WELCOME TO TODAY'S WEDNESDAY
AFTERNOON LECTURE SERIES
PRESENTATION.
I'M THE CHIEF OF THE SECTION
ON MICROBIAL PATHOGENESIS OF
NICHD.
ON BEHALF OF THE
PROTEIN TRAFFICKING DYNAMICS
INTEREST GROUP I WOULD LIKE TO
WELCOME BACK TO THE NIH TODAY'S
WALS SPEAKER, FRIEND AND FORMER
COLLEAGUE DR. MANU HEGDE.
MANY OF YOU REMEMBER MANU FROM
HIS TIME AT NIH, AS A MEMBER OF
THE NCI SCHOLARS PROGRAM, LATER
STARTING IN 2002 AS INVESTIGATOR
IN WHAT WAS FORMERLY KNOWN AS
THE CELL BIOLOGY METABOLISM
BRANCH OF NICHD.
SAME PROGRAM I JOINED A FEW
YEARS LATER.
MANU RECEIVED TENURE IN 2008,
2011 SADLY MOVED HIS LABORATORY
TO THE UNIVERSITY OF CAMBRIDGE
IN ENGLAND TO JOIN THE MEDICAL
RESEARCH COUNCIL LABORATORY OF
MOLECULAR BIOLOGY WHERE HE'S
BEEN SINCE THEN.
MANU IS AN ABSOLUTE LEADER IN
QUALITY PROTEIN CONTROL, AT THE
FOREFRONT OF STUDYING PROTEINS,
MEMBRANE PROTEINS IN PARTICULAR,
FROM THE MOMENT THEY ARE
SYNTHESIZED OR BORN AT THE
RIBOSOME TO DELIVERED TO THE
FINAL DESTINATION COMPARTMENT
WITHIN CELLS AND HOW CELLS
RECOGNIZE ERRORS THAT IF NOT
CORRECTED WOULD SIGNIFICANTLY
IMPACT HUMAN HEALTH.
MANU'S CONTRIBUTIONS TO THE
FIELD OF PROTEIN BIOGENESIS AND
QUALITY CONTROL ARE EXCEPTIONAL
IN ORIGINALITY, AND MORE THAN
130 PUBLICATIONS TODAY EVIDENCE
HE'S IN A CLASS OF HIS OWN,
REALLY.
SO FUN FACT, MANU SENT HIS HIS
CV, 13 PAGES LONG, PUBLICATION
RECORD COVERED 11 OF THE 13
PAGES.
WHAT MAKES HIM AN OUTSTANDING
SCIENTIST IS INSTINCT FOR
IMPORTANT BIOLOGICAL QUESTIONS,
AND HIS CLEVERNESS IN DEVELOPING
ELEGANT EXPERIMENTAL APPROACHES
TO FIND THE CORRECT ANSWER.
MANY DISCOVERIES MANU HAS MADE
OVER THE YEARS ONE MIGHT
DRAG
ADJACENT PART OF HYDROPHILIC POP
POLLY PEPTIDE ACROSS, THIS
DIDN'T MAKE SENSE TO SOME
PEOPLE, AND FELT STRONGLY THE
MOVEMENT ACROSS THE MEMBRANE
MUST GO THROUGH A CHANNEL.
AND SO AROUND 1980 OR SO IT WAS
PROPOSED THE WAY A MEMBRANE
PROTEIN GETS MADE IS YOU HAVE
THESE HYDROPHOBIC SEGMENTS OF
POLYPEPTIDE OUT OF THE RIBOSOME
INSERTED THROUGH A CHANNEL.
WHEN I WAS A GRADUATE STUDENT, I
WROTE A REVIEW ON HOW MEMBRANE
PROTEINS GET MADE, OF COURSE THE
PICTURE HADN'T CHANGED AT ALL IN
THE 17 YEARS SINCE IT WAS
PROPOSED, AND WHEN I WROTE THIS
REVIEW, THE IDEA IS THAT A
RIBOSOME IS DOCKED AT
TRANSLOCATION CHANNEL OR
TRANSLOCON, AS THESE BLACK BARS
COME OUT OF THE RIBOSOME THEY RD
THINGS FORWARD WAS THE IDENTITY
OF THIS, AND THE CENTRAL
COMPONENT AROSE FROM A
COMBINATION OF YEAST AND
BACTERIAL GENETICS AND
CHEMISTRY, AND A REAL ADVANCE
WAS TO GET THE STRUCTURE OF AN
ARC HEEL VERSION IN 2004, AND
THE PROTEIN WASN'T ACTUALLY IN
THE PROCESS OPEN INTO THE
LIPID BILAYER THROUGH THE
LATERAL GATE FOR INSERTION OF
HYDROPHOBIC ELEMENTS, THE IDEA
TURNS OUT TO BE TRUE, OUR
CONTRIBUTION WAS TO LEVERAGE
ADVANCES IN CryoEM TO STAGE
THE PROCESS OF INSERTION OF
HYDROPHOBIC SEGMENT BEFORE AND
AFTER IT ENGAGES THIS SEC61
TRANSLOCATION CHANNEL AND ONE
CAN SEE HOW THIS MIGHT WORK.
SO, THIS IS JUST A MORPH BETWEEN
AN INACTIVE AND ACTIVE VERSION
OF SEC61, ACTIVE MEANING ENGAGED
BY HYDROPHOBIC SEGMENT, IN THIS
CASE A SIGNAL PEPTIDE, AS
PREDICTED ORIGINALLY FROM
BIOCHEMICAL AND STRUCTURAL WORK
IS SITTING AT THIS LATERAL GATE.
IN ORDER TO SIT THERE, HALF OF
THE MOLECULE MOVES APART FROM
THE OTHER HALF, AND IN THAT
PROCESS THE CENTRAL OROIFICE
OPENS.
IN THIS TYPE OF PICTURE HOW THIS
TRANSLOCATION CHANNEL WORKS, YOU
HAVE A HYDROPHOBIC SEGMENT OF
POLYPEPTIDE THAT ENGAGES LATERAL
PART OF THIS CHANNEL, DRAGGING
THE HYDROPHILIC SEGMENT
DOWNSTREAM AND AT A LATER STEP
MOVED INTO THE BILAYER AND
THROUGH THE MEMBRANE THROUGH
THIS CHANNEL.
YOU CAN IMAGINE THEN, AGAIN I
EMPHASIZE THE FACT THAT IT
REALLY IS A WORKING MODEL, OF
HOW YOU WOULD THEN HAVE AS MORE
AND MORE HYDROPHOBIC SEGMENTS
COME OUT OF THE RIBOSOME THEY DO
THE SAME THING.
YOU INSERT THEM ALL ONE BY ONE
THROUGH THE LATERAL GATE OF THE
SEC61 CHANNEL, THE WORKING MODEL
IN REVIEWER TEXT BOOK THAT
YOU'LL SEE.
NOW, THAT MAKES A LOT OF SENSE.
THERE'S A GOOD REASON WHY THIS
MODEL HAS BEEN EXTREMELY
DURABLE.
BUT WHEN YOU LOOK AT A REAL
MEMBRANE PROTEIN AS OPPOSED TO
MODELS, YOU START TO SEE SOME
PROBLEMS.
SO, WHAT I'VE DEPICTED HERE IS A
G-PROTEIN COUPLED RECEPTOR, BETA
ADRENERGIC RECEPTOR.
WHEN THE PROTEIN IS MADE, AND
THIS IS A STRUCTURE OF IT, THE
HYDROPHOBIC PARTS OF THE
POLYPEPTIDE ARE IN THE MEMBRANE
AND ALL THE HYDROPHOBIC SURFACES
FACE THE LIPID BILAYER, HERE
HYDROPHOBIC IS RED.
THE INTERIOR OF THAT IS FILLED
WITH LOTS OF HYDROPHILIC AMINO
ACIDS, AND THOSE ARE
FUNCTIONALLY IMPORTANT FOR
THINGS LIKE BINDING TO LIGANDS,
IN THIS CASE ADRENALINE.
SO IF YOU LOOK AT KIND OF A
DECONSTRUCTED VERSION OF THIS,
YOU CAN SEE THE INDIVIDUAL
SEGMENTS OF POLYPEPTIDE THAT
SPAN THE MEMBRANE ARE HIGHLY
VARIABLE.
SO SOME ARE REMARKABLE HIGH
DOUGH FILL -- HYDROPHILIC.
IF YOU TEST WHETHER THEY CAN
ENGAGE SEC61 AND INSERT INTO THE
LIPID BILAYER, SEVERAL DON'T.
THREE OUT OF SEVEN DON'T.
IN MANY, MANY MULTI-PASS
MEMBRANE PROTEINS AROUND HALF OF
THE MEMBRANE PROTEIN SEGMENTS
CAN'T ENGAGE SEC61 WHEN TESTED
IN A MODEL SYSTEM RAISING THE
PROBLEM OF HOW THESE SEGMENTS
GET INSERTED.
THE SECOND PROBLEM OF
HYDROPHILIC PARTS OF A PROTEIN
LIKE THIS IN THE MEMBRANE IS
THESE PROTEINS ARE SYNTHESIZED
CO-TRANSLATIONALLY, INSERTED
CO-TRANSLATIONALLY.
TRANSLATION IS RELATIVELY SLOW,
YOU MAKE FIVE OR SIX AMINO ACIDS
A SECOND.
