 00:50:33,240 --> 00:50:35,000 A CHANNEL ACROSS THE MEMBRANE. 1161 00:50:35,000 --> 00:50:45,480 SO IT'S JUST REDUCING THE 1162 00:50:47,040 --> 00:50:48,600 BARRIER, E. COLI SEEMS TO BE 1163 00:50:48,600 --> 00:50:51,880 ABOUT 50 AMINO ACIDS SO 1164 00:50:51,880 --> 00:50:53,720 TRANSMEMBRANE SEGMENTING WITH 1165 00:50:53,720 --> 00:50:57,720 FLANKING DOMAIN OF 50 AMINO 1166 00:50:57,720 --> 00:50:59,200 ACIDS USE THE OXA 1 FAMILIAR. 1167 00:50:59,200 --> 00:51:04,240 IN THE MAMMALIAN E.R. THERE ARE 1168 00:51:04,240 --> 00:51:08,000 FAMILY MEMBERS, BACTERIA HAVE 1169 00:51:08,000 --> 00:51:10,600 EMC, INTERMITOCHONDRIAAL 1170 00:51:10,600 --> 00:51:12,360 MEMBRANE HAS OXA 1, SO ALL THESE 1171 00:51:12,360 --> 00:51:14,080 MEMBRANES THAT NEED TO MAKE 1172 00:51:14,080 --> 00:51:17,640 MEMBRANE PROTEINS HAVE A MEMBER 1173 00:51:17,640 --> 00:51:18,560 OF THIS FAMILY. 1174 00:51:18,560 --> 00:51:21,040 AND SO WE BELIEVE THAT THIS 1175 00:51:21,040 --> 00:51:23,280 FAMILY CAN ALSO INSERT A PAIR OF 1176 00:51:23,280 --> 00:51:24,240 TRANSMEMBRANE SEGMENTS AS LONG 1177 00:51:24,240 --> 00:51:27,280 AS THE LOOP IS SHORT. 1178 00:51:27,280 --> 00:51:29,720 IF YOU NEED TO GET A LONG LOOP 1179 00:51:29,720 --> 00:51:32,640 ACROSS THE MEMBRANE YOU NEED A 1180 00:51:32,640 --> 00:51:32,880 CHANNEL. 1181 00:51:32,880 --> 00:51:34,160 THIS IS OBVIOUS BECAUSE, YOU 1182 00:51:34,160 --> 00:51:35,480 KNOW, THE ENERGETIC BARRIER TO 1183 00:51:35,480 --> 00:51:37,920 MOVING A LONG SEGMENT OF 1184 00:51:37,920 --> 00:51:39,840 POLYPEPTIDE IS TOO HIGH, YOU 1185 00:51:39,840 --> 00:51:41,400 NEED AN AQUEOUS CHANNEL TO MOVE 1186 00:51:41,400 --> 00:51:44,440 IT ACROSS THE MEMBRANE, WE THINK 1187 00:51:44,440 --> 00:51:45,160 THE TRANSMEMBRANE SEGMENT 1188 00:51:45,160 --> 00:51:48,080 ADJACENT TO THE LONG DOMAIN 1189 00:51:48,080 --> 00:51:51,440 ENGAGES AND OPENS LATERAL GATE, 1190 00:51:51,440 --> 00:51:52,600 PULLS THE PROTEIN THROUGH AND 1191 00:51:52,600 --> 00:51:54,160 YOU GET THE LONG LOOP ACROSS. 1192 00:51:54,160 --> 00:51:55,840 IF YOU LOOK AT THE DIVERSITY OF 1193 00:51:55,840 --> 00:51:57,800 MEMBRANE PROTEINS AS I SHOWED 1194 00:51:57,800 --> 00:51:59,320 YOU, WHAT I THINK IS GOING 1195 00:51:59,320 --> 00:52:02,080 HAPPEN AND WE'VE TESTED PARTS 1196 00:52:02,080 --> 00:52:04,200 BUT NOT ALL, IS THAT PROTEINS 1197 00:52:04,200 --> 00:52:06,000 THAT HAVE A SHORT SEGMENT THAT 1198 00:52:06,000 --> 00:52:07,880 NEEDS TO BE TRANSLOCATED ACROSS 1199 00:52:07,880 --> 00:52:10,960 ARE INSERTED BY AN OXA FAMILY 1200 00:52:10,960 --> 00:52:15,520 MEMBER, FOR EXAMPLE THE TAIL 1201 00:52:15,520 --> 00:52:18,200 ANCHORED FAMILY IS INSERTED BY 1202 00:52:18,200 --> 00:52:20,360 GET OR EMC, RIGOROUSLY 1203 00:52:20,360 --> 00:52:21,240 DEMONSTRATED. 1204 00:52:21,240 --> 00:52:22,880 GPCR, ALL THE PARTS ACROSS THE 1205 00:52:22,880 --> 00:52:24,640 MEMBRANE ARE SHORT, MEDIATED BY 1206 00:52:24,640 --> 00:52:26,920 OXA FAMILY MEMBERS SUCH AS EMC 1207 00:52:26,920 --> 00:52:28,480 FOR THE FIRST SEGMENT, GEL FOR 1208 00:52:28,480 --> 00:52:29,360 THE OTHERS. 