Once the autophagosomes and Cvt vesicles have fused with the vacuolar membrane, the interior single-membrane vesicles are released into the vacuolar lumen. These vesicles, now termed autophagic and Cvt bodies, are subsequently consumed by hydrolases (Fig. 1). This breakdown requires normal acidification of the vacuole (77), presumably to maintain the optimal pH of the degradative enzymes but also because it is essential for the autocatalytic cleavage and subsequent activation of proteinase A (Pep4) and proteinase B (the product of the PRB1 gene, allelic to CVT1) (43, 124). These proteases are at the apex of a proteolytic cascade that leads to the activation of most vacuolar hydrolases. Mutations in the PEP4 or PRB1 genes stabilize autophagic and Cvt bodies as well as peroxisomes that have been delivered to the vacuole (6, 31, 39, 58, 113, 117).
In addition to proteases, the complete degradation of lumenal vesicles requires the action of lipases. The cvt17/aut5 mutant exhibits an accumulation of both Cvt and autophagic bodies inside the vacuole (32, 97). The CVT17 gene was recently cloned and shown to code for an integral membrane protein with an essential domain conserved among lipases (116). One model is that Cvt17 enters the inner lipid bilayer of Cvt and autophagic bodies, probably at the place of their formation, and is successively degraded with this membrane in the vacuolar lumen (116). It is not clear if Cvt17 is required for the direct lysis of subvacuolar vesicles. It is also possible that its lipase activity is necessary to alter the lipid composition of the subvacuolar vesicles as they form, a modification that may be essential to target other hydrolases inside the bodies or to render the lipid bilayer more susceptible to other vacuolar lipases.
Another protein that has also been shown to play a role in the degradation of subvacuolar vesicles is Aut4, a multispanning transmembrane protein (112). The fact that Aut4 is completely immersed in membranes suggests a function linked to the lipid composition of subvacuolar vesicles (lipid synthase, lipase, or flipase); however, a role in targeting hydrolases to autophagic bodies cannot be excluded. Aut4 function seems to be distinct from that of Cvt17 for two reasons. First, Aut4 is autophagy specific, and aut4
cells grown under nutrient-rich conditions show normal prApe1 processing (112). Second, Aut4 localizes to perivacuolar punctate structures and to the vacuolar membrane but does not enter autophagic bodies (112).
Both Cvt17 and Aut4 have homologues only in bacteria or other fungi (112, 116) (Table 1), reflecting a possible involvement with a particular lipid. In order for lipases to recognize membranes destined for degradation, the lipid bilayer composition of autophagic and Cvt bodies should be different from that of the delimiting vacuolar membrane. Multivesicular bodies are another population of subvacuolar vesicles that are degraded within the vacuolar lumen (67, 83). In mammalian cells, they are reported to have a unique lipid composition, being enriched in the unusual lipid lyso-bis-phosphatidic acid (60). Cvt17 and Aut4 may play a role in modifying transport membranes to differentiate them from the vacuolar membrane and allow for their specific degradation.
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