A 350 AMINO ACID PASSED PROTEIN
WILL TAKE OFF SECONDS TO GET
MADE, THEY ARE WAITING FOR QUITE
A LONG TIME, ON THE SCALE OF
MOLECULAR DYNAMICS TENS OF
SECONDS IS AN ENORMITY, A VERY
LONG TIME.
HOW HAD HYDROPHILIC SEGMENTS
STABILIZED UNTIL YOU MAKE THE --
WE TRIED TO UNDERSTAND THIS, ONE
CLASSIC APPROACH TO TAKE A
BIOCHEMICAL STRATEGY, AND THE
APPROACH HERE IS TO TAKE A CRUDE
EXTRACT THAT CAN SYNTHESIZE
PROTEINS, IN THIS CASE AN
EXTRACT MADE FROM LYSATE, ADD
AN mRNA THAT CODES FOR PROTEIN
YOU'RE INTERESTED IN, THE IDEA
IS THAT WOULD GET
GPCR.
SO IF YOU TRANSLATE THE PROTEIN,
IN THIS IN VITRO REACTION, YOU
PRODUCE A PRODUCT THAT CODES FOR
THE GPCR, IN THIS WAYS BETA 1
RECEPTOR, IN PRESENCE YOU CAN
SEE POLYPEPTIDEA FAIR AMOUNT,
SHIFTS TO HIGHER MOLECULAR
WEIGHT, THAT TURNS OUT IS
GLYCOSYLATION, THAT TELLS YOU
THAT AT LEAST THE BEGINNING PART
OF THE PROTEIN GOT INSERTED INTO
THE LIPID BILAYER.
WE SHOWED THE OTHER PARTS WERE S
NOT ONLY BEEN INSERTED BUT
FOLDED CORRECTLY.
IF YOU IMMOBILIZE A LIGAND ON
THIS CLASS FOR SOME BEADS AND DO
A PULLDOWN, YOU CAN APPRECIATE
THAT ONLY THE INSERTED AND
GLYCOSYLATED POPULATION GETS
PULLED OUT.
SO, WHAT THIS TELLS YOU IS THAT
IN THIS IN VITRO REACTION, EVEN
THOUGH IT DOESN'T NECESSARILY
WORK TO 100% EFFICIENCY, ALL THE
COMPONENTS THAT ARE NEEDED TO
SYNTHESIZE THE PROTEIN TAKE THE
RIBOSOME TO THE ER, INSERT
TRANSMEMBRANE SEGMENTS, THIS IS
AT LEAST A COMPLETE SYSTEM AND
THE TRICK IS TO FIGURE OUT
WHAT'S INVOLVED.
WE FACE TWO BIG PROBLEMS.
HOW DO YOU ACTUALLY FIND THE
FACTORS OF A PROCESS THAT OCCURS
CO-TRANSLATIONALLY AND IS VERY
DYNAMIC?
THE SECOND AND MORE CHALLENGING
PROBLEM IS HOW DO YOU CONVINCE
SOMEBODY TO LOOK FOR FACTORS
WHEN ALL OF THE TEXT BOOKS TELL
YOU THAT EVERYTHING IS ALREADY
KNOWN, RIGHT?
AND HERE WE BASICALLY WERE
STUMPED, RIGHT?
SO IT WAS DIFFICULT TO KIND OF
CONVINCE PEOPLE TO WORK ON THIS
AND OF COURSE WE DIDN'T HAVE
GREAT IDEAS HOW TO FIGURE THIS
OUT.
AT THIS POINT I'M REMINDED OF A
BOOK GIVEN TO ME BY MY DAD
GROWING UP, "HOW TO SOLVE IT,"
IT'S A MATHEMATICS BOOK, HE WAS
A MATHEMATICIAN.
IT'S HOW TO SOLVE MATHEMATICAL
PROBLEMS.
AND ONE OF THE KEY PRINCIPLES IN
THIS BOOK IS IF YOU CAN'T SOLVE
THE PROBLEM YOU'RE INTERESTED
IN, TRY TO SOLVE A SIMPLER
RELATED PROBLEM.
AND THE HOPE IS THAT IF THE
PROBLEM IS RELATED ENOUGH,
INSIGHTS FROM THE SIMPLER
VERSION WILL OF COURSE GIVE YOU
SOME TOE HOLD INTO THE PROBLEM
THAT WILL HELP YOU SOLVE THE
MORE COMPLICATED ONE.
WE OF COURSE USE THIS ALL THE
TIME.
THIS IS THE BASIS OF MODEL
SYSTEMS, WHY WE MAKE SIMPLE
VERSIONS OF CONSTRUCTS AND SO
FORTH BUT WORTH REMINDING
OURSELVES ONCE IN A WHILE.
MEMBRANE PROTEINS COME IN
VARIETIES AND FLAVORS, A COUPLE
LABELED HERE, GPCR IN THE
MIDDLE.
ABC TRANSPORTERS, RECEPTOR
TYROSINE KINASES, AND SO FORTH.
A NUMBER OF YEARS AGO WE GOT
INTERESTED IN UNDERSTANDING HOW
THE CLASS OF PROTEINS GET MADE,
AN IMPORTANT SUBSET ARE SNARE
PROTEIN INVOLVED IN PROTEIN
TRAFFICKING WITH A SINGLE
SEGMENT, IT WASN'T CLEAR HOW
THEY GET TARGETED TO THE
MEMBRANE AND INSERTED.
IT MIGHT SEEM LIKE INVOLVING
THAT PROBLEM WOULD GIVE US
INSIGHT INTO THESE OTHERS WAS A
BIT FANCIFUL, THAT'S THE CASE
AND I'LL EXPLAIN WHY.
SO, THIS GOES BACK QUITE MANY
YEARS NOW, BUT HOW THIS WORKS
WAS REALLY UNCLEAR, AND WHAT WE
DID WAS RESEARCHED FOR FACTORS
THAT RECOGNIZED THIS HYDROPHOBIC
TRANSMEMBRANE SEGMENT AND
DELIVER PROTEIN TO THE MEMBRANE
WHERE IT NEEDS TO BE INSERTED.
USING A BIOCHEMICAL STRATEGY WE
IDENTIFIED A FACTOR IN MAMMALIAN
CELLS WHICH IS ATPase WIDELY
CONSERVED, IT SEEMED OF SEEMS TO
BE TARGETING FACTOR THAT
DELIVERS IT TO THE E.R. FOR
INSERTION.
AND THE REASON THIS WAS AN
IMPORTANT ADVANCE IS BECAUSE
ONCE YOU KNOW THE FIRST
COMPONENT OF ANY PATHWAY, YOU
CAN APPLY LOTS OF OTHER TOOLS,
GENETIC TOOLS, BIOCHEMICALS
TOOLS AND SO FORTH TO WORK OUT
PATHWAY.
WE NOW HAVE A COMPLETE PICTURE
OF HOW THIS WORKS INCLUDING
STRUCTURES, RECONSTITUTED WITH
PURIFIED COMPONENTS AND SO FORTH
BUT ONE OF THE KEY INSIGHTS IS
AT THE MEMBRANE RECEPTOR FOR
THIS TARGETING FACTOR IS A
TWO-PROTEIN COMPLEX THAT
RELEASES THE SUBSTRATE FROM THIS
TARGETING FACTOR AND THEN
FACILITATES ITS INSERTION INTO
THE BILAYER.
THE REASON THIS WAS IMPORTANT TO
UNDERSTAND AND THESE ARE THE
NAMES OF THE YEAST PROTEINS, GET
1, GET 2, GET 3, I SHOULD SAY A
NUMBER OF GROUPS AROUND THE
WORLD CONTRIBUTED TO WORKING OUT
THIS PATHWAY, IS THAT THESE TWO
PROTEINS ALTHOUGH INITIALLY
DIFFICULT TO FIND HOMOLOGS OF
IT, WITH BETTER METHODS OF
SEARCHING SUCH AS STRUCTURE
BASED METHODS LIKE HH PRED COULD
APPRECIATE THERE WERE HOMOLOGS,
IN THE MAMMALIAN TWO OTHER
HOMOLOGS, TWO THE CENTRAL PART
OF A MUCH LARGER COMPLEX, EMC,
AND THERE WAS A VERY POORLY
UNDERSTOOD PAIR OF PROTEINS THAT
LATER WERE SHOWN TO FORM A
COMPLEX, AND THAT WE NAMED THE
GEL COMPLEX, AND I'LL COME BACK
TO THAT.
OF COURSE, IF THIS IS AN
INSERTION FACTOR FOR INSERTING
TRANSMEMBRANE SEGMENT INTO THE
BILAYER, IT STANDS TO REASON
THESE OTHERS MIGHT HAVE A
SIMILAR FUNCTION IF THEY ARE
STRUCTURALLY SIMILAR.
AND SO, MY LAB DECIDED TO
INVESTIGATE THIS FOR EMC, AND
OUR COLLABORATOR BOB KEENAN WENT
TO WORK TO STUDY WHAT THE OTHER
COMPLEX, THE GEL COMPLEX, DOES.
AND THIS WINDS UP CONVERGING A
BIT LATER.
SO, WHAT WE FOUND, THE OTHER
IMPORTANT THING TO SAY, I SHOULD
GO BACK HERE, IS THAT THESE ARE
THE MAMMALIAN PROTEINS AND
NAMES.