1209 00:52:29,360 --> 00:52:32,960 AND SOME PROTEINS SUCH AS THIS 1210 00:52:32,960 --> 00:52:35,600 ONE WHICH REPRESENTS SUBUNIT OF 1211 00:52:35,600 --> 00:52:36,760 THE PENTAMERIC ION CHANNEL, 1212 00:52:36,760 --> 00:52:38,680 FIRST PART IS MEDIATED BY SEC, 1213 00:52:38,680 --> 00:52:41,400 IN FACT THERE'S A CLEAVABLE 1214 00:52:41,400 --> 00:52:42,920 SIGNAL PEPTIDE ENCODED THERE. 1215 00:52:42,920 --> 00:52:45,680 OTHER PARTS ARE MEDIATED BY OXA, 1216 00:52:45,680 --> 00:52:46,600 THAT ARE SHORT. 1217 00:52:46,600 --> 00:52:50,200 SO NOW WHAT STARTED OFF AS THIS 1218 00:52:50,200 --> 00:52:53,040 SORT OF BEWILDERING DIVERSITY OF 1219 00:52:53,040 --> 00:52:55,240 MEMBRANE PROTEINS I THINK CAN BE 1220 00:52:55,240 --> 00:53:00,240 SIMPLIFIED TO A SIMPLE CONCEPT 1221 00:53:00,240 --> 00:53:03,280 WHICH IS THAT EVOLUTIONARY THERE 1222 00:53:03,280 --> 00:53:13,840 ARE TWO CLASSIFICATIONS, OXA FOR 1223 00:53:14,120 --> 00:53:17,400 INHERITING HYDROPHOBIC, AND SEC 1224 00:53:17,400 --> 00:53:18,320 FOR SECRETION, MOVING 1225 00:53:18,320 --> 00:53:20,400 POLYPEPTIDE ACROSS THE LIPID 1226 00:53:20,400 --> 00:53:21,600 BILAYER, FOR SOME MEMBRANE 1227 00:53:21,600 --> 00:53:24,560 PROTEINS BUT NOT OTHERS. 1228 00:53:24,560 --> 00:53:27,080 AND SO I WILL LEAVE IT THERE AND 1229 00:53:27,080 --> 00:53:29,680 JUST REMIND YOU THAT WE THINK 1230 00:53:29,680 --> 00:53:31,680 THAT IN ALL ORGANISMS THERE 1231 00:53:31,680 --> 00:53:33,200 WOULD BE THESE FACTORS THAT 1232 00:53:33,200 --> 00:53:34,440 MEDIATE INSERTION OF DIFFERENT 1233 00:53:34,440 --> 00:53:35,920 PARTS OF THE MEMBRANE PROTEIN 1234 00:53:35,920 --> 00:53:38,080 AND BECAUSE AT LEAST IN THE 1235 00:53:38,080 --> 00:53:38,880 MAMMALIAN SYSTEM THAT WE'VE 1236 00:53:38,880 --> 00:53:45,920 STUDIED THESE FACTORS CAN BE 1237 00:53:45,920 --> 00:53:46,760 ASSEMBLED IN CONTEXT-DEPENDENT 1238 00:53:46,760 --> 00:53:48,560 OR SUBSTRATE-DRIVEN MANNER, AS 1239 00:53:48,560 --> 00:53:50,320 THE POLYPEPTIDE COMES OUT OF THE 1240 00:53:50,320 --> 00:53:51,280 RIBOSOME THE CAPACITY TO USE 1241 00:53:51,280 --> 00:53:56,400 EITHER MEMBERS OF THE FAMILY FR 1242 00:53:56,400 --> 00:53:59,160 INSERTION BUTTRESSED WITH THE 1243 00:53:59,160 --> 00:54:00,720 PAT COMPLEX THAT MAY FACILITATE 1244 00:54:00,720 --> 00:54:03,160 CHAPERONING OF THE PROTEIN IN A 1245 00:54:03,160 --> 00:54:04,920 PROTECTED SPACE BEHIND SEC61 1246 00:54:04,920 --> 00:54:07,680 WHERE WE THINK THE PROTEIN CAN 1247 00:54:07,680 --> 00:54:08,880 FOLD WITHOUT BEING INTERFERED 1248 00:54:08,880 --> 00:54:11,080 WITH BY, YOU KNOW, AGGREGATION 1249 00:54:11,080 --> 00:54:13,040 OR INTERACTION WITH QUALITY 1250 00:54:13,040 --> 00:54:13,600 CONTROL FACTORS. 