POSSIBLE ROLE OF VAMP7 IN AUTOPHAGOSOME FORMATION

FADER CM; COLOMBO MI

Puerto Madryn

Congreso; XLVI Reunión Anual Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2010

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called autophagosome which finally fuses with the lysosome to degrade the sequestered material. The protein LC3 is an autophagic marker present in eukaryotic cells as a soluble form (LC3-I) and a membrane-associated form (LC3-II). LC3-I is conjugated to a lipid molecule to generate LC3-II, which localizes to autophagosomes. We have analyzed by fluorescence microscopy and Western blot the participation of the fusion protein VAMP7 (VSNARE) in autophagosome formation. Our results indicate that a population of LC3 positive structures is decorated with endogenous VAMP7. Furthermore, overexpression of the N-terminal domain of VAMP7, which inhibits the SNARE complex formation, alters the RFP-LC3 pattern from a punctate to a diffuse distribution in several cell types, indicating that LC3 is not associated to autophagosomal membranes. Moreover, silencing VAMP7 caused a marked decrease in the number of LC3 labeled structures. These results were confirmed by Western blot observing a decrease in the levels of the LC3-II when endogenous VAMP7 was knocked down. Taken together, our results suggest that VAMP7 is involved at the initial steps of autophagosome formation, probably allowing the delivery of ER membranes to the pre-autophagosomal structures.