Yeast cells are able to break down numerous membrane-enclosed autophagic bodies in their vacuoles, leaving the limiting vacuolar membrane intact. Except for our knowledge of the need of vacuolar acidification (23
) and vacuolar proteinases (33
), components of this inevitably highly specific membrane disintegration machinery remain mysterious. We here identified Aut5p, a component essential for disintegration of autophagic bodies and demonstrated the relevance of its lipase active-site motif via site-directed mutagenesis.
Since autophagic bodies are lysed inside the vacuole, it seems conceivable that Aut5p is targeted to the vacuole, probably simultaneously with autophagic bodies. Indeed, our pulse-chase analysis indicates a rapid (half-life, 50 to 70 min) turnover of Aut5-Ha dependent on the presence of vacuolar proteinase A. Consistently, in cells defective in vacuolar proteolysis, indirect immunofluorescence (Fig. B and C) and immunogold electron microscopy (Fig. B to D) visualized significant amounts of Aut5-Ha in the vacuole. However, in wild-type cells this vacuolar pool of Aut5-Ha was lacking (Fig. A). This finding is in contrast to findings in the study of Cvt17p, for which degradation “independent of the major vacuole proteases” was reported (35
). We believe this discrepancy might be due to the use of an antiserum raised against synthetic peptides, since in our hands the protein was hard to detect with two individual antisera against synthetic peptides (not shown), but a reliable strong signal was detectable using the Ha-tagged species.
How does Aut5p reach the vacuole? The breakdown of Aut5-Ha in autophagy-deficient aut3Δ cells (Fig. D), together with the vacuolar localization in starved (Fig. C) and logarithmically growing (not shown) aut3Δ pep4Δ cells in immunofluorescence, argues against the use of the autophagic pathway. This conclusion is consistent with our statistical analysis of immunogold microscopy. We cannot exclude, however, the possibility that a small portion of Aut5p travels to the vacuole via autophagy at autophagic bodies and then induces their breakdown, but our data clearly indicate that the major part of Aut5-Ha reaches the vacuole independent of autophagy.
The observed glycosylation of Aut5-Ha demonstrates that Aut5-Ha follows the ER-to-Golgi sorting pathway. Immunofluorescence suggests a localization of Aut5-Ha in the vacuolar lumens of pep4
Δ cells (Fig. B and C). Since Aut5-Ha in these cells behaves as an integral membrane protein (Fig. C), we expected a localization at membranous structures inside the vacuole. Immunogold electron microscopy in fact displayed localization at ~50-nm-diameter vesicles (Fig. B to D) in the vacuolar lumen. The appearance of 50-nm-diameter vesicles in the vacuolar lumen is reminiscent of the function of the MVB pathway (24
). The MVB pathway at the prevacuolar endosome (compartment) diverges from the biosynthetic ER-to-Golgi vacuolar protein sorting pathway. Integral membrane proteins, which are cargo molecules of the MVB pathway, are specifically directed to invaginations of the membrane of the prevacuolar endosome. Then vesicles carrying these cargo molecules branch off to the interior of the organelle, and a so-called MVB is formed. Fusion of the MVB with the vacuole finally releases the ~50-nm-diameter vesicles to the vacuolar lumen, where they are thought to be broken down (24
Vps class E mutants affect both the biosynthetic and MVB traffic to the vacuole. In these mutants proCPS, which is a cargo molecule of the MVB pathway, is retarded at the prevacuolar compartment and, due to a defect in sorting to internal vesicles at the prevacuolar endosome, is mislocalized to the limiting vacuolar membrane. The phosphatidylinositol 3-phosphate 5-kinase Fab1p is specifically required for MVB sorting but not for biosynthetic vacuolar protein sorting, and therefore a lack of this protein causes no retardation at the prevacuolar endosome but only mislocalization to the vacuolar membrane (41
). We observed in both mutant types a mislocalization of Aut5-Ha similar to that described for proCPS (Fig. ), suggesting a sorting similar to that of the MVB pathway. Taken together our findings suggest that Aut5-Ha reaches the vacuolar lumen after transit through the ER and Golgi apparatus in a way similar to that of the MVB pathway.