1251 00:54:13,600 --> 00:54:15,520 AND SO THE LAST THING I'LL SAY 1252 00:54:15,520 --> 00:54:21,320 IS THAT THAT REVIEW I WROTE 25 1253 00:54:21,320 --> 00:54:24,200 YEARS AGO, LAST FIGURE 1254 00:54:24,200 --> 00:54:25,800 SPECULATED HOW THIS MIGHT WORK. 1255 00:54:25,800 --> 00:54:28,200 ALTHOUGH IT WAS PURE 1256 00:54:28,200 --> 00:54:30,320 SPECULATION, SOMETHING IN 1257 00:54:30,320 --> 00:54:32,400 REVIEWS BACK THEN, WE IN FACT 1258 00:54:32,400 --> 00:54:33,840 SPECULATED THAT THERE'S NO WAY 1259 00:54:33,840 --> 00:54:35,400 IT'S POSSIBLE FOR A SMALL 1260 00:54:35,400 --> 00:54:37,960 CHANNEL TO INSERT SO MANY 1261 00:54:37,960 --> 00:54:39,080 MEMBRANE PROTEINS. 1262 00:54:39,080 --> 00:54:40,040 SANDY SIMON HAD INDIRECT 1263 00:54:40,040 --> 00:54:42,320 EVIDENCE THERE WAS THE CAPACITY 1264 00:54:42,320 --> 00:54:43,320 TO HAVE MULTIPLE TRANSMEMBRANE 1265 00:54:43,320 --> 00:54:45,920 SEGMENTS THAT HAD NOT QUITE BEEN 1266 00:54:45,920 --> 00:54:48,040 INSERTED BUT SOMEHOW HELD IN THE 1267 00:54:48,040 --> 00:54:51,080 MEMBRANE, WE DREW THIS VERY 1268 00:54:51,080 --> 00:54:56,880 LARGE MACHINE, SITTING ON THE 1269 00:54:56,880 --> 00:54:59,960 RIBOSOME. 1270 00:54:59,960 --> 00:55:09,160 SATISFYING BUT I TAKE NO CREDIT, 1271 00:55:09,160 --> 00:55:11,640 THE SIZE MATCHES TO THE LOCON 1272 00:55:11,640 --> 00:55:12,040 STRUCTURE. 1273 00:55:12,040 --> 00:55:13,680 I WILL LEAVE IT THERE AND THANK 1274 00:55:13,680 --> 00:55:15,200 YOU FOR YOUR ATTENTION. 1275 00:55:15,200 --> 00:55:17,360 >> THAT WAS FANTASTIC TALK. 1276 00:55:17,360 --> 00:55:18,640 TWO QUESTIONS. 1277 00:55:18,640 --> 00:55:21,400 SO, FIRST ONE, GIVEN THE FACT 1278 00:55:21,400 --> 00:55:27,640 THAT THIS SYSTEM IS DEPENDENT ON 1279 00:55:27,640 --> 00:55:28,360 THE PARTIAL HYDROPHILICITY, 1280 00:55:28,360 --> 00:55:30,280 THERE'S A HETEROGENEITY OF THAT 1281 00:55:30,280 --> 00:55:32,440 IN THE MEMBRANE PROTEIN WALL, SO 1282 00:55:32,440 --> 00:55:35,080 YOU IMAGINE THAT THERE'S A 1283 00:55:35,080 --> 00:55:37,160 HETEROGENEITY OF OTHER CHAPERONE 1284 00:55:37,160 --> 00:55:39,920 PROTEINS WHEN DIFFERENT MEMBRANE 1285 00:55:39,920 --> 00:55:40,880 PROTEINS ARE INSERTED, THAT'S 1286 00:55:40,880 --> 00:55:44,680 THE FIRST QUESTION. 1287 00:55:44,680 --> 00:55:45,360 SECOND ONE, CROSS-LINKING SYSTEM 1288 00:55:45,360 --> 00:55:47,440 IS VERY BEAUTIFUL BUT DID YOU 1289 00:55:47,440 --> 00:55:49,840 EVER TRY TO GET SOME OF THE 1290 00:55:49,840 --> 00:55:52,360 INITIAL DATA JUST USING 1291 00:55:52,360 --> 00:55:53,000 CROSS-LINKING MASS SPECTROMETRY 1292 00:55:53,000 --> 00:55:55,320 AND DID YOU FIND ANYTHING? 1293 00:55:55,320 --> 00:55:58,440 >>YEAH, GREAT QUESTION. 1294 00:55:58,440 --> 00:56:01,680 SO, THE FIRST QUESTION, ALTHOUGH 1295 00:56:01,680 --> 00:56:05,920 THERE'S A LARGE DIVERSITY OF 1296 00:56:05,920 --> 00:56:07,120 HYDROPHILICICT IN TRANSMEMBRANE 1297 00:56:07,120 --> 00:56:08,520 DOMAINS ALMOST ALL MULTI-PASS 1298 00:56:08,520 --> 00:56:12,800 PROTEINS HAVE EITHER THE FIRST 1299 00:56:12,800 --> 00:56:14,240 OR SECOND DOMAIN BEING 1300 00:56:14,240 --> 00:56:16,760 RELATIVELY HYDROPHILIC. 