Vesicle formation starts with the enwrapping of the selected cargo, organelle, or cytosol by a membrane. This process ends with the fusion of the extremities of the surrounding membrane, leading to the completion of a double-membrane vesicle, an autophagosome or a Cvt vesicle, depending on size and on nutritional conditions (Fig. 1).
Several components are necessary for this process and are shared by macroautophagy, pexophagy, and the Cvt pathway. In particular, some of the factors are parts of two different ubiquitin-like (UBL) systems that are essential for vesicle biogenesis (84). Ubiquitin is first activated by binding to a ubiquitin-activating enzyme (also called E1). Then it is transferred to a ubiquitin-conjugating enzyme (also called E2). Then a ubiquitin protein ligase enzyme (also called E3) catalyzes its covalent binding to the target substrate (136). The specificity of substrate recognition is modulated by the E2 and E3 enymes, but other factors are also implicated.
The first UBL protein shown to be involved in macroautophagy was Apg12 (72). As in the ubiquitin system, Apg12 is first activated by the E1 enzyme Apg7/Cvt2 in an ATP-dependent reaction that leads to the formation of a high-energy thioester bond between the carboxy-terminal glycine of Apg12 and cysteine 507 of Apg7 (50, 61, 72, 114). Subsequently, Apg12 is transferred to the E2 enzyme Apg10, forming a new thioester bond with its cysteine 133 (72, 105). The last step of this sequence of reactions is the covalent linkage of Apg12 to lysine 149 of Apg5 (46, 72). This conjugation system is constitutive and does not possess the counterpart of an E3 enzyme (84); this function is probably accomplished by Apg10. The Apg12-Apg5 conjugate then associates with Apg16, forming a trimeric complex that is able to multimerize (71). The function of this large complex is unknown, but the proteins seem to be localized at the site of autophagosome formation (28).
The second UBL protein involved in macroautophagy is Aut7/Apg8/Cvt5. Aut7 is a soluble protein, and its carboxy-terminal arginine is removed by the Aut2/Apg4 cysteine protease, leaving a glycine residue at the carboxy terminus (51, 57). As with Apg12, Aut7 is activated by the E1 enzyme Apg7/Cvt2 through a thioester bond between its carboxy-terminal glycine and cysteine 507 of Apg7 (40, 57, 61). Aut7 is subsequently transferred to the E2 enzyme Aut1/Apg3 via a new thioester bond between those two proteins (40, 51, 95). Aut7 is finally covalently conjugated to a phosphatidylethanolamine (PE) molecule, becoming tightly membrane associated (40). Until the step when they form their respective E2 conjugates, the Aut7 and Apg12 conjugation systems proceed independently but may be coordinated through the dual requirement for Apg7. The Apg12-Apg5 complex forms in the absence of the Aut7 conjugation system (28, 72), whereas the linkage between Aut7 and PE is blocked in the absence of the Apg12 system (51).
The Aut2 protease, in addition to removing the carboxy-terminal arginine of newly synthesized Aut7, is also able to cleave the amide bond linking Aut7 to PE, changing it back to a soluble form (57). This second cleavage event is part of the dynamic utilization of Aut7 during autophagosome and Cvt vesicle formation and is required as part of the normal itinerary of this protein. Under nutrient-rich growth conditions, Aut7 localizes to unidentified dot structures dispersed in the cytoplasm (51, 56). After shifting of cells to a minimal medium that induces starvation, Aut7 becomes concentrated to punctate perivacuolar structures (56). These structures appear to be autophagosomes in the process of formation. Aut7 localizes along the entire preautophagosomal membrane during expansion (56). Upon completion of the autophagosome, the Aut7 pool on the surface dissociates from the membrane, becoming soluble, an event probably catalyzed by the action of Aut2 (56). The Aut7 pool trapped inside the autophagosome is successively degraded in the vacuolar lumen together with the autophagic body (37, 56). The behavior of Aut7 suggests that it is a structural element necessary for autophagosome and Cvt vesicle formation. This idea is also supported by the fact that Aut7 is induced by starvation (37, 56) and is required for expansion of the membrane that forms the autophagosome (2).
Aut7, like the coat proteins involved in other vesicular trafficking events, may have a double role in coordinating autophagosome biogenesis. As discussed above, Aut7 plays a structural role in vesicle formation. In addition, the presence of Aut7 during this event may have the function of preventing the premature fusion of an unfinished autophagosome with the vacuole. This activity can be achieved through the binding of Aut7 in an inhibitory manner to one of the SNARE proteins required for the fusion of the autophagosome with the vacuole (66). The result is that only complete, Aut7-uncoated autophagosomes can fuse with the vacuole. The signal that triggers Aut7 release upon completion of the autophagosome is not yet known.
PE is an abundant lipid present in all cellular membranes, but Aut7 is specifically conjugated only to PE present in the forming autophagosome. It is unclear how the Aut7-Aut1 activated complex is recruited to the right place. It is known that the binding of Aut7 to PE depends on the Apg12 conjugation system (51). It is possible that one of the events stimulated by this system is the supply of a docking point for the Aut7-Aut1 complex. In addition, an E3 enzyme implicated in the transfer of Aut7 to PE has not yet been identified. It is conceivable that this enzyme provides such a landmark.
Apg9/Cvt7/Aut9 is the only characterized multispanning transmembrane protein essential for autophagosome and Cvt vesicle formation (64, 79). It localizes to perivacuolar punctate structures but appears to be excluded from the membranes composing autophagosomes and Cvt vesicles (79). Several components involved in the induction and formation of Cvt and macroautophagy transport vesicles are peripheral membrane proteins, but the nature of their targeting to membranes is unknown. Apg9 shares a localization pattern similar to that of those proteins, and it is likely that it is used as a docking point by some of them. This seems to be the case at least for Apg2, a protein colocalizing with Apg9 and implicated in the process of autophagosome and Cvt vesicle formation as well as in pexophagy (106, 132). Apg2 directly binds Apg9 (132) and, like Apg9, is excluded from autophagosomes (106). In the absence of Apg9, Apg2 becomes cytosolic (106, 132). The association between Apg9 and Apg2 requires the presence of Apg1 but not its kinase activity (106). Apg2 also colocalizes with Aut7, but its distribution does not depend on Aut7 and vice versa, suggesting that these two proteins are independently recruited to the same perivacuolar structures (51, 106).
Cvt18/Gsa12 is a cytosolic protein that contains two WD40 domains and that is also essential for double-membrane vesicle formation in macroautophagy, the Cvt pathway, and pexophagy (29). Cvt18 localizes to perivacuolar punctate structures and to the vacuolar rim, but its association with membranes does not appear to depend on the characterized APG/CVT/AUT genes. The function of Cvt18 is not known, but it is required to target Apg2 to punctate structures adjacent to the vacuole (29).
Answers to all your Biology Questions