BUT IT TURNS OUT A REALLY
IMPORTANT INSIGHT FROM THIS KIND
OF BIOINFORMATIC ANALYSIS IS
THAT IT APPEARED THAT THE GET1
WAS RELATED TO A BACTERIAL
PROTEIN, AND THE BACTERIAL
PROTEIN EMC, LINKED COMPONENTS
IN BACTERIA WITH COMPONENTS IN
EUKARYOTES, AND SEEMED LIKE THIS
WAS A VERY UNIVERSAL FAMILY AND
IT'S NOW KNOWN AS THE OXA 1
FAMILY, NOMENCLATURE IS TERRIBLE
BUT NOT THAT IMPORTANT FOR OUR
DISCUSSION.
WHAT'S KNOWN FROM STUDYING THIS
FAMILY, THE E. COLI PROTEIN
LOOKS LIKE THIS, A BEAUTIFUL
STRUCTURE, THE WAY IT MEDIATES
TRANSMEMBRANE DOMAIN INSERTION
IS HYPOTHESIZED TO BE THE
FOLLOWING.
REMEMBER THAT AT THE BEGINNING I
MENTIONED THAT HYDROPHOBIC
DOMAINS WILL SPONTANEOUSLY
INSERT INTO THE LIPID BILAYER
BUT BARRIER IS THE TRANSLOCATION
OF THE HYDROPHILIC PART ACROSS
THE MEMBRANE SO THE ENERGY
GAINED BY PARTITIONING THIS THS
HYDROPHOBIC, REDUCES THE ENERGY
BARRIER FOR THE HYDROPHOBIC
PART.
THE PROTEIN IS STRUCTURED THAT
IT HAS THIS VESTIBULE THAT GOES
PART OF THE WAY ACROSS THE
MEMBRANE SO YOU CAN IMAGINE THE
LIPID BILAYER WILL BE A BIT
THINNER NEAR THE VESTIBULE.
SO IT REDUCES THE BARRIER TO
TRANSLOCATION WHICH FACILITATES
THIS INSERTION REACTION.
THERE'S REASONABLE SUPPORT BUT
RIGOROUS PROOF STILL AWAITS MORE
STUDIES.
EMC MEDIATES THIS REACTION.
WE COULD SHOW THIS IN DIFFERENT
CONTEXTS BUT THE ONE IMPORTANT
FOR GPCR DOES THE JOB FOR THE
FIRST SEGMENT.
PATRICK CHITWOOD SHOWS EMC IS
REQUIRED TO INSERT INTO THE
LIPID BILAYER AS LONG AS THE
PART THAT GOES ACROSS THE
MEMBRANE IS SHORT, THE CASE FOR
90% OF GPCRs.
THIS DEMONSTRATES THIS WHERE WE
MADE CELL LINES THAT ARE LACKING
EMC BY DELETING A
SUBOPPORTUNITY, AND THIS IS A
FLOW CYTOMETRY ASSAY THAT
MEASURES HOW THE OF THE BETA 1
ADRENERGIC RECEPTOR GETS
PRODUCED, IN THE PRODUCTION IS
REDUCED BY 50%, IN EMC
KNOCKOUTS, THAT CAN BE RESCUED
BY REINTRODUCING COMPLEX BACK
INTO THE CELLS.
WE CAN RECONSTITUTE WITH
PURIFIED COMPONENTS, EMC WAS
SUFFICIENT AND LATER STUDIES
COULD MUTATE CERTAIN RESIDUES TO
SEE HOW THIS WORKS.
EMC MEDIATES THE FIRST SEGMENT
OF THE GPCR, FOR EXAMPLE, AND
THE RIBOSOME DOCKS ON THIS SEC
61 WHERE ADDITIONAL STUFF
HAPPENS.
IT WAS IMPORTANT TO ASSIGN A
FUNCTION TO EMC, IMPORTANT FOR
GETTING INSIGHT 2000 GPCRs,
MORE IMPORTANT FOR ILLUSTRATING
SEC61 IS NOT THE ONLY COMPONENT
THAT DOES EVERYTHING.
I THINK THAT IF YOU WOULD HAVE
ASKED ANYONE, THEY WOULD HAVE
SAID THIS TRANSMEMBRANE DOMAIN
IS INSERTED BY SEC61 BUT
INHIBITORS BLOCK THAT LATERAL
DATE AND SEGMENTS GET INSERTED
FINE IN THE PRESENCE OF
INHIBITORS.
THAT PSYCHOLOGICALLY CHANGED
QUITE A BIT, RIGHT?
IT ALLOWED FOR THE POSSIBILITY
THAT THERE WERE FACTORS DESPITE
THIS FIELD HAVING EXISTED FOR
DECADES, THAT THERE WERE FACTORS
WE DIDN'T KNOW ABOUT.
THAT MADE IT MUCH EASIER FOR US
TO BE MOTIVATED TO FIND
ADDITIONAL COMPONENTS.
AND SO THE NEXT QUESTION WAS
AFTER YOU GET THAT FIRST
TRANSMEMBRANE SEGMENT INSERTED
WHAT THEN HAPPENS?
AND WHAT WE SPECULATED IS THAT
REMEMBER THAT MANY OF THESE
HYDROPHOBIC SEGMENTS ARE
PEPPERED WITH HYDROPHILIC
RESIDUES, THERE MUST BE A
CHAPERONE THAT TEMPORARY
STABILIZED INTERMEDIATES WHILE
YOU SYNTHESIZE THE REST OF THE
PROTEIN UNTIL IT HOLDS TOGETHER,
ANALOGOUS IN THE CYTOSOL, WHERE
THEY BIND UNTIL YOU FOLD THE
WHOLE THING, MAKE THE WHOLE
PROTEIN AND THEN EVENTUALLY FOLD
CHAPERONES IN THE MEMBRANE WOULD
MAKE SENSE BUT WE DIDN'T KNOW
WHAT THEY WERE.
SO, PATRICK MOTIVATED BY THE
POSSIBILITY OF ADDITIONAL
FACTORS USED A CROSS-LINKING
STRATEGY TO IDENTIFY SUCH
CHAPERONES.
THE EXPERIMENT IS A
STRAIGHTFORWARD ONE.
HE USED THIS IN VITRO
TRANSLATION SYSTEM TO SYNTHESIZE
A PROTEIN UP TO THE POINT WHERE
THE FIRST TRANSMEMBRANE SEGMENT
HAD BEEN SYNTHESIZED AND
INHERITED BUT NO FURTHER.
AND THERE'S SOME TRICKS ONE CAN
USE IN IN VITRO TRANSLATION TO
GENERATE SUCH A STALLED
INTERMEDIATE USING TRUNCATED
mRNA WITHOUT A STOP CODE.
WHAT IS ADJACENT TO THE SEGMENT,
USING CYSTEINE REACTIVE
CROSS-LINKING, THE YELLOW
CIRCLE, BUT LATER COULD SHOW
THIS USING SITE-SPECIFIC PHOTO
CROSS-LINKING USING AMINO ACIDS.
THE EXPERIMENT IS BEAUTIFUL IN
SIMPLICITY.
ONE ASSEMBLED WITH OR WITHOUT
THE CYSTEINE IN THIS
TRANSMEMBRANE SEGMENT, ADD A
CYSTEINE REACTIVE CROSS-LINKER,
ONE CAN SEE THERE'S A VERY
PROMINENT UNKNOWN CROSS-LINKED
THAT CROSS-LINKS TO GLYCOSYLATED
INSERTED TRANSMEMBRANE SEGMENT.
YOU SEE SOME CROSS-LINKS TO
SEC61 WHICH WE KNOW IS NEARBY.
IT TOOK A WHILE TO FIGURE OUT
WHAT THIS CROSS-LINK IS, SKILLED
UP, MASS SPEC, DIDN'T INITIALLY
IDENTIFY THE CROSS-LINK WE
IDENTIFIED A PARTNER THAT IT WAS
ASSOCIATED WITH AND THEN USED
THE PARTNER TO FIGURE OUT WHAT
THE COMPLEX WAS.
AND IT TURNS OUT THAT IT
CROSS-LINKS TO A TWO-PROTEIN
COMPLEX VIA SMALL SUBUNIT,
ASTERIX, AND LARGE IS CCD47, THE
FUNCTION OF NEITHER WAS KNOWN AT
THE TIME.
WHAT WE COULD SHOW BY THIS
CROSS-LINKING IS IT'S ADJACENT
TO A TRANSMEMBRANE SEGMENT
THAT'S JUST BEEN INSERTED INTO
THE LIPID BILAYER, AND OBVIOUSLY
ITS FUNCTION WASN'T CLEAR.
WHAT WE COULD FURTHER SHOW IS
THAT IF WE MOVE THAT
CROSS-LINKER, FOR EXAMPLE,
PHOTOCROSS-LINKER TO VARIOUS
POSITIONS ALONG THE POLYPEPTIDE
CROSS-LINKS ARE ONLY SEEN IN THE
TRAN MEMBRANE SEGMENT,
RECOGNIZING IN THE LIPID
BILAYER, OBVIOUSLY THE KIND OF
PROTEIN WE WERE LOOKING FOR, A
CHAPERONE IN THE MEMBRANE.
THE SECOND THING IS THAT FIRST
TRANSMEMBRANE SEGMENT TURNS OUT
HAS A COUPLE HYDROPHILIC
RESIDUES, IF YOU CHANGE THOSE TO
HYDROPHOBIC, THEN YOU NO LONGER
SEE THIS INTERACTION.