1301 00:56:16,760 --> 00:56:18,200 THAT'S INTERESTING BECAUSE IT 1302 00:56:18,200 --> 00:56:21,080 DOES SUGGEST THE PAT COMPLEX AND 1303 00:56:21,080 --> 00:56:22,560 OTHER COMPONENTS GET RECRUITED 1304 00:56:22,560 --> 00:56:23,120 QUITE EARLY. 1305 00:56:23,120 --> 00:56:25,840 IT'S NOT THAT SURPRISING IN 1306 00:56:25,840 --> 00:56:27,840 HINDSIGHT BECAUSE PACKING OF 1307 00:56:27,840 --> 00:56:28,680 MULTIPLE TRANSMEMBRANE SEGMENTS 1308 00:56:28,680 --> 00:56:33,440 INTO A BUNDLE WHICH IS WHAT MOST 1309 00:56:33,440 --> 00:56:36,080 MEMBRANES DO IS MEDIATED 1310 00:56:36,080 --> 00:56:40,320 PRIMARILY BY EITHER SHORT SIDE 1311 00:56:40,320 --> 00:56:45,440 CHAINS LIKE LYSINES OR 1312 00:56:45,440 --> 00:56:46,600 HYDROPHILIC RESIDUES, ENRICHMENT 1313 00:56:46,600 --> 00:56:47,640 MAKES SENSE. 1314 00:56:47,640 --> 00:56:48,960 MASS SPEC, IT'S A GOOD QUESTION. 1315 00:56:48,960 --> 00:56:50,760 I DIDN'T TALK ABOUT SOME OF THE 1316 00:56:50,760 --> 00:56:53,840 REALLY NICE WORK THAT BOB KEENAN 1317 00:56:53,840 --> 00:56:57,920 DID, BUT BOB WAS FOCUSED ON THE 1318 00:56:57,920 --> 00:56:58,440 GEL COMPLEX. 1319 00:56:58,440 --> 00:57:01,320 AND BEFORE WE KNEW IT WAS A 1320 00:57:01,320 --> 00:57:02,640 COMPLEX. 1321 00:57:02,640 --> 00:57:05,560 HE PULLED ON TMCL1, A SUBUNIT, 1322 00:57:05,560 --> 00:57:07,960 DID MASS SPEC AND CROSSLINKING 1323 00:57:07,960 --> 00:57:10,360 MASS SPEC. 1324 00:57:10,360 --> 00:57:11,760 IN HINDSIGHT WITH STRUCTURES, 1325 00:57:11,760 --> 00:57:13,440 ONCE CAN MAKE A FAIR AMOUNT OF 1326 00:57:13,440 --> 00:57:14,760 SENSE OF THIS. 1327 00:57:14,760 --> 00:57:20,400 BUT I THINK THAT IT WAS A BIT 1328 00:57:20,400 --> 00:57:22,080 DIFFICULT WITHOUT THAT AS KIND 1329 00:57:22,080 --> 00:57:24,480 OF A DISCOVERY VEHICLE. 1330 00:57:24,480 --> 00:57:25,120 BUT YES. 1331 00:57:25,120 --> 00:57:27,560 >> THANK YOU. 1332 00:57:27,560 --> 00:57:29,400 >> YEAH, BEAUTIFUL TALK. 1333 00:57:29,400 --> 00:57:30,760 ODDLY SPECIFIC QUESTION. 1334 00:57:30,760 --> 00:57:33,000 I WAS INTERESTED IN THIS ASTRIX 1335 00:57:33,000 --> 00:57:36,200 PROTEIN IN THE PAT COMPLEX, IT 1336 00:57:36,200 --> 00:57:38,920 HAS A UNIQUE HYDROPHILIC FACE. 1337 00:57:38,920 --> 00:57:40,440 HAVE YOU STUDIED HOW IT'S 1338 00:57:40,440 --> 00:57:41,720 INSERTED IN THE MEMBRANE AND 1339 00:57:41,720 --> 00:57:43,640 WHAT'S THE STABILITY LIKE IF YOU 1340 00:57:43,640 --> 00:57:45,480 HAVE THE HYDROPHILIC FACE 1341 00:57:45,480 --> 00:57:47,640 IMPORTANT FOR ANCHORING THESE 1342 00:57:47,640 --> 00:57:48,600 UNIQUE SEGMENTS? 1343 00:57:48,600 --> 00:57:51,520 >> YEAH, I HAVE TO SAY WHAT I 1344 00:57:51,520 --> 00:57:52,480 SHOWED, THIS BEAUTIFUL 1345 00:57:52,480 --> 00:57:55,680 HYDROPHILIC FACE, WE HAVE TO 1346 00:57:55,680 --> 00:57:57,760 REMEMBER THAT'S A MODEL. 