The molecular mechanism of autophagy

Abstract

Autophagy is a conserved trafficking pathway that is highly regulated by environmental conditions. During autophagy, portions of cytoplasm are sequestered into a double-membrane autophagosome and delivered to a degradative organelle, the vacuole in yeast and the lysosome in mammalian cells, for breakdown and recycling. Autophagy is induced under starvation conditions and in mammalian cells is also invoked in response to specific hormones. In yeast, under nutrient-rich conditions, a constitutive biosynthetic pathway, termed the cytoplasm to vacuole targeting (Cvt) pathway, utilizes most of the same molecular machinery and topologically similar vesicles for the delivery of the resident hydrolase aminopeptidase I to the vacuole. Both autophagy and the Cvt pathway have been extensively studied and comprehensively reviewed in the past few years. In this review, we focus on the yeast system, which has provided most of the insight into the molecular mechanism of autophagy and the Cvt pathway, and highlight the most recent additions to our current knowledge of both pathways.

Figure 1

Morphology of autophagy pathways in yeast. Autophagy is the membrane-trafficking pathway that delivers cytoplasmic material to the vacuole for degradation and recycling. Macroautophagy involves the formation of a cytosolic double-membrane vesicle, an autophagosome, which sequesters bulk cytoplasm. Upon completion, autophagosomes fuse with the vacuole membrane releasing a single membrane autophagic body inside the vacuole lumen. The autophagic body is degraded by vacuolar hydrolases. During microautophagy, the sequestration event occurs directly at the vacuole surface. The process also results in a single-membrane vesicle that is ultimately degraded inside the vacuole. Peroxisomes can be selectively taken into the vacuole for degradation by the pexophagy pathway, a specific type of autophagy. Whereas macropexophagy requires the formation of a sequestering vesicle in the cytosol, micropexophagy occurs directly at the vacuole surface.

Figure 2

Schematic model of the steps involved in autophagy. Under starvation conditions a signal transduction event results in the inactivation of Tor kinase and the induction of autophagy. Membrane from an unknown source nonspecifically sequesters cytoplasm. The resulting double-membrane autophagosome targets to and fuses with the lysosome/vacuole. Fusion allows the release of the single-membrane autophagic body that is subsequently degraded in the lysosome/vacuole lumen allowing the recycling of cytoplasmic constituents.

Figure 3

Working models for autophagy and the Cvt pathway in the yeast S. cerevisiae. Both pathways require a membrane nucleation step followed by the formation of double-membrane vesicles. The type of vesicles that are produced depends on the nutrient conditions. Autophagosomes (300 to 900 nm diameter) form during autophagy under conditions of nutrient deprivation. Cvt vesicles (140 to 160 nm diameter) are generated through the Cvt pathway under nutrient-rich conditions. The subsequent fusion of the vesicle with the vacuole membrane results in autophagic bodies or Cvt bodies that are ultimately degraded. The degradation process allows the precursor form of the resident hydrolase aminopeptidase I (prApe1) to be processed into its mature form (mApe1). While the biosynthetic Cvt pathway selectively packages and transports prApe1 under nutrient rich conditions, prApe1 is able to be transported through autophagy during starvation.

Figure 4

The Tor kinase is a key regulatory component that controls induction of autophagy. Tor-mediated control of autophagy may occur through at least 2 different mechanisms: direct phosphorylation of Apg13 and regulation of transcription by controlling the activation of phosphatases that regulate downstream transcriptional activators. As a general example, Tor phosphorylates the regulatory subunit Tap42 allowing it to bind and inhibit the PP2A-related Sit4 phosphatase. Inhibition of Tor allows the dissociation of Tap42 from Sit4; the activated Sit4 can now dephosphorylate and activate a regulatory protein that controls transcription of components that are required for autophagy. In autophagic induction, however, this process may be controlled by a regulatory subunit other than Tap42. Tor also causes the hyperphosphorylation of Apg13, which reduces its affinity for Apg1 and inhibits autophagy.

Figure 5

Schematic models for cargo selection and Cvt vesicle formation. The Cvt pathway selectively transports the vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1) into the yeast vacuole. Precursor Ape1 (prApe1) is synthesized in the cytosol, forms dodecamers, and is further assembled into an Ape1 complex composed of multiple dodecamers. The propeptