Autophagosome formation depends on early secretory pathway

ZOPPINO, FCM Y COLOMBO MI

Carlos Paz Cordoba Argentina

Workshop; Workshop de transporte de membranas; 2005

SBC

Autophagy is a process by which portions of cytoplasm and/or organelles are sequestered by membranes to form vesicles called autophagosomes, which finally fuse with lysosomes. The origin of autophagosome  membranes has not been clearly determined. Intracellular tubulovesicular trafficking is regulated by members of the Rab GTPase family. It is well known the function of Rab1 promoting the anterograde transport between ER and Golgi membranes. In this study we have analyzed by fluorescence and confocal microscopy, the behavior of GFP-Rab1b wt and mutants, during autophagy by using autophagosome markers such as GFP/RFP-LC3 and monodansylcadaverine (MDC). Cells were incubated under different conditions that stimulate autophagy such as starvation or rapamycin treatment. Our data suggest that Rab1-labeled vesicles partially colocalized with RFP-LC3, whereas a marked colocalization was observed in cells overexpressing the GTPase deficient mutant Q67L. The Rab protein remained associated to these vacuoles until a later stage (e.g. autophagolysosome), as indicated by the acquisition of cathepsin D, a lysosomal enzyme. Moreover, Rab1 requires its active conformation to anchor on autophagic membranes, since the mutant S22N with impaired capacity to bind GTP, did not colocalize with MDC marked vesicles. Our results suggest that transport vesicles containing Rab1 may contribute to the generation of autophagic vacuoles.