1347 00:57:57,760 --> 00:58:00,280 AND SO ONE POSSIBILITY IS THAT 1348 00:58:00,280 --> 00:58:01,840 THERE'S A DIFFERENT CONFIRMATION 1349 00:58:01,840 --> 00:58:03,200 THAT OBSCURES THAT HYDROPHILIC 1350 00:58:03,200 --> 00:58:05,600 FACE WHEN IT'S NOT BEING USED. 1351 00:58:05,600 --> 00:58:08,800 THAT'S MY FAVORITE IDEA OF 1352 00:58:08,800 --> 00:58:09,960 WHAT'S HAPPENING. 1353 00:58:09,960 --> 00:58:12,000 AND SO -- AND THEN -- BUT WE 1354 00:58:12,000 --> 00:58:13,960 DON'T HAVE ANY INFORMATION ABOUT 1355 00:58:13,960 --> 00:58:14,560 THAT. 1356 00:58:14,560 --> 00:58:16,680 THE OTHER ASPECT OF THE 1357 00:58:16,680 --> 00:58:19,920 QUESTION, IT TURNS OUT MOST -- 1358 00:58:19,920 --> 00:58:21,480 MANY PROTEINS INVOLVED IN 1359 00:58:21,480 --> 00:58:22,720 BIOGENESIS OFTEN NEED THEMSELVES 1360 00:58:22,720 --> 00:58:24,920 TO BE MADE. 1361 00:58:24,920 --> 00:58:27,680 SO, FOR EXAMPLE, SEC61 IS GOING 1362 00:58:27,680 --> 00:58:30,160 TO USE IN PART SEC61 AND THESE 1363 00:58:30,160 --> 00:58:32,440 OTHER COMPONENTS TO BE PRODUCED, 1364 00:58:32,440 --> 00:58:33,560 AND PRESUMABLY ASTRIX WILL ALSO 1365 00:58:33,560 --> 00:58:36,800 NEED SOME OF THESE COMPONENTS TO 1366 00:58:36,800 --> 00:58:39,360 BE PRODUCED. 1367 00:58:39,360 --> 00:58:43,040 CYTOSOL, FOR EXAMPLE, HSB7 0 1368 00:58:43,040 --> 00:58:43,680 PROBABLY NEEDS THAT. 1369 00:58:43,680 --> 00:58:44,480 >> CHAPERONE HELPS CHAPERONE. 1370 00:58:44,480 --> 00:58:46,240 DO YOU THINK THAT'S RELATED TO 1371 00:58:46,240 --> 00:58:48,000 TRANSITION FROM EARLY TO LATE? 1372 00:58:48,000 --> 00:58:48,960 >> YEAH, THE TRANSITION FROM 1373 00:58:48,960 --> 00:58:52,280 EARLY TO LATE IS SOMETHING WE 1374 00:58:52,280 --> 00:58:53,560 DON'T FULLY UNDERSTAND. 1375 00:58:53,560 --> 00:58:57,920 PART OF THE EQUATION CLEARLY IS 1376 00:58:57,920 --> 00:58:59,640 HYDROPHILICITY OF THE EARLY 1377 00:58:59,640 --> 00:59:01,880 TRANSMEMBRANE SEGMENT. 1378 00:59:01,880 --> 00:59:07,800 IF WE MAKE THAT HYDROPHILIC, ALL 1379 00:59:07,800 --> 00:59:11,960 LEUCINES, THE PAT COMPLEXES AND 1380 00:59:11,960 --> 00:59:14,520 OTHER COMPLEXES DON'T GET 1381 00:59:14,520 --> 00:59:15,040 RECRUITED. 1382 00:59:15,040 --> 00:59:22,880 THEY CAN CLEARLY BIND THE 1383 00:59:22,880 --> 00:59:24,760 RIBOSOME, THAT AFFINITY IS NOT 1384 00:59:24,760 --> 00:59:25,480 ENOUGH. 1385 00:59:25,480 --> 00:59:27,360 >> A QUICK QUESTION FROM ONLINE 1386 00:59:27,360 --> 00:59:28,960 COMMUNITY. 1387 00:59:28,960 --> 00:59:33,560 HOW DOES THE ENGAGEMENT BETWEEN 1388 00:59:33,560 --> 00:59:34,160 SEC61 AND HYDROPHILIC SEGMENT 1389 00:59:34,160 --> 00:59:36,000 OUT OF THE RIBOSOME HAPPEN? 1390 00:59:36,000 --> 00:59:38,320 IS THERE A SIGNAL OR CHEMICAL 1391 00:59:38,320 --> 00:59:40,120 REACTION THAT OPENS UP THE 1392 00:59:40,120 --> 00:59:40,360 CHANNEL? 1393 00:59:40,360 --> 00:59:42,440 >> YEAH, SO THIS IS A GREAT 1394 00:59:42,440 --> 00:59:42,800 QUESTION. 1395 00:59:42,800 --> 00:59:44,320 AND WE HAVE A SNAPSHOT OF WHAT 1396 00:59:44,320 --> 00:59:46,200 IT LOOKS LIKE AFTER IT'S 1397 00:59:46,200 --> 00:59:49,640 ENGAGED, WE DO NOT KNOW HOW 1398 00:59:49,640 --> 00:59:50,880 SEC61 GETS OPENED. 