Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation

Many organelles change their shape in the course of the cell cycle or in re sponse to environmental conditions. Lysosomes undergo drastic changes of sh ape during microautophagocytosis, which include the invagination of their b oundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocyto sis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion . During the uptake reaction, vacuolar membrane invaginations, called autop hagic tubes, were observed. The reaction resulted in the transient formatio n of autophagic bodies in the vacuolar lumen, which were degraded upon prol onged incubation. Under starvation conditions, the system reproduced the in duction of autophagocytosis and depended on specific gene products, which w ere identified in screens for mutants deficient in autophagocytosis. Microa utophagic uptake depended on the activity of the vacuolar ATPase and was se nsitive to GTP gammaS, indicating a requirement for GTPases and for the vac uolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, sc ission of the invaginated membrane must occur via a novel mechanism distinc t from the homotypic fusion of vacuolar membranes.