The lysis of membranes implies a potential high risk for the cell. Very interesting questions therefore are what prevents a premature activation of such a membrane lysis machinery and what impedes lysis of the vacuolar membrane itself? Our observation that two distinct pathways are used for vacuolar targeting of autophagic bodies and Aut5-Ha, respectively, opens up new vistas in answering these questions. Probably Aut5p, a putative lipase, becomes activated only upon direct interaction with a component present exclusively at autophagic bodies. It would be tempting to speculate that this interacting partner acts similarly to a colipase. This idea would explain not only the specificity in disintegrating autophagic bodies without the integrity of the vacuolar membrane being affected but also how the cell prevents untimely activation of Aut5p on its transit to the vacuole by keeping both partners apart by two different transport pathways, which converge at their final destination, the vacuolar lumen. Since Aut5p might remain in its active form even after lysis of the autophagic body, the observed rapid vacuolar degradation of Aut5-Ha would prevent further trouble. This idea implies that Aut5p exhibits its active site and a putative interaction domain towards the vacuolar lumen and not to the interior of the 50-nm-diameter vesicle. The potential glycosylation sites and the lipase active-site motif of Aut5p are located after its predicted transmembrane domain (Fig. B). Since Aut5-Ha is glycosylated, this part of the protein including the lipase active-site motif would be estimated to be exposed to the ER lumen. Assuming that the membrane topology is preserved during sorting, one would therefore indeed expect that the lipase active-site motif together with most of Aut5p is exposed to the outside of the 50-nm-diameter vesicle membrane, i.e., to the vacuolar lumen. Another possibility would be that Aut5p selectively attacks lipid molecules present only at the membranes of autophagic bodies and not at the limiting vacuolar membrane.
Interestingly, Aut5-Ha is localized at the ER (nuclear envelope) in wild-type cells and also in pep4Δ cells. This localization is indicated by four lines of evidence: (i) glycosylation, which points to entry in the ER to the Golgi secretory pathway; (ii) indirect immunofluorescence of both starved (Fig. ) and logarithmic (not shown) cells, which shows a typical ring-like staining around the nucleus; (iii) sucrose density gradient fractionation of starved wild-type cells, in which the ER marker Kar2p cosedimented with Aut5-Ha (not shown); and (iv) immunogold electron microscopy, which showed beside the vacuolar localization a significant localization at the nuclear envelope and ER and at the cortical ER (Fig. ).
Detection of Aut5-Ha at the ER raises the possibility that instead of the vacuolar lumen Aut5p might alternatively function at the ER by hydrolyzing specific lipids, thus rendering autophagic bodies competent for breakdown. The membrane source of autophagic bodies remains unknown, although there are some hints pointing to the ER (8
) or the Golgi apparatus (18
). One might therefore speculate that lipids modified at the ER by Aut5p are specifically targeted to autophagosomes. Finally, yet unknown components in the vacuole might catalyze the breakdown of autophagic bodies dependent on the presence of these lipid molecules. In this scenario Aut5p would be catalytically active at the ER and most likely also on its transit to the vacuole. To avoid unspecific hydrolysis of membranes, which surely would lead to cell death, Aut5p must therefore exhibit a very high substrate specificity. When Aut5p functions at the ER, the rapid transport to the vacuolar lumen would reflect only protein degradation. Further extensive studies are necessary to distinguish between these models of Aut5p function.
Aut5p shares significant homologies with several proteins of unknown function (Fig. B); the homologies very interestingly include the putative lipase active-site motif. This finding might point to a common function of these proteins. The pSI-7 protein from C. fulvum
was identified for its induced expression during starvation and its pathogenic growth on tomato plants (5
). Probably our work on Aut5p in yeast therefore might also contribute in the future to the understanding of the pathogenic interaction between C. fulvum