1399 00:59:50,880 --> 00:59:55,480 AND SO ONE POSSIBILITY IS THAT 1400 00:59:55,480 --> 00:59:57,080 MAYBE IT INITIALLY KIND OF 1401 00:59:57,080 --> 00:59:59,720 ENGAGES A LITTLE BIT OF THE 1402 00:59:59,720 --> 01:00:02,240 LATERAL GATE THAT'S PARTIALLY 1403 01:00:02,240 --> 01:00:07,600 CRACKED, AND SORT OF AS MORE 1404 01:00:07,600 --> 01:00:12,640 COMES OUT IT WEAVES INTO THE 1405 01:00:12,640 --> 01:00:17,160 LATERAL GATE AND FORMS A HELIX. 1406 01:00:17,160 --> 01:00:18,000 CO-TRANSLATIONAL PROCESSES HAVE 1407 01:00:18,000 --> 01:00:22,440 BEEN DIFFICULT TO STUDY. 1408 01:00:22,440 --> 01:00:24,640 >> IF YOU HAVE A C-TERMINAL 1409 01:00:24,640 --> 01:00:26,120 TRANSMEMBRANE SEGMENT WHICH IS 1410 01:00:26,120 --> 01:00:27,640 ATTACHED TO A LARGE HYDROPHILIC 1411 01:00:27,640 --> 01:00:30,080 SEGMENT THAT HAS TO BE 1412 01:00:30,080 --> 01:00:32,880 TRANSLOCATED INTO THE LUMEN OF 1413 01:00:32,880 --> 01:00:35,200 THE E.R. AND THEREFORE THAT 1414 01:00:35,200 --> 01:00:36,200 PARTICULAR TRANSMEMBRANE SEGMENT 1415 01:00:36,200 --> 01:00:39,000 WOULD GO THROUGH THE SEC61 1416 01:00:39,000 --> 01:00:43,120 CHANNEL, WOULD THERE BE A 1417 01:00:43,120 --> 01:00:44,520 PROBLEM JOINING THE N-TERMINAL 1418 01:00:44,520 --> 01:00:47,480 PORTION OF THE PROTEIN WHICH IS 1419 01:00:47,480 --> 01:00:52,400 ASSOCIATED WITH PAT, GEL, AND 1420 01:00:52,400 --> 01:00:52,600 BOS? 1421 01:00:52,600 --> 01:00:53,680 >> YEAH, I THINK THAT WHAT 1422 01:00:53,680 --> 01:00:54,920 NEEDS TO HAPPEN IS THE 1423 01:00:54,920 --> 01:00:55,520 FOLLOWING. 1424 01:00:55,520 --> 01:00:59,080 WE DON'T HAVE EVIDENCE FOR THIS 1425 01:00:59,080 --> 01:01:00,480 YET, MY FAVORITE HYPOTHESIS. 1426 01:01:00,480 --> 01:01:10,880 REMEMBER THERE ARE THESE TWO 1427 01:01:10,880 --> 01:01:13,920 HELLICICES THAT WEDGE BETWEEN. 1428 01:01:13,920 --> 01:01:15,360 THE TWO HELICES HAVE TO MOVE OUT 1429 01:01:15,360 --> 01:01:17,080 OF THE WAY, OTHERWISE THERE'S NO 1430 01:01:17,080 --> 01:01:18,440 WAY TO OPEN SEC61. 1431 01:01:18,440 --> 01:01:19,880 THE QUESTION IS WHAT IS THE 1432 01:01:19,880 --> 01:01:23,200 TRIGGER FOR THAT TO HAPPEN? 1433 01:01:23,200 --> 01:01:26,160 AND WE THINK -- IF YOU -- I 1434 01:01:26,160 --> 01:01:27,200 DIDN'T GO INTO DETAILS OF THE 1435 01:01:27,200 --> 01:01:31,360 STRUCTURE BUT THE END OF THOSE 1436 01:01:31,360 --> 01:01:33,240 TWO HELICES PROTRUDES INTO THE 1437 01:01:33,240 --> 01:01:36,680 TUNNEL OF THE RIBOSOME, SO WE 1438 01:01:36,680 --> 01:01:41,520 THINK THAT PAT COMPLEX 1439 01:01:41,520 --> 01:01:42,640 SPECIFICALLY THE CCD47 SUBUNIT 1440 01:01:42,640 --> 01:01:43,880 CAN SOMEHOW SENSE WHAT'S COMING 1441 01:01:43,880 --> 01:01:46,920 OUT OF THE RIBOSOME SO ONCE YOU 1442 01:01:46,920 --> 01:01:49,120 START SYNTHESIZING A SEGMENT OF 1443 01:01:49,120 --> 01:01:52,800 HYDROPHILIC POPPY PEPTIDE THAT 1444 01:01:52,800 --> 01:01:55,000 NEEDS TO BE TRANSLOCATED MY 1445 01:01:55,000 --> 