Cell-Free Reconstitution of Microautophagic Vacuole Invagination and Vesicle Formation

Abstract

Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary 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 microautophagocytosis 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 autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPγS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.

Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae

“…That observation supports our analysis that fails to detect any t-SNARE at the PAS (Figure 2, D and E), and it suggests a different mechanism takes place during double-membrane vesicle sealing. This suggestion would be in agreement with the finding that microautophagy, another process that requires the fusion of double membranes, does not use the SNARE proteins involved in homotypic vacuole fusion (Sattler and Mayer, 2000); that is, a different type of fusion event is taking place that is independent of SNAREs.…”

“…That observation supports our analysis that fails to detect any t-SNARE at the PAS (Figure 2, D and E), and it suggests a different mechanism takes place during double-membrane vesicle sealing. This suggestion would be in agreement with the finding that microautophagy, another process that requires the fusion of double membranes, does not use the SNARE proteins involved in homotypic vacuole fusion (Sattler and Mayer, 2000); that is, a different type of fusion event is taking place that is independent of SNAREs.Previous studies have shown that part of the machinery involved in COPII vesicle formation is also required for autophagy but not the Cvt pathway (Klionsky et al, 1992;Ishihara et al, 2001). One problem is that those studies used conditional alleles, and it is not clear how long it takes to interfere with either the preexisting or the forming PAS after the block is induced by the temperature shift.…”

Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance

“…The process has been studied mainly in yeast and cell free systems, and little is known about the molecular mechanism of microautophagy in mammals 147,148 . It is generally accepted that microautophagy is a constitutively active, nonselective catabolic pathway in the cell.…”

The Vacuolar Transporter Chaperone (VTC) Complex Is Required for Microautophagy

“…Fusion of the outer autophagosomal layer with the vacuolar membrane liberates autophagic bodies (single-layered intravacuolar vesicles) into the vacuolar lumen for degradation (Takeshige et al, 1992). Relevant actors of macroautophagy (Atg proteins; Klionsky et al, 2003) have been identified, mainly by genetic screens (Tsukada and Ohsumi, 1993;Thumm et al, 1994;Harding et al, 1995Harding et al, , 1996Titorenko et al, 1995) and have been studied intensively over the last decade.Little is known about microautophagy, a process consisting of a direct invagination of the vacuolar boundary membrane and budding of autophagic bodies into the vacuolar lumen (Muller et al, 2000;Sattler and Mayer, 2000;Kunz et al, 2004). Microautophagy of soluble cytosolic components is topologically equivalent to invaginations occurring during multivesicular body (MVB) formation at the endosome, piecemeal microautophagy of the nucleus (PMN) (Roberts et al, 2003) into the yeast vacuole and micropexophagy in methylotrophic yeasts (Veenhuis et al, 1983;Tuttle et al, 1993;Tuttle and Dunn, 1995;Sakai et al, 1998;Mukaiyama et al, 2002Mukaiyama et al, , 2004.…”

“…Also nascent autophagosomes are virtually free of intramembraneous particles, suggesting that membrane removal by microautophagy might compensate macroautophagic membrane influx both in terms of quantity and quality. Microautophagic vacuole invagination could be reconstituted in a cell-free system composed of purified vacuoles and cytosolic extracts (Muller et al, 2000;Sattler and Mayer, 2000). Using a pharmacological approach, the in vitro uptake reaction could be dissected into different kinetic stages (Kunz et al, 2004).…”