NIH VideoCast - 2013 Nobel Prize Winner will speak on "Biogenesis and Function of the Autophagosome"

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2013 Nobel Prize Winner will speak on "Biogenesis and Function of the Autophagosome"

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Air date:	Monday, November 4, 2013, 11:00:00 AM Time displayed is Eastern Time, Washington DC Local
Views:	Total views: 2,083 (456 Live, 1,627 On-demand)
Category:	Special
Runtime:	01:08:33
Closed captioning:	Check box to display CC outside of the video Note: You can drag the captioning window around and resize it
Description:	The NIH Cell Biology and Metabolism Program will host Randy Schekman, co-winner of the 2013 Nobel Prize for Physiology or Medicine. The pathway of autophagy has assumed an important position in the analysis of mammalian cellular response to stress, hypoxia and pathogen infection. Autophagosomes mature by growth and envelopment of cytosolic proteins and organelles that are trapped within the inner membrane of a two-membrane organelle. Trapped proteins are delivered by autophagosome fusion to the lysosome where protein and polysaccharide degradation permit amino acids and sugars to be recycled. A pre-autophagsosomal membrane matures by the addition of membrane material from various intracellular sources and the attachment of peripheral proteins that remain bound through a covalent lipidation reaction. The origin and the mechanism of generation of the pre-autophagic membrane are poorly understood. Published evidence suggests an origin of this membrane on the ER, on the mitochondrial surface or possibly the Golgi or plasma membrane. We have addressed these issues with the development and analysis of a cell-free reaction that reproduces the lipidation of a major peripheral autophagosomal protein, LC3. Mouse embryonic fibroblasts (MEFs) taken from a mouse strain deficient in Atg5 are unable to lipidate LC3 and other targets of lipidation involved in the autophagic process. A crude membrane fraction isolated from these MEFs was mixed with cytosol harvested from normal cells that were untreated or subjected to a stress regimen known to induce autophagy. On addition of ATP, incubation of the mixture resulted in the formation of lipidated endogenous LC3 or exogenous recombinant LC3. The reaction required both membranes and cytosol and was stimulated 2-5 fold when the cytosol was taken from stress-induced cells, but was inactive when cytosol was used from stressed atg5 mutant MEFs. Autophagosome maturation requires a class III PI-3 kinase (VPS34 homolog); LC3 lipidation in our cell-free reaction was inhibited by wortmannin and 3-methyladenine, known inhibitors of this kinase, and by the addition of a peptide containing a PI3P-bnding sequence, the FYVE domain. Using cell fractionation techniques including differential centrifugation and buoyant density sedimentation, we have identified an ERGIC fraction enriched in Sec22 and ERGIC-53 that has the highest specific activity for LC3 lipidation in comparison to other cellular membranes. Membranes isolated from cells depleted of the ERGIC by use of selective inhibitors of traffic did not retain lipidation activity, but the activity reappeared quickly when traffic and the ERGIC was restored by removal of the inhibitor. Other early markers of the autophagosome membrane, Atg14L and DFCP1, are recruited to the ERGIC in cultured cells and in our cell-free reaction. This approach may now be exploited to understand the molecular mechanism of growth, closure and cargo capture by the maturing autophagosomal membrane.

NLM Title:	Biogenesis and function of the autophagosome / Randy Schekman ; NIH Cell Biology and Metabolism Program.
Author:	Schekman, Randy W. Eunice Kennedy Shriver National Institute of Child Health and Human Development (U.S.). Cell Biology and Metabolism Program,
Publisher:
Abstract:	(CIT): The NIH Cell Biology and Metabolism Program will host Randy Schekman, co-winner of the 2013 Nobel Prize for Physiology or Medicine. The pathway of autophagy has assumed an important position in the analysis of mammalian cellular response to stress, hypoxia and pathogen infection. Autophagosomes mature by growth and envelopment of cytosolic proteins and organelles that are trapped within the inner membrane of a two-membrane organelle. Trapped proteins are delivered by autophagosome fusion to the lysosome where protein and polysaccharide degradation permit amino acids and sugars to be recycled. A pre-autophagsosomal membrane matures by the addition of membrane material from various intracellular sources and the attachment of peripheral proteins that remain bound through a covalent lipidation reaction. The origin and the mechanism of generation of the pre-autophagic membrane are poorly understood. Published evidence suggests an origin of this membrane on the ER, on the mitochondrial surface or possibly the Golgi or plasma membrane. We have addressed these issues with the development and analysis of a cell-free reaction that reproduces the lipidation of a major peripheral autophagosomal protein, LC3. Mouse embryonic fibroblasts (MEFs) taken from a mouse strain deficient in Atg5 are unable to lipidate LC3 and other targets of lipidation involved in the autophagic process. A crude membrane fraction isolated from these MEFs was mixed with cytosol harvested from normal cells that were untreated or subjected to a stress regimen known to induce autophagy. On addition of ATP, incubation of the mixture resulted in the formation of lipidated endogenous LC3 or exogenous recombinant LC3. The reaction required both membranes and cytosol and was stimulated 2-5 fold when the cytosol was taken from stress-induced cells, but was inactive when cytosol was used from stressed atg5 mutant MEFs. Autophagosome maturation requires a class III PI-3 kinase (VPS34 homolog); LC3 lipidation in our cell-free reaction was inhibited by wortmannin and 3-methyladenine, known inhibitors of this kinase, and by the addition of a peptide containing a PI3P-bnding sequence, the FYVE domain. Using cell fractionation techniques including differential centrifugation and buoyant density sedimentation, we have identified an ERGIC fraction enriched in Sec22 and ERGIC-53 that has the highest specific activity for LC3 lipidation in comparison to other cellular membranes. Membranes isolated from cells depleted of the ERGIC by use of selective inhibitors of traffic did not retain lipidation activity, but the activity reappeared quickly when traffic and the ERGIC was restored by removal of the inhibitor. Other early markers of the autophagosome membrane, Atg14L and DFCP1, are recruited to the ERGIC in cultured cells and in our cell-free reaction. This approach may now be exploited to understand the molecular mechanism of growth, closure and cargo capture by the maturing autophagosomal membrane.
Subjects:	Autophagy--physiology Organelle Biogenesis Phagosomes--physiology
Publication Types:	Lecture Webcast

NLM Classification:	QU 375
NLM ID:	101622742
CIT Live ID:	13289
Permanent link:	https://videocast.nih.gov/watch=13289

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