01:01:56,400 HYPOTHESIS IT STARTS 1446 01:01:56,400 --> 01:01:58,200 ACCUMULATING IN THE LIMITED 1447 01:01:58,200 --> 01:02:00,360 STAYS BETWEEN THE RIBOSOME AND 1448 01:02:00,360 --> 01:02:03,000 MEMBRANE COMPONENTS, AND THAT 1449 01:02:03,000 --> 01:02:08,880 DISPLACES THESE TWO HELICES OF 1450 01:02:08,880 --> 01:02:10,880 CCD47, ALLOWING SEC61 TO HOPE. 1451 01:02:10,880 --> 01:02:17,000 I THINK IT'S AN ATTRACTIVE 1452 01:02:17,000 --> 01:02:17,320 HYPOTHESIS. 1453 01:02:17,320 --> 01:02:18,920 WE DO KNOW IT THAT HAPPENS 1454 01:02:18,920 --> 01:02:20,280 BECAUSE WE CAN DESIGN SUBSTRATES 1455 01:02:20,280 --> 01:02:23,120 AND SHOW IT DOES SHIFT OVER FROM 1456 01:02:23,120 --> 01:02:25,080 ENGAGING THE PAT COMPLEX TO THE 1457 01:02:25,080 --> 01:02:25,680 SEC COMPLEX. 1458 01:02:25,680 --> 01:02:28,200 NOW, WE DON'T THINK THE PAT 1459 01:02:28,200 --> 01:02:29,720 COMPLEX DIFFUSES AWAY OR 1460 01:02:29,720 --> 01:02:30,600 DISASSEMBLES BECAUSE IT'S 1461 01:02:30,600 --> 01:02:31,800 ASSOCIATED WITH THE SUBSTRATE. 1462 01:02:31,800 --> 01:02:34,560 THAT'S WHY I FOCUS ON MOVEMENT 1463 01:02:34,560 --> 01:02:36,840 OF THESE HELICES OUT OF THE WAY 1464 01:02:36,840 --> 01:02:43,840 TO ALLOW SEC TO OPEN. 1465 01:02:43,840 --> 01:02:44,360 >> GREAT TALK. 1466 01:02:44,360 --> 01:02:46,440 THE QUESTION IS ABOUT YOU 1467 01:02:46,440 --> 01:02:49,520 STARTED WITH SHOWING THAT THE 1468 01:02:49,520 --> 01:02:51,600 FIRST TRANSMEMBRANE IS INSERTED 1469 01:02:51,600 --> 01:02:54,040 BY THE EMC. 1470 01:02:54,040 --> 01:02:56,760 AND YET SHOWED ALSO THAT THE 1471 01:02:56,760 --> 01:02:57,960 FIRST TRANSMEMBRANE IS 1472 01:02:57,960 --> 01:03:02,400 HYDROPHILIC AND STABILIZED WITH 1473 01:03:02,400 --> 01:03:03,960 INTERACTION BETWEEN SEC61 AND 1474 01:03:03,960 --> 01:03:08,120 PAT SO HOW IS IT STABILIZED WHEN 1475 01:03:08,120 --> 01:03:09,080 INSERTED AFTER EMC? 1476 01:03:09,080 --> 01:03:13,600 >> YEAH, SO I THINK IT MAY BE 1477 01:03:13,600 --> 01:03:16,880 THAT THE TIME IT IS IN THE 1478 01:03:16,880 --> 01:03:17,960 MEMBRANE IS RELATIVELY SHORT 1479 01:03:17,960 --> 01:03:20,320 BETWEEN WHEN INSERTED BY EMC AND 1480 01:03:20,320 --> 01:03:21,920 ENGAGES THE PAT COMPLEX. 1481 01:03:21,920 --> 01:03:23,840 I FORGOT TO MENTION, MAYBE I DID 1482 01:03:23,840 --> 01:03:25,880 MENTION IN PASSING, THE PAT 1483 01:03:25,880 --> 01:03:28,160 COMPLEX IS FAIRLY ABUNDANT AS 1484 01:03:28,160 --> 01:03:28,360 WELL. 1485 01:03:28,360 --> 01:03:32,000 WE THINK THAT PERHAPS IT SIMPLY 1486 01:03:32,000 --> 01:03:32,800 CAPTURES FAIRLY QUICKLY AND 1487 01:03:32,800 --> 01:03:35,520 THERE ISN'T A BIG PROBLEM. 1488 01:03:35,520 --> 01:03:38,280 THE OTHER MORE ATTRACTIVE IDEA 1489 01:03:38,280 --> 01:03:40,360 OBVIOUSLY THERE'S A PRECISE 1490 01:03:40,360 --> 01:03:41,520 HANDOVER FROM ONE COMPLEX TO 1491 01:03:41,520 --> 01:03:41,880 ANOTHER. 