AND IF YOU PUT HYDROPHILIC
RESIDUES IN OTHER PARTS OF THE
TRANSMEMBRANE SEGMENT YOU CAN
GET BACK THE INTERACTION.
WHAT WE LEARNED FROM THAT IS
THAT THIS FACTOR IS RECOGNIZING
TRANSMEMBRANE SEGMENTS IN THE
LIPID BILAYER VIA PARTIAL
HYDROPHILICICT, HELPING WITH
UNDERSTANDING HOW THESE
PARTIALLY HYDROPHOBIC SEGMENTS
MAY BE STABILIZED UNTIL YOU MAKE
THE PROTEIN.
THE COMPLEX IS CONSERVED IN
EUKARYOTES.
IF YOU KNOCK IT OUT, THE CELLS
ARE STILL ALIVE, AND AS ARE
YEAST, BUT SEEMS TO CAUSE ER
STRESS, WHAT YOU WOULD EXPECT
FOR PROTEIN FOLDING PROBLEMS, IF
YOU CAN'T ASSEMBLE MEMBRANE
PROTEINS CORRECTLY.
KNOCKOUT OF CCCD47 MOUSE IN
EMBRYONIC LETHAL, AND THERE ARE
POINT MUTANTS THAT HAVE BEEN
DESCRIBED IN HUMANS PRESUMABLY
HYPOMORPHIC ALLELES, APPEARS TO
BE AN IMPORTANT WIDELY
CONSERVES.
THAT'S A GOOD SIGN.
AND THEN IN FUNCTIONAL
EXPERIMENTS, USING AGAIN OUR
FLOW CYTOMETRY ASSAY, IT SEEMS
TO REDUCE THE BIOGENESIS OF
MULTI-PASS MEMBRANE PROTEINS,
DIFFERENT CLASSES SHOWN HERE,
BUT NOT SINGLE PASS MEMBRANE
PROTEINS.
REMEMBER, THIS CLASS IS THE ONE
WE STARTED WITH, IT DEPENDS ON
THE GET PATHWAY, AND SO THE
MULTI-PASS PROTEINS ARE
SELECTIVELY AFFECTED, THAT MAKES
SENSE FOR THE KIND OF FACTORS
WE'RE LOOKING FOR.
SO, WHAT WHAT WE'RE LEFT WITH AT
THIS POINT THERE'S SOME FACTOR
THAT ENGAGES THE EARLY PARTS OF
A MEMBRANE PROTEIN AND WE COULD
SHOW IN EXPERIMENTS IT SEEMS TO
REMAIN ASSOCIATED OR AT LEAST
NEARBY UNTIL YOU MAKE THE REST
OF THE PROTEIN AND THEN ONCE THE
PROTEIN FOLDS IT'S NO LONGER
ASSOCIATED.
SO THAT KIND OF FITS THE IDEA
THAT IT IS SOME TYPE OF
CHAPERONE THAT OPERATES INSIDE
THE MEMBRANE.
IT SEEMS TO DO SO, IT'S NOT AN
ENERGY DEPENDENT FACTORS, SEEMS
TO DO SO BY BINDING EXPOSED
HYDROPHILICITY IN THE MEMORY.
THAT'S THE WORKING MODEL.
HOW DOES IT WORK?
WHAT DOES IT LOOK LIKE AND SO
FORTH?
SO, LUCCA, A Ph.D. STUDENT IN
THE LAB, DECIDED TO INVESTIGATE.
WHAT SHE DECIDED TO DO WAS FIRST
ASK WHEN IS IT STABLY RECRUITED
TO THE RIBOSOME TRANSLOCON
COMPLEX.
TWO LENGTHS ARE SHOWN HERE, IN
THE FIRST ONE THE TRANSMEMBRANE
SEGMENT HAS JUST BEEN INSERTED,
IN THE SECOND IT'S BEEN INSERTED
AND MORE POLYPEPTIDE IS
SYNTHESIZED, NOW COMING OUT OF
THE RIBOSOME.
YOU CAN AFFINITY PURIFY EACH
SEQUENTIAL INTERMEDIATES,
ANALYZE BY MASS SPEC, BLOTTING,
AND SO FORTH.
SHOWN IS AN EXPERIMENT LIKE
THAT, WHERE WE HAVE INCREASING
LENGTHS OF POLYPEPTIDE
DOWNSTREAM OF THE FIRST
TRANSMEMBRANE SEGMENT.
AND I THINK WHAT YOU CAN
APPRECIATE IS THAT ALL OF THEM
ASSOCIATE OF COURSE WITH THE
RIBOSOME, WHAT IS SYNTHESIZING,
AND THE SEC61 PROTEIN TO WHICH
THE RIBOSOME IS DOCKED.
THE PAT COMPLEX IS RECRUITED
AFTER YOU MAKE A CERTAIN AMOUNT
OF POLYPEPTIDE, AND IN THIS
EXPERIMENT ONE HAS SOLUBILIZED
MEMBRANE AND AFFINITY PURIFIED
IT, TELLING YOU WHERE IT
RECRUITS THE PAT COMPLEX THAT'S
A FAIRLY STABLE COMPLEX, THERE'S
NO CROSS-LINKING OR ANYTHING
HERE.
IT'S BEEN STABLY RECRUITED TO
THE TRANSLOCON, ANALYZE BY
SINGLE PARTICLE CryoEM.
ANOTHER MEMBER OF THE LAB, MIN,
DECIDED TO DO THIS.
IT WAS DISAPPOINTING INITIALLY
BECAUSE ALTHOUGH WE COULD GET
GOOD DENSITY FOR THE SEC61
COMPLEX, GREEN HERE, AND SOME
OTHER PARTS, THE PART THAT'S
NEXT TO THE RIBOSOME IS WELL
ORDERED BUT THE PART IN THE
MEMBRANE WAS RATHER BLOBY.
YOU CAN'T REALLY SEE MUCH THERE.
ALPHA FOLD IS PUBLISHED AT THIS
PUBLISHED, TURNS OUT IT'S
PARTICULARLY GOOD AT PREDICTING
STRUCTURES OF MEMBRANE PROTEINS.
AND SO ASTRIX IS 10 KILODALTONS,
AND CCCD47 IS BIGGER, IT PREDICT
AS BEAUTIFUL STRUCTURE, TOGETHER
IT PREDICTS A STRUCTURE OF THE
COMPLEX THAT FITS NICELY INTO
THIS DENSITY.
YOU HAVE A SMALL THREE-HELIX
BUNDLE OF THIS ASTRIX PROTEIN,
ADJACENT TO WHICH IS THE SINGLE
TRANSMEMBRANE DOMAIN OF CCD47,
LARGE GLOBULAR DOMAIN AND OTHER
PARTS THAT BIND THE RIBOSOME.
IT'S IMPORTANT OF COURSE TO KNOW
THAT THIS IS SOME SEMBLANCE OF
REALITY, AS OPPOSED TO COMPLETE
ARTIFACT, BUT OF COURSE FITTING
INTO THIS LOW RESOLUTION DENSITY
WAS A GOOD START.
WHAT LUCCA DID WAS PUT
PHOTOCROSS-LINKERS IN DIFFERENT
PARTS OF ASTRIX, ESSENTIALLY
RECONSTITUTED ASTRIX KNOCKOUT
CELLS WITH VERSIONS IN WHICH ONE
USED AMBER SUPPRESSION TO PUT A
PHOTOCROSS-LINKER IN ANY ONE
LOCATION.
I'M COMPARING A
PHOTOCROSS-LINKER AT POSITION 42
VERSES 62 SHOWN BY THIS RED AND
BLUE STAR.
AND THEN YOU IRRADIATE WITH UV
LIGHT, WHAT ONE CAN SEE IS THIS
IS THE ASTRIX AT THE BOTTOM OF
THE GEL, IN UV DEPENDENT MANNER
YOU SEE ADDITIONAL BAN
IMMUNOPRECIPITATED WITH
ANTIBODIES AGAINST CCD47
REPRESENTING A CROSS-LINK AS
EXPECTED FROM STRUCTURAL MODEL.
THE OTHER DOESN'T CROSS-LINK TO
CCD47, IT'S ON THE OTHER SIDE.
HEE THEE PLACES THE PROBE IN --
SHE PLACES THE PROBE IN A DOZEN
PLACES, IT'S A SMALL PROTEIN,
AND SO WE CAN, ONE, VERIFY WHICH
PART IS ADJACENT TO CCCD47,
VALIDATING THE ALPHAFOLD MODEL.
TWO, WE CAN THEN FIND THAT THIS
PART OF ASTRIX IS THE PART THAT
RECOGNIZES SUBSTRATE.
SO, KNOWING KIND OF THAT THIS IS
A REASONABLE PLAUSIBLE MODEL,
STRUCTURAL MODEL, WHAT DO WE
LEARN FROM THIS?
HERE'S THE LATERAL GATE OF
SEC61.
IT'S CLOSED.
SO, ALTHOUGH THIS COMPLEX IS IN
THE PROCESS OF MAKING A MEMBRANE
PROTEIN AND YOU MIGHT IMAGINE
THAT THE MEMBRANE PROTEIN WOULD
GET INSERTED BILATERAL GATE,
THAT'S NOT HAPPENING.
THE CHAPERONE PAT COMPLEX IS NOT
LOCATED NEAR THE LATERAL GATE,
IT'S BEHIND SEC61, QUITE FAR
AWAY.
