INDUCTION OR CARGO PACKAGING
With macroautophagy, pexophagy, and the Cvt pathway, cells use largely the same machinery to accomplish a similar goal, delivery of cytoplasmic components into the interior of the vacuole. Growth conditions dictate which components must be targeted for delivery.
Under conditions of nutrient stress, it becomes necessary for the cell to eliminate nonutilized energy-consuming cytosolic proteins and organelles. These components are delivered by macroautophagy to the vacuole, where they are degraded in order to generate an internal supply of nutrients (113). Tor is a serine/threonine kinase that, in response to amino acids and growth factors, coordinates different aspects of cell growth, such as transcription, translation, tRNA and ribosome biogenesis, actin organization, and protein kinase C signaling. Starvation inhibits Tor activity, provoking various cellular responses, including cell arrest in the early G1 phase, inhibition of protein synthesis, nutrient transporter turnover, transcriptional changes, and autophagy (81, 87, 90, 96). An identical cell reaction can also be triggered by treatment with rapamycin, a specific Tor inhibitor (34, 63).
Tor inactivation induces autophagy at two different levels: transcription and autophagosome formation (2). Gln3 is a transcriptional regulator of nitrogen source utilization genes, and it normally resides in the cytoplasm. Its localization is due to Tor-dependent phosphorylation that promotes its binding to the cytoplasmic repressor Ure2 (9). Tor inactivation leads to the dephosphorylation and dissociation of the inhibitory subunits of protein phosphatase 2A (PP2A), a phosphatase that acts on several Tor substrates, including Gln3 (90, 96). Dephosphorylation of Gln3 by PP2A promotes its dissociation from Ure2 and its successive translocation into the nucleus, where it activates the transcription of several genes (9, 14). Among those genes, some are part of the macroautophagy machinery (see below; genes shown in bold type have been shown to be induced only through microarray studies): APG1/AUT3, AUT1/APG3, AUT2/APG4, APG5, AUT7/APG8, APG7/CVT2, APG12, APG13, and APG14 (15, 33, 37, 56, 78). After treatment with rapamycin, macroautophagy is not blocked even if the de novo biosynthesis of induced proteins is inhibited by cycloheximide, suggesting that up-regulation of those factors is not essential for this process (2). Autophagosomes formed under those conditions are significantly smaller than normal, however, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion (2).
An important role in switching from the Cvt pathway to macroautophagy in response to nutrient conditions seems to be played by the cytosolic Apg1-Apg13 complex. Apg1/Aut3 is a serine/threonine kinase required for both the Cvt pathway and macroautophagy (70, 99, 109), and its activity is modulated by Apg13 (27, 45). Tor signaling negatively regulates the association between Apg1 and Apg13. Under nutrient-rich conditions, active Tor causes hyperphosphorylation of Apg13, preventing or moderating its association with Apg1 (45). It is not known whether Tor directly phosphorylates Apg13. Tor inactivation by starvation or rapamycin treatment promotes the rapid dephosphorylation of Apg13, a process that seems to be independent of PP2A (45, 100). Dephosphorylated Apg13 binds to Apg1; this association promotes autophosphorylation and activation of Apg1, leading to the induction of macroautophagy (45, 70).
In addition to Apg13, Apg1 also interacts with three other proteins whose function is specific either for macroautophagy or the Cvt pathway: Apg17, Cvt9, and Vac8 (45, 53, 100). APG17 is one of the small group of known genes with a function exclusively restricted to macroautophagy, and its gene product seems to participate in the formation or stabilization of the Apg1-Apg13 complex (45). It is possible that Apg17 has a role in cellular physiology other than autophagy because it seems to be a general factor required for the coordination of several cellular processes (20). Vac8 and Cvt9 are phosphoproteins specific for the Cvt pathway (53, 100, 134). It is possible that Apg1 and Apg13, both essential for macroautophagy and Cvt transport (27, 45, 70, 99, 100, 109), are the core of a regulatory system that controls conversion between those two pathways. Modulation of this system is accomplished in part through phosphorylation or dephosphorylation reactions and through interactions with factors specific for macroautophagy or for the Cvt pathway (45, 53, 100). However, this general scenario is complicated by the fact that other protein kinases, such as Snf1 and Pho85, recently were shown to have a role in the regulation of autophagy, probably via Apg1 and Apg13 (135). It remains to be determined whether all these components are assembled into a single large protein conglomerate or whether they form separate complexes depending on nutritional conditions.
Autophagy is a nonspecific degradative process and is used to deliver bulk cytoplasmic components to the vacuole. In contrast, the Cvt pathway is biosynthetic and selects specific cargo components for transport. Two hydrolases are known to use this route: Ape1 (58) and 
-mannosidase (Ams1) (38). After translation, Ams1 and prApe1 rapidly assemble into large oligomers (38, 55) that cannot be translocated to the endoplasmic reticulum. Thus, these proteins are unable to follow the normal route through a portion of the secretory pathway that most vacuolar proteins use to reach their final localization. The presence of an alternative route to the vacuole, such as the Cvt pathway, may have developed to allow the transport of large resident protein complexes.
Cvt19 is the receptor required for the transport of prApe1 and Ams1 to the vacuole via the Cvt pathway (65, 98). Cvt19 is not required for macroautophagy, but in its absence, prApe1 is not efficiently delivered to the vacuole under starvation conditions (98). Cvt19 specifically binds the propeptide of prApe1 and travels along with this protein to the vacuole, where it is finally degraded by vacuolar proteases. The association of prApe1 with Cvt19 promotes the inclusion of both proteins into the forming Cvt vesicles (98), but it is not clear yet if the formation of this complex is the event that triggers Cvt vesicle formation. The absence of Cvt19 destabilizes but does not prevent the association of prApe1 with membranes, indicating that prApe1 itself is able to bind a specific lipid or another protein (98). The lack of Cvt9 interferes with the proper association of prApe1 with membranes (53), but it is not known if this protein binds prApe1 or Cvt19. Interestingly, both Cvt9 and Cvt19 are peripheral membrane proteins that localize in a single punctate structure near the vacuole, possibly the site of Cvt vesicle formation (53, 98). It is tempting to speculate that an interaction of prApe1, Cvt19, and Cvt9 modulates the activity of the Apg1-Apg13 complex and consequently also the induction of Cvt vesicle formation.
Pexophagy, like autophagy, is a degradative process (Fig. 1). However, it is not induced by starvation conditions per se. When yeast cells are grown in the presence of oleic acid as the sole carbon source, peroxisome biogenesis is induced in order to carry out fatty acid ß oxidation (128). After shifting of cells to a glucose-containing medium, the excess peroxisomes are degraded in the vacuole (17, 24, 39). The degradation of superfluous peroxisomes by micropexophagy has been shown to require most of the Apg and Cvt proteins, including the Cvt transport-specific component Cvt9/Gsa9 (39, 53). This requirement suggests that pexophagy may be induced in a manner similar to the Cvt pathway. It is not surprising that both the Cvt pathway and micropexophagy require Cvt9, because these two processes are active under the same growth conditions. Peroxisome degradation is rapid and highly specific (39), but it does not use the Cvt19 receptor (98). It is not known how peroxisomes are selected by the enwrapping membranes, but Cvt9/Gsa9 probably plays an important role (53).
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