Several genes essential for macroautophagy, the Cvt pathway, and pexophagy have now been identified and ordered in functional groups. This classification is based on the step in the import pathway in which they act and on their interactions with other proteins. A future task will be to try to find the connections among these clusters of genes in order to obtain the complete picture of these processes. For example, it has been determined that Apg1 kinase activity is essential for the induction of autophagosomes and Cvt vesicles. Many proteins interacting with Apg1 are also known, but the target of this cluster of proteins remains unidentified. The binding of cargo molecules to the Cvt19 receptor also seems to be important for the induction step, but at the moment, no connection between such complexes and other known elements has been made. Similarly, three groups of genes are involved in the biogenesis of double-membrane vesicles: the Apg12 and Aut7 conjugation systems and the Apg2-Apg9 complex. The interactions among these sets of proteins remain largely unknown. Identification of the connections among these groups of genes will help to order the events that lead to the formation and completion of these vesicles.
The origin(s) of the sequestering membranes in the Cvt, macroautophagy, and macropexophagy pathways is not known. In mammalian cells, it is generally believed that autophagosomes are derived from the endoplasmic reticulum (22). It is now evident that in yeast cells, double-membrane vesicles are formed in a perivacuolar punctate structure. The only known structure with this characteristic location is the late endosome or prevacuolar compartment (88, 131). Several observations argue against the participation of this organelle in the formation of double-membrane vesicles. For example, Pep12 is a SNARE protein that localizes to the late endosome (8, 36). Gradient analyses have shown that Pep12 does not completely cofractionate with markers of the perivacuolar structure involved in the macroautophagy and Cvt pathways (53, 79, 106, 132). Moreover, nonfunctional PEP12 alleles that block the vacuolar delivery of proteins passing through the late endosome do not affect the transport of prApe1 (1, 8). Finally, mutations that affect the morphology of the late endosome do not interfere with that of the macroautophagy and Cvt pathway perivacuolar structure (79). Conversely, other lines of evidence implicate the endosome or a domain of the endosome in these pathways. The csc1 mutant is constitutive for macroautophagy. CSC1 is allelic with the gene encoding endosomal protein Vps4 (107). Another protein that has been implicated in endosomal function, Vps30/Apg6, is part of two similar complexes, one required for sorting of carboxypeptidase Y and the other required for the macroautophagy and Cvt pathways (47, 49, 102). In addition to Apg6, these complexes contain two other common subunits: the PI 3-kinase Vps34 and the protein kinase Vps15 (49). The specificity for the pathway is given by the fourth subunit: Apg14 is essential for the macroautophagy and Cvt pathways, whereas Vps38 is necessary for the Vps pathway (49). Recent studies showed that Apg14 colocalizes with many of the other Apg/Cvt proteins (52). It will now be interesting to determine where the mammalian homologues of the yeast proteins in this newly discovered perivacuolar structure are localized.
The presence of a newly discovered organelle in the yeast endosomal system or the involvement of a subdomain of the endosome raises new questions connected with subcellular protein trafficking. Three of the components localizing to this structure are integral membrane proteins (64, 79, 112, 116). One of those, Cvt17, is glycosylated, indicating passage through the Golgi complex (116). After translation, all the transmembrane proteins of the endosomal system are translocated to the endoplasmic reticulum and follow the secretory route until the late Golgi compartment (18, 67). In this compartment, they are recognized by coat proteins and specifically packaged into vesicles directed to their final destinations (18, 67). Which class of vesicles is involved in the transport of Cvt17, Apg9, and Aut4 to the perivacuolar structure involved in the macroautophagy and Cvt pathways? How are these proteins recognized and segregated from other proteins present in the endoplasmic reticulum? The involvement in macroautophagy of a subset of components required for vesicle coat formation at the endoplasmic reticulum (41) may provide some insight into these questions.
Finally, vesicle fusion with the target compartment is catalyzed by the interaction of SNARE proteins present on the two approaching membranes. The best candidate for a SNARE protein on the perivacuolar organelle is Tlg2. This tSNARE protein localizes to punctate structures (36) and is required for the normal processing of prApe1 (1). TLG2 deletion strains block the formation and completion of Cvt vesicles (1). However, Tlg2 is specific for the Cvt pathway and is unnecessary for macroautophagy (1). These findings leave open the question of which SNARE proteins are essential for autophagosome formation. Are there different perivacuolar structures, some necessary for Cvt vesicle formation and others necessary for autophagosome biogenesis? Analysis of the trafficking of integral membrane proteins under different growth conditions will help to answer this question.
Autophagy, the Cvt pathway, and pexophagy are complex processes involving dynamic rearrangements of membranes to deliver proteins and organelles from the cytoplasm to the vacuole. These processes are conserved in eukaryotes. Future studies will continue to provide molecular details about these alternative vacuolar targeting pathways.
ACKNOWLEDGMENTS
F.R. is supported by a long-term fellowship from the European Molecular Biology Organization, and D.J.K. is supported by Public Health Service grant GM53396 from the National Institutes of Health.
FOOTNOTES
* Corresponding author. Mailing address: University of Michigan, Department of Molecular, Cellular, and Developmental Biology, Ann Arbor, MI 48109-1048. Phone: (734) 615-6556. Fax: (734) 647-0884. E-mail: klionsky@umich.edu
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