YOU COULD EXPECT A CHAPERONE TO
CAPTURE MEMBRANE PROTEINS AS
THEY COME OUT OF THE SEC61
TRANSLOCON, THIS DOESN'T MAKE
SENSE WITH THAT.
NOW IF YOU HAVE A CLOSER LOOK AT
ASTRIX, SOME THINGS DO MAKE
SENSE.
SO THIS IS THE SURFACE OF ASTRIX
THAT BY CROSS-LINKING ENGAGES
SUBSTRATE.
AND ONE THING YOU SEE IS IT'S
HIGHLY CONSERVED, SO PURPLE IS
CONSERVED HERE, BACK SIDE WHICH
ISN'T SHOWN IS NOT WELL
CONSERVED BUT THE PART THAT
BINDS SUBSTRATE IS CONSERVED.
SECOND, IT'S HYDROPHILIC.
THE JOB BIOCHEMICAL IS TO
RECOGNIZE HYDROPHILIC SEGMENTS,
SO HAVING A HYDROPHILIC SURFACE
SEEMS TO MAKE SENSE.
IF YOU MUTATE THIS SURFACE TO
MAKE IT LESS HYDROPHILIC, IT NO
LONGER ENGAGES SUBSTRATE.
THE HYDROPHILICITY IS IMPORTANT.
MANY SURFACES ARE MADE OF
METHININES, MANY PROTEINS
INVOLVED IN TRANSMEMBRANE DOMAIN
RECOGNITION ARE METHININES RICH,
A FEATURE OF THIS PROTEIN AS
WELL, THOUGHT THAT PART OF THE
REASON IS BECAUSE IT IS THE MOST
FLEXIBLE SIDE CHAIN AND SO MAYBE
IT CAN ACCOMMODATE A WIDE RANGE
OF SEQUENCES THROUGH THAT
PROPERTY.
WHAT WE LEARN FROM THIS
STRUCTURE IS THAT ASTRIX HAS A
SUBSTRATE BINDING REGION, THAT
ENGAGES PARTIALLY HYDROPHILIC
SUBSTRATES.
BUT THE PARTS THAT ARE PUZZLING,
IT DOES SO BEHIND SEC61.
WHAT IT MEANS IS THAT THE
POLYPEPTIDE IS REROUTED OVER
HERE TO THE LEFT IN THIS GREEN,
AWAY FROM THE LATERAL GATE.
IN THE CONVENTIONAL MODEL OF HOW
MEMBRANE INSERTION SHOULD WORK
THIS NEXT TRANSMEMBRANE SEGMENT
NEEDS TO ENGAGE THE LATERAL GATE
BUT IS BEING PULLED AWAY.
SO THAT IS REALLY PUZZLING.
AS IF THAT WASN'T BAD ENOUGH, A
STUDENT IN THE LAB HAPPENED TO
BE LOOKING AT OUR STRUCTURE, HE
SAID THERE'S ANOTHER PROBLEM.
THAT'S BECAUSE THE CCD47, THIS
PINK-COLORED POLY PEPTIDE, SITS
CLOSE TO SEC61 AND WHAT AARON
NOTICED IS THAT THAT PART OF
CCD47 IS WEDGED BETWEEN SEC61
AND RIBOSOME.
YOU CAN APPRECIATE IN THIS
SPACE-FILLING MODEL IT'S A TIGHT
FIT.
THE CONSEQUENCE IS SEC61 CANNOT
OPEN.
I'M SHOWING AGAIN WHAT THE MOVIE
OF SEC61 OPENING LOOKS LIKE.
I THINK YOU CAN APPRECIATE THIS
IS THE SAME VIEW HERE THAT THIS
KIND OF MOVEMENT SIMPLY CANNOT
HAPPEN WHEN THIS WEDGE IS
SITTING HERE.
SO, WHAT HAS HAPPENED WHEN YOU
RECRUITED THE PAT COMPLEX YOU
WEDGE SEC61 TO A CLOSED
CONFIRMATION AND PULL THE
SUBSTRATE AWAY FROM THE LATERAL
GATE.
AND SO THAT IS NOT CONDUCIVE TO
INSERTING THE NEXT TRANSMEMBRANE
SEGMENT INTO THE LATERAL GATE OF
SEC61 AS CONVENTIONAL MOD WOMAN
-- MODEL WOULD HAVE YOU
LEAVE.
THE ONLY PLAUSIBLE MODEL IS THAT
AS YOU ELONGATE MORE, YOU MAKE
THE NEXT TRANSMEMBRANE SEGMENT,
YOU HAVE TWO TRANSMEMBRANE
SEGMENTS THAT WOULD INSERT AS A
PAIR INTO THE SPACE BEHIND SEC
61 BETWEEN THE CHAPERONE AND
BACK OF SEC61.
AND SO THE QUESTION IS, IS THAT
A PLAUSIBLE SCENARIO?
AND IF IT IS, THEN IS THERE
MACHINERY THAT CATALYZEs THAT
REACTION?
SO I WILL TELL YOU THAT IT IS
PLAUSIBLE IN THAT THIS LOOP
BETWEEN THE TWO TRANSMEMBRANE
SEGMENTS, IF YOU PUT A FOLDED
DOMAIN THERE, THIS INSERTION
REACTION IN FACT DOESN'T WORK
VERY EFFICIENTLY, SUGGESTING IN
FACT THAT MIGHT BE WHAT'S
HAPPENING.
BUT TO FIGURE OUT WHAT WAS GOING
ON, WE THEN ISOLATED THESE
INTERMEDIATES, AT THE POINT
WHERE THE NEXT TWO TRANSMEMBRANE
SEGMENTS WOULD BE INSERTING.
AND THEN AFFINITY PURIFIED, DID
MASS SPEC, VARIOUS BLOTTING
EXPERIMENTS AND CryoEM TO SEE
WHAT WAS GOING ON.
AND THIS TURNS OUT TO BE
REVEALING.
THIS IS A COMPLICATED SLIDE WITH
A FAIRLY SIMPLE CONCLUSION.
THIS IS A MASS SPEC EXPERIMENT
WHERE WE'RE COMPARING AN EARLY
INTERMEDIATE, WHERE THE FIRST
TRANSMEMBRANE SEGMENT HAS BEEN
INSERTED, BUT THE PAT COMPLEX
HAS NOT BEEN RECRUITED.
COMPARED TO A LATE INTERMEDIATE,
WHERE THE PAT COMPLEX IS
RECRUITED, NEXT TWO SEGMENTS ARE
RIGHT ABOUT THE POINT WHERE THEY
ARE INSERTING IN THE BILAYER.
AND BY COMPARING THESE TWO
COMPLEXES USING QUANTITATIVE
MASS SPEC, THERE ARE ONLY A
HANDFUL OF PROTEINS THAT ARE
ENRICHED IN THE LATE COMPLEX.
ONE OF THEM -- ALL OF THEM TURN
OUT TO BE PARTS OF LARGER
COMPLEXES THAT OUR LAB AND BOB
KEENAN'S LAB CHARACTERIZED.
THIS IS A BLOT VERIFYING IT'S
RECRUITED TO THE LATE COMPLEX,
ASTRIX IS NOT IDENTIFIED BY MASS
SPEC BECAUSE IT HAS VERY FEW
CRYPTIC PEPTIDES.
THIS PROTEIN NICALIN AND NOMO
ARE PART OF 47, PART OF AN
OBLIGATE COMPLEX, WE CALLED THIS
THE BACK OF SEC61 COMPLEX, OR
BOS
COMPLEX, NO IDEA WHAT IT DOES
BUT WE KNOW WHERE IT IS.
THIRD IS TWO-PROTEIN COMPLEX
TMCL 1 AND INTERACTION PARTNER,
AND THIS IS THE COMPLEX THAT I
MENTIONED BRIEFLY AT THE
BEGINNING, HOMOLOGOUS TO THE GET
COMPLEX, AND EMC, ALSO MEDIATING
TRANSMEMBRANE DOMAIN INSERTION,
CALL IT THE GEL COMPLEX, FOR GET
AND EMC-LIKE.
THIS FACTORS SEEMS LIKE IT WOULD
BE MEDIATING TRANSMEMBRANE
DOMAIN INSERTION.
WE HAVE THREE COMPLEXES HERE,
REALLY NICE WORK FROM THE KEENAN
LAB HAS SHOWN THESE ARE THREE
INDEPENDENT COMPLEXES ASSEMBLED
TOGETHER ON THE RIBOSOME BUT
OTHERWISE DON'T INTERACT WITH
EACH OTHER.
SO, ONE COULD OF COURSE THEN
ANALYZE THIS BY EM, RESOLUTION
AGAIN IS MEAGER.
HOWEVER, THE ABILITY TO DOCK IN
REASONABLY GOOD PREDICTIONS IS
VERY GOOD.
AND SO WHAT YOU'RE LOOKING AT
HERE IS THE VIEW OF THIS
TRANSLOCON FROM THE LUMEN OF THE
E.R.
YOU'RE LOOKING TOWARDS THE
RIBOSOME, AND THE PROTEIN THAT'S
BEING SYNTHESIZED IS COMING OUT
OF THE RIBOSOME TOWARDS YOU.
AND WHAT WE WIND UP SEEING IS
THE THIRD TRANSMEMBRANE DOMAIN
OF THE INSERTING POLYPEPTIDE BUT
CAN'T SEE THE OTHER TWO.