1492 01:03:41,880 --> 01:03:45,960 AND I WILL SAY THAT WHEN WE 1493 01:03:45,960 --> 01:03:47,240 FIRST PURIFIED EMC FOR 1494 01:03:47,240 --> 01:03:48,320 RECONSTITUTION STUDIES AND SO 1495 01:03:48,320 --> 01:03:51,120 FORTH AND DID MASS SPEC ON IT, 1496 01:03:51,120 --> 01:03:53,080 CCD47 WAS ONE OF THE COMPONENTS. 1497 01:03:53,080 --> 01:03:57,520 AND SO I'VE ALWAYS LIKED THIS 1498 01:03:57,520 --> 01:03:58,040 IDEA. 1499 01:03:58,040 --> 01:04:00,400 HOWEVER, SEEING A VERY ABUNDANT 1500 01:04:00,400 --> 01:04:06,520 MEMBRANE PRO IN A MASS SPEC 1501 01:04:06,520 --> 01:04:08,000 EXPERIMENT MAKES ME NERVOUS SO 1502 01:04:08,000 --> 01:04:09,440 I'VE BEEN HESITANT TO PUT TOO 1503 01:04:09,440 --> 01:04:10,240 MUCH STOCK IN IT. 1504 01:04:10,240 --> 01:04:11,800 >> A MORE GENERAL QUESTION. 1505 01:04:11,800 --> 01:04:15,160 AFTER THE FIRST STAGE YOU NOW 1506 01:04:15,160 --> 01:04:17,240 HAVE TO TRANSFER TWO 1507 01:04:17,240 --> 01:04:17,960 TRANSMEMBRANE DOMAINS, THAT 1508 01:04:17,960 --> 01:04:19,960 STILL SEEMS KIND OF PAINFUL. 1509 01:04:19,960 --> 01:04:22,040 IS THERE EVIDENCE OR SUGGESTION 1510 01:04:22,040 --> 01:04:23,360 PERHAPS THE MEMBRANE COMPOSITION 1511 01:04:23,360 --> 01:04:25,240 IN THIS AREA IS DIFFERENT IN 1512 01:04:25,240 --> 01:04:29,200 SOME WAY TO FACILITATE THE 1513 01:04:29,200 --> 01:04:29,600 TRANSFER? 1514 01:04:29,600 --> 01:04:30,480 >>YEAH, I PERSONALLY REALLY 1515 01:04:30,480 --> 01:04:31,680 LIKE THIS IDEA BUT CANNOT THINK 1516 01:04:31,680 --> 01:04:34,760 OF A GOOD WAY TO LIKE TEST THIS 1517 01:04:34,760 --> 01:04:35,360 CAREFULLY. 1518 01:04:35,360 --> 01:04:37,720 AND I WOULD LOVE TO HEAR 1519 01:04:37,720 --> 01:04:38,320 SUGGESTIONS. 1520 01:04:38,320 --> 01:04:42,520 >> SO THERE WERE NO OTHER 1521 01:04:42,520 --> 01:04:44,040 PROTEINS THAT MIGHT MODIFY THE 1522 01:04:44,040 --> 01:04:45,000 PROTEINS IN YOUR SCHEME? 1523 01:04:45,000 --> 01:04:46,440 >> NO, NOT THAT WE CAN THINK 1524 01:04:46,440 --> 01:04:46,720 OF. 1525 01:04:46,720 --> 01:04:48,320 WHAT I SORT OF IMAGINE IS THAT 1526 01:04:48,320 --> 01:04:50,080 THE KIND OF -- I DIDN'T SLOW 1527 01:04:50,080 --> 01:04:54,960 THIS BUT THE INTERIOR OF THE 1528 01:04:54,960 --> 01:04:58,480 HORSESHOE CAVITY IS PARTIALLY 1529 01:04:58,480 --> 01:05:00,720 HYDROPHILIC AND HYDROPHOBIC, ONE 1530 01:05:00,720 --> 01:05:01,520 POSSIBILITY IS THOSE 1531 01:05:01,520 --> 01:05:03,320 POSSIBILITIES RECRUIT A CERTAIN 1532 01:05:03,320 --> 01:05:04,880 SUBSET OF LIPIDS THAT FACILITATE 1533 01:05:04,880 --> 01:05:05,320 INSERTION. 1534 01:05:05,320 --> 01:05:06,440 I LIKE THAT IDEA. 1535 01:05:06,440 --> 01:05:12,720 AND MAYBE SOME TYPE OF LIPID 1536 01:05:12,720 --> 01:05:13,680 CROSS-LINKING EXPERIMENTS COULD 1537 01:05:13,680 --> 01:05:14,040 BE DONE TO SEE IF THERE ARE 1538 01:05:14,040 --> 01:05:15,920 NON-BULK LIPIDS THERE. 1539 01:05:15,920 --> 01:05:18,720 >>THANK YOU VERY MUCH, 1540 01:05:18,720 --> 01:05:19,840 MANU, NOR GIVING A FANTASTIC 1541 01:05:19,840 --> 01:05:20,160 PRESENTATION. 1542 01:05:20,160 --> 00:00:00,000 [APPLAUSE]