HOWEVER, WE CAN DEDUCE WHICH
PARTS OF THE SUBSTRATE IS
ADJACENT TO THESE OTHER
COMPLEXES USING
PHOTOCROSS-LINKING.
THIS IS SEEN ON THE RIGHT.
AND THEN YOU IRRADIATE WITH UV
LIGHT.
WHAT YOU CAN SEE IS THAT HERE'S
A CROSS-LINK TO THE FIRST
TRANSMEMBRANE SEGMENT, PRIMARY
CROSS-LINK.
AND THAT TURNS OUT TO BE ASTRIX.
I'M NOT SHOWING THE IPS HERE BUT
THAT PLACES THE FIRST
TRANSMEMBRANE SEGMENT HERE.
THE SECOND ONE DOES NOT
CROSS-LINK TO ASTRIX BUT TO
SOMETHING SLIGHTLY BIGGER, WHICH
TURNS OUT TO BE A COMPONENT OF
THE GEL COMPLEX, TMCL1 SUBUNIT.
THE THIRD SEGMENT CROSS-LINKS TO
THE SEC61 COMPLEX AND DEPENDING
ON WHERE YOU PUT THE
PHOTOCROSS-LINKER, IT
CROSS-LINKS TO ALPHA OR BETA
SUBUNIT, BETA IS RIGHT HERE,
THIS NICELY ILLUSTRATES HOW
SPECIFIC PHOTOCROSS-LINKER,
INCREDIBLY SPECIFIC.
WE FEEL REASONABLE COULD HAVE
DENT DRAWING THE SUBSTRATE HERE.
WE THINK WE'RE LOOKING AT
INTERMEDIATE IN WHICH THE SECOND
AND THIRD TRANSMEMBRANE SEGMENTS
HAVE JUST BEEN INSERTED, AND
OBVIOUSLY THEY ARE NOT AT THE
LATERAL GATE OF SEC 61.
THEY ARE BEHIND SEC 61, ADJACENT
TO POORLY CHARACTERIZED
COMPLEXES THAT FORM THIS
HORSESHOE SHAPE BEHIND SEC61,
WHY WE CALL THIS THE MULTI-PASS
TRANSLOCON, IT SEEMS TO BE
INVOLVED IN INSERTING MULTI-PASS
PROTEINS.
THE IDEA THEN IS THAT AFTER YOU
INSERT THE FIRST TRANSMEMBRANE
SEGMENT, THE PARTIAL HYDROPHILIC
CHARACTER SEEMS TO PLAY A ROLE
IN RECRUITING PAT COMPLEX, WHICH
SEEMS TO BE CO-RECRUITED WITH
THE OTHER TWO COMPLEXES, GEL AND
BOS.
WHAT THE PAT COMPLEX DOES, SEEMS
TO LOCK SEC61 CLOSED BY WEDGING
WITH THE RIBOSOME PULLING THE
NASCENT CHAIN.
AS YOU MAKE MORE SEGMENTS OF
POLY PEPTIDE WE GET INSERTED
BEHIND SEC61.
WE THINK THIS REACTION IS
MEDIATED BY GEL COMPLEX.
AND ONCE YOU GET THAT INSERTION
THEN YOU CAN IMAGINE THAT AS YOU
SYNTHESIZE MORE POLYPEPTIDE THEY
CAN THEN CONTINUE TO BE INSERTED
BEHIND SEC61, UNTIL YOU MAKE THE
WHOLE PROTEIN, IT FOLDS, AND
ONCE ALL THE HYDROPHILIC PARTS
OF THE POLYPEPTIDE ARE BURIED
THERE'S NOTHING FOR COMPLEXES TO
BIND TO ANYMORE AND WE IMAGINE
THE COMPLEX DISASSEMBLES AND
YOU'VE MADE YOUR PROTEIN.
SO, THIS OBVIOUSLY IS A
COMPLETELY DIFFERENT WAY OF
THINKING ABOUT HOW MEMBRANE
INSERTION WORKS THAN THE KIND OF
SEQUENTIAL INSERTION VIA THE
LATERAL GATE OF SEC61.
THE FIRST TRANSMEMBRANE SEGMENT
IS INSERTED BY THIS PROTEIN
COMPLEX, EMC, WHICH ISN'T SHOWN
IN THE DIAGRAM.
AND LATER SEGMENTS OF THIS GPCR
ARE INSERTED BY OTHER COMPONENTS
BEHIND SEC61.
SO NONE OF THEM ARE INSERTED BY
THE LATERAL GATE OF SEC61.
SO THAT MAKES A PREDICTION WHICH
IS THAT IF YOU BLOCK THE LATERAL
GATE OF SEC61, INSERTION OF THIS
GPCR SHOULD STILL WORK.
AND ONE THING THAT'S HAPPENED IN
THE PAST NUMBER OF YEARS, IS
THAT THERE HAVE BEEN A NUMBER OF
GOOD INHIBITORS OF SEC61 LATERAL
GATE, AND NICE WORK BY PARK AND
JACK TAUNTON HAVE GOTTEN
STRUCTURES OF SUCH INHIBITORS
AND BIND RIGHT AT THE LATERAL
GATE AND BLOCK IT.
AND SO WE CAN THEN TEST THIS
IDEA.
SO, HERE'S A POSITIVE CONTROL
WHICH IS A SINGLE PASS PROTEIN
THAT IS KNOWN TO INSERT VIA
LATERAL GATE OF SEC61, AND THIS
IS A FLOW CYTOMETRY ASSAY IN
WHICH THE GRAY IS CONTROL CELLS,
NO INHIBITORS, NOTHING KNOCKED
DOWN.
OBVIOUSLY IF YOU KNOCK DOWN
SEC61 INSERT DOESN'T WORK.
BUT IF YOU ADD THE INHIBITOR OF
THE LATERAL GATE INSERTION
DOESN'T WORK AS WELL.
THAT MAKES COMPLETE SENSE.
BUT HERE'S RHODOPSIN.
OBVIOUSLY IF YOU KNOCK DOWN
SEC61, INSERTION DOESN'T WORK,
BUT THAT'S THE BINDING SITE FOR
THE RIBOSOME.
RIBOSOME HAS NOTHING TO BIND TO
AT THE MEMBRANE.
THE INHIBITOR IS COMPLETELY
INEFFECTIVE AGAINST INHIBITING
RHODOPSIN INSERTION.
I HAVE TO SAY, SURPRISINGLY
CLEAN HOW GOOD THIS RESULT IS.
WE DID THIS WITH THREE
COMPLETELY UNRELATED CHEMICALLY
UNRELATED INHIBITORS, AND WE'VE
ALSO DONE THIS WITH MULTIPLE
DIFFERENT GPCRs.
AND SO THIS CLASS OF PROTEINS
DOESN'T SEEM TO USE THE LATERAL
GATE OF SEC61.
YOU MIGHT ASK WHAT EXACTLY IS
SEC61 NEEDED FOR?
OBVIOUSLY IT'S NEEDED HERE IN
THIS CASE FOR BINDING TO THE
RIBOSOME AND SERVING AS
TEMPLATES.
IF YOU'RE IN THE PROCESS OF
MAKING A MEMBRANE PROTEIN,
SUPPOSE THAT THERE'S A LOOP IN
THE MIDDLE THAT'S REALLY LONG.
THE COMPONENTS I TOLD YOU ABOUT,
THEY ARE NOT WELL SUITED FOR
TRANSLOCATING LONG SEGMENTS OF
POLYPEPTIDE BECAUSE THEY DON'T
HAVE A CHANNEL IN THEM LIKE THE
CHANNEL THAT'S IN SEC61.
AND IT TURNS OUT THAT IF YOU
HAVE SUCH A PROTEIN AND THIS IS
THE C3A COMPLEMENT RECEPTOR, THE
LOOP IS 150 AMINO ACIDS LONG,
TRANSLOCATION OF THE LOOP
REQUIRES SEC61.
HERE IS THE EXPERIMENT TO SHOW
IT.
THIS PROTEIN HAS TWO
GLYCOSYLATION SITES.
AND THE SECOND ONE IS IN THIS
LOOP.
SO HERE'S AN EXPERIMENT WITHOUT
SEC61 INHIBITOR.
THE PROTEIN GETS GLYCOSYLATED
ONCE AND TWICE, RIGHT HERE.
BUT IF YOU HAVE SEC61 INHIBITOR,
THE FIRST GLYCOSYLATION STILL
OCCURS, AND THAT'S OCCURRING AT
THE N N-TERMINUS, IT'S MEDIATEY
EMC, BUT THE SECOND IS
PREFERENTIALLY GONE.
AND THAT'S BECAUSE YOU CANNOT
TRANSLOCATE THAT LOOP.
WE CAN THEN SHOW THIS USING
PROTEASE PROTECTION, IF YOU
DISSOLVE WITH PROTEASE, FIRST
THREE ARE PROTECTED FROM
PROTEASE, WHAT THE FRAGMENT HERE
REPRESENTS.
AND AMAZINGLY ENOUGH, IF YOU
SHORTEN THAT LOOP TO BE 20 OR SO
AMINO ACIDS, NOW WHETHER YOU
HAVE SEC61 INHIBITOR OR NOT,
INSERTION WORKS PERFECTLY FINE.
SO, WHAT ONE THEN IS LEFT WITH
IS THIS IDEA THAT THE OXA
FAMILY, WHICH I REMIND YOU IS
COULD BE SERVED ACROSS ALL
ORGANISMS, MEDIATES INSERTION OF
TRANSMEMBRANE SEGMENTS WHEN THE
ADJACENT FLANKING DOMAIN IS
RELATIVELY SHORT.
THAT'S BECAUSE IT SEEMS TO WORK
BY USING A VESTIBULE THAT IS NOT
A CHANNEL ACROSS THE MEMBRANE.
SO IT'S JUST REDUCING THE
BARRIER, E. COLI SEEMS TO BE
ABOUT 50 AMINO ACIDS SO
TRANSMEMBRANE SEGMENTING WITH
FLANKING DOMAIN OF 50 AMINO
ACIDS USE THE OXA 1 FAMILIAR.
IN THE MAMMALIAN E.R. THERE ARE
FAMILY MEMBERS, BACTERIA HAVE
EMC, INTERMITOCHONDRIAAL
MEMBRANE HAS OXA 1, SO ALL THESE
MEMBRANES THAT NEED TO MAKE
MEMBRANE PROTEINS HAVE A MEMBER
OF THIS FAMILY.
AND SO WE BELIEVE THAT THIS
FAMILY CAN ALSO INSERT A PAIR OF
TRANSMEMBRANE SEGMENTS AS LONG
AS THE LOOP IS SHORT.
IF YOU NEED TO GET A LONG LOOP
ACROSS THE MEMBRANE YOU NEED A
CHANNEL.
THIS IS OBVIOUS BECAUSE, YOU
KNOW, THE ENERGETIC BARRIER TO
MOVING A LONG SEGMENT OF
POLYPEPTIDE IS TOO HIGH, YOU
NEED AN AQUEOUS CHANNEL TO MOVE
IT ACROSS THE MEMBRANE, WE THINK
THE TRANSMEMBRANE SEGMENT
ADJACENT TO THE LONG DOMAIN
ENGAGES AND OPENS LATERAL GATE,
PULLS THE PROTEIN THROUGH AND
YOU GET THE LONG LOOP ACROSS.
IF YOU LOOK AT THE DIVERSITY OF
MEMBRANE PROTEINS AS I SHOWED
YOU, WHAT I THINK IS GOING
HAPPEN AND WE'VE TESTED PARTS
BUT NOT ALL, IS THAT PROTEINS
THAT HAVE A SHORT SEGMENT THAT
NEEDS TO BE TRANSLOCATED ACROSS
ARE INSERTED BY AN OXA FAMILY
MEMBER, FOR EXAMPLE THE TAIL
ANCHORED FAMILY IS INSERTED BY
GET OR EMC, RIGOROUSLY
DEMONSTRATED.
GPCR, ALL THE PARTS ACROSS THE
MEMBRANE ARE SHORT, MEDIATED BY
OXA FAMILY MEMBERS SUCH AS EMC
FOR THE FIRST SEGMENT, GEL FOR
THE OTHERS.
AND SOME PROTEINS SUCH AS THIS
ONE WHICH REPRESENTS SUBUNIT OF
THE PENTAMERIC ION CHANNEL,
FIRST PART IS MEDIATED BY SEC,
IN FACT THERE'S A CLEAVABLE
SIGNAL PEPTIDE ENCODED THERE.
OTHER PARTS ARE MEDIATED BY OXA,
THAT ARE SHORT.
SO NOW WHAT STARTED OFF AS THIS
SORT OF BEWILDERING DIVERSITY OF
MEMBRANE PROTEINS I THINK CAN BE
SIMPLIFIED TO A SIMPLE CONCEPT
WHICH IS THAT EVOLUTIONARY THERE
ARE TWO CLASSIFICATIONS, OXA FOR
INHERITING HYDROPHOBIC, AND SEC
FOR SECRETION, MOVING
POLYPEPTIDE ACROSS THE LIPID
BILAYER, FOR SOME MEMBRANE
PROTEINS BUT NOT OTHERS.
AND SO I WILL LEAVE IT THERE AND
JUST REMIND YOU THAT WE THINK
THAT IN ALL ORGANISMS THERE
WOULD BE THESE FACTORS THAT
MEDIATE INSERTION OF DIFFERENT
PARTS OF THE MEMBRANE PROTEIN
AND BECAUSE AT LEAST IN THE
MAMMALIAN SYSTEM THAT WE'VE
STUDIED THESE FACTORS CAN BE
ASSEMBLED IN CONTEXT-DEPENDENT
OR SUBSTRATE-DRIVEN MANNER, AS
THE POLYPEPTIDE COMES OUT OF THE
RIBOSOME THE CAPACITY TO USE
EITHER MEMBERS OF THE FAMILY FR
INSERTION BUTTRESSED WITH THE
PAT COMPLEX THAT MAY FACILITATE
CHAPERONING OF THE PROTEIN IN A
PROTECTED SPACE BEHIND SEC61
WHERE WE THINK THE PROTEIN CAN
FOLD WITHOUT BEING INTERFERED
WITH BY, YOU KNOW, AGGREGATION
OR INTERACTION WITH QUALITY
CONTROL FACTORS.
AND SO THE LAST THING I'LL SAY
IS THAT THAT REVIEW I WROTE 25
YEARS AGO, LAST FIGURE
SPECULATED HOW THIS MIGHT WORK.
ALTHOUGH IT WAS PURE
SPECULATION, SOMETHING IN
REVIEWS BACK THEN, WE IN FACT
SPECULATED THAT THERE'S NO WAY
IT'S POSSIBLE FOR A SMALL
CHANNEL TO INSERT SO MANY
MEMBRANE PROTEINS.
SANDY SIMON HAD INDIRECT
EVIDENCE THERE WAS THE CAPACITY
TO HAVE MULTIPLE TRANSMEMBRANE
SEGMENTS THAT HAD NOT QUITE BEEN
INSERTED BUT SOMEHOW HELD IN THE
MEMBRANE, WE DREW THIS VERY
LARGE MACHINE, SITTING ON THE
RIBOSOME.
SATISFYING BUT I TAKE NO CREDIT,
THE SIZE MATCHES TO THE LOCON
STRUCTURE.
I WILL LEAVE IT THERE AND THANK
YOU FOR YOUR ATTENTION.
>> THAT WAS FANTASTIC TALK.
TWO QUESTIONS.
SO, FIRST ONE, GIVEN THE FACT
THAT THIS SYSTEM IS DEPENDENT ON
THE PARTIAL HYDROPHILICITY,
THERE'S A HETEROGENEITY OF THAT
IN THE MEMBRANE PROTEIN WALL, SO
YOU IMAGINE THAT THERE'S A
HETEROGENEITY OF OTHER CHAPERONE
PROTEINS WHEN DIFFERENT MEMBRANE
PROTEINS ARE INSERTED, THAT'S
THE FIRST QUESTION.
SECOND ONE, CROSS-LINKING SYSTEM
IS VERY BEAUTIFUL BUT DID YOU
EVER TRY TO GET SOME OF THE
INITIAL DATA JUST USING
CROSS-LINKING MASS SPECTROMETRY
AND DID YOU FIND ANYTHING?
>>YEAH, GREAT QUESTION.
SO, THE FIRST QUESTION, ALTHOUGH
THERE'S A LARGE DIVERSITY OF
HYDROPHILICICT IN TRANSMEMBRANE
DOMAINS ALMOST ALL MULTI-PASS
PROTEINS HAVE EITHER THE FIRST
OR SECOND DOMAIN BEING
RELATIVELY HYDROPHILIC.
THAT'S INTERESTING BECAUSE IT
DOES SUGGEST THE PAT COMPLEX AND
OTHER COMPONENTS GET RECRUITED
QUITE EARLY.
IT'S NOT THAT SURPRISING IN
HINDSIGHT BECAUSE PACKING OF
MULTIPLE TRANSMEMBRANE SEGMENTS
INTO A BUNDLE WHICH IS WHAT MOST
MEMBRANES DO IS MEDIATED
PRIMARILY BY EITHER SHORT SIDE
CHAINS LIKE LYSINES OR
HYDROPHILIC RESIDUES, ENRICHMENT
MAKES SENSE.
MASS SPEC, IT'S A GOOD QUESTION.
I DIDN'T TALK ABOUT SOME OF THE
REALLY NICE WORK THAT BOB KEENAN
DID, BUT BOB WAS FOCUSED ON THE
GEL COMPLEX.
AND BEFORE WE KNEW IT WAS A
COMPLEX.
HE PULLED ON TMCL1, A SUBUNIT,
DID MASS SPEC AND CROSSLINKING
MASS SPEC.
IN HINDSIGHT WITH STRUCTURES,
ONCE CAN MAKE A FAIR AMOUNT OF
SENSE OF THIS.
BUT I THINK THAT IT WAS A BIT
DIFFICULT WITHOUT THAT AS KIND
OF A DISCOVERY VEHICLE.
BUT YES.
>> THANK YOU.
>> YEAH, BEAUTIFUL TALK.
ODDLY SPECIFIC QUESTION.
I WAS INTERESTED IN THIS ASTRIX
PROTEIN IN THE PAT COMPLEX, IT
HAS A UNIQUE HYDROPHILIC FACE.
HAVE YOU STUDIED HOW IT'S
INSERTED IN THE MEMBRANE AND
WHAT'S THE STABILITY LIKE IF YOU
HAVE THE HYDROPHILIC FACE
IMPORTANT FOR ANCHORING THESE
UNIQUE SEGMENTS?
>> YEAH, I HAVE TO SAY WHAT I
SHOWED, THIS BEAUTIFUL
HYDROPHILIC FACE, WE HAVE TO
REMEMBER THAT'S A MODEL.
AND SO ONE POSSIBILITY IS THAT
THERE'S A DIFFERENT CONFIRMATION
THAT OBSCURES THAT HYDROPHILIC
FACE WHEN IT'S NOT BEING USED.
THAT'S MY FAVORITE IDEA OF
WHAT'S HAPPENING.
AND SO -- AND THEN -- BUT WE
DON'T HAVE ANY INFORMATION ABOUT
THAT.
THE OTHER ASPECT OF THE
QUESTION, IT TURNS OUT MOST --
MANY PROTEINS INVOLVED IN
BIOGENESIS OFTEN NEED THEMSELVES
TO BE MADE.
SO, FOR EXAMPLE, SEC61 IS GOING
TO USE IN PART SEC61 AND THESE
OTHER COMPONENTS TO BE PRODUCED,
AND PRESUMABLY ASTRIX WILL ALSO
NEED SOME OF THESE COMPONENTS TO
BE PRODUCED.
CYTOSOL, FOR EXAMPLE, HSB7 0
PROBABLY NEEDS THAT.
>> CHAPERONE HELPS CHAPERONE.
DO YOU THINK THAT'S RELATED TO
TRANSITION FROM EARLY TO LATE?
>> YEAH, THE TRANSITION FROM
EARLY TO LATE IS SOMETHING WE
DON'T FULLY UNDERSTAND.
PART OF THE EQUATION CLEARLY IS
HYDROPHILICITY OF THE EARLY
TRANSMEMBRANE SEGMENT.
IF WE MAKE THAT HYDROPHILIC, ALL
LEUCINES, THE PAT COMPLEXES AND
OTHER COMPLEXES DON'T GET
RECRUITED.
THEY CAN CLEARLY BIND THE
RIBOSOME, THAT AFFINITY IS NOT
ENOUGH.
>> A QUICK QUESTION FROM ONLINE
COMMUNITY.
HOW DOES THE ENGAGEMENT BETWEEN
SEC61 AND HYDROPHILIC SEGMENT
OUT OF THE RIBOSOME HAPPEN?
IS THERE A SIGNAL OR CHEMICAL
REACTION THAT OPENS UP THE
CHANNEL?
>> YEAH, SO THIS IS A GREAT
QUESTION.
AND WE HAVE A SNAPSHOT OF WHAT
IT LOOKS LIKE AFTER IT'S
ENGAGED, WE DO NOT KNOW HOW
SEC61 GETS OPENED.
AND SO ONE POSSIBILITY IS THAT
MAYBE IT INITIALLY KIND OF
ENGAGES A LITTLE BIT OF THE
LATERAL GATE THAT'S PARTIALLY
CRACKED, AND SORT OF AS MORE
COMES OUT IT WEAVES INTO THE
LATERAL GATE AND FORMS A HELIX.
CO-TRANSLATIONAL PROCESSES HAVE
BEEN DIFFICULT TO STUDY.
>> IF YOU HAVE A C-TERMINAL
TRANSMEMBRANE SEGMENT WHICH IS
ATTACHED TO A LARGE HYDROPHILIC
SEGMENT THAT HAS TO BE
TRANSLOCATED INTO THE LUMEN OF
THE E.R. AND THEREFORE THAT
PARTICULAR TRANSMEMBRANE SEGMENT
WOULD GO THROUGH THE SEC61
CHANNEL, WOULD THERE BE A
PROBLEM JOINING THE N-TERMINAL
PORTION OF THE PROTEIN WHICH IS
ASSOCIATED WITH PAT, GEL, AND
BOS?
>> YEAH, I THINK THAT WHAT
NEEDS TO HAPPEN IS THE
FOLLOWING.
WE DON'T HAVE EVIDENCE FOR THIS
YET, MY FAVORITE HYPOTHESIS.
REMEMBER THERE ARE THESE TWO
HELLICICES THAT WEDGE BETWEEN.
THE TWO HELICES HAVE TO MOVE OUT
OF THE WAY, OTHERWISE THERE'S NO
WAY TO OPEN SEC61.
THE QUESTION IS WHAT IS THE
TRIGGER FOR THAT TO HAPPEN?
AND WE THINK -- IF YOU -- I
DIDN'T GO INTO DETAILS OF THE
STRUCTURE BUT THE END OF THOSE
TWO HELICES PROTRUDES INTO THE
TUNNEL OF THE RIBOSOME, SO WE
THINK THAT PAT COMPLEX
SPECIFICALLY THE CCD47 SUBUNIT
CAN SOMEHOW SENSE WHAT'S COMING
OUT OF THE RIBOSOME SO ONCE YOU
START SYNTHESIZING A SEGMENT OF
HYDROPHILIC POPPY PEPTIDE THAT
NEEDS TO BE TRANSLOCATED MY
HYPOTHESIS IT STARTS
ACCUMULATING IN THE LIMITED
STAYS BETWEEN THE RIBOSOME AND
MEMBRANE COMPONENTS, AND THAT
DISPLACES THESE TWO HELICES OF
CCD47, ALLOWING SEC61 TO HOPE.
I THINK IT'S AN ATTRACTIVE
HYPOTHESIS.
WE DO KNOW IT THAT HAPPENS
BECAUSE WE CAN DESIGN SUBSTRATES
AND SHOW IT DOES SHIFT OVER FROM
ENGAGING THE PAT COMPLEX TO THE
SEC COMPLEX.
NOW, WE DON'T THINK THE PAT
COMPLEX DIFFUSES AWAY OR
DISASSEMBLES BECAUSE IT'S
ASSOCIATED WITH THE SUBSTRATE.
THAT'S WHY I FOCUS ON MOVEMENT
OF THESE HELICES OUT OF THE WAY
TO ALLOW SEC TO OPEN.
>> GREAT TALK.
THE QUESTION IS ABOUT YOU
STARTED WITH SHOWING THAT THE
FIRST TRANSMEMBRANE IS INSERTED
BY THE EMC.
AND YET SHOWED ALSO THAT THE
FIRST TRANSMEMBRANE IS
HYDROPHILIC AND STABILIZED WITH
INTERACTION BETWEEN SEC61 AND
PAT SO HOW IS IT STABILIZED WHEN
INSERTED AFTER EMC?
>> YEAH, SO I THINK IT MAY BE
THAT THE TIME IT IS IN THE
MEMBRANE IS RELATIVELY SHORT
BETWEEN WHEN INSERTED BY EMC AND
ENGAGES THE PAT COMPLEX.
I FORGOT TO MENTION, MAYBE I DID
MENTION IN PASSING, THE PAT
COMPLEX IS FAIRLY ABUNDANT AS
WELL.
WE THINK THAT PERHAPS IT SIMPLY
CAPTURES FAIRLY QUICKLY AND
THERE ISN'T A BIG PROBLEM.
THE OTHER MORE ATTRACTIVE IDEA
OBVIOUSLY THERE'S A PRECISE
HANDOVER FROM ONE COMPLEX TO
ANOTHER.
AND I WILL SAY THAT WHEN WE
FIRST PURIFIED EMC FOR
RECONSTITUTION STUDIES AND SO
FORTH AND DID MASS SPEC ON IT,
CCD47 WAS ONE OF THE COMPONENTS.
AND SO I'VE ALWAYS LIKED THIS
IDEA.
HOWEVER, SEEING A VERY ABUNDANT
MEMBRANE PRO IN A MASS SPEC
EXPERIMENT MAKES ME NERVOUS SO
I'VE BEEN HESITANT TO PUT TOO
MUCH STOCK IN IT.
>> A MORE GENERAL QUESTION.
AFTER THE FIRST STAGE YOU NOW
HAVE TO TRANSFER TWO
TRANSMEMBRANE DOMAINS, THAT
STILL SEEMS KIND OF PAINFUL.
IS THERE EVIDENCE OR SUGGESTION
PERHAPS THE MEMBRANE COMPOSITION
IN THIS AREA IS DIFFERENT IN
SOME WAY TO FACILITATE THE
TRANSFER?
>>YEAH, I PERSONALLY REALLY
LIKE THIS IDEA BUT CANNOT THINK
OF A GOOD WAY TO LIKE TEST THIS
CAREFULLY.
AND I WOULD LOVE TO HEAR
SUGGESTIONS.
>> SO THERE WERE NO OTHER
PROTEINS THAT MIGHT MODIFY THE
PROTEINS IN YOUR SCHEME?
>> NO, NOT THAT WE CAN THINK
OF.
WHAT I SORT OF IMAGINE IS THAT
THE KIND OF -- I DIDN'T SLOW
THIS BUT THE INTERIOR OF THE
HORSESHOE CAVITY IS PARTIALLY
HYDROPHILIC AND HYDROPHOBIC, ONE
POSSIBILITY IS THOSE
POSSIBILITIES RECRUIT A CERTAIN
SUBSET OF LIPIDS THAT FACILITATE
INSERTION.
I LIKE THAT IDEA.
AND MAYBE SOME TYPE OF LIPID
CROSS-LINKING EXPERIMENTS COULD
BE DONE TO SEE IF THERE ARE
NON-BULK LIPIDS THERE.
>>THANK YOU VERY MUCH,
MANU, NOR GIVING A FANTASTIC
PRESENTATION.
[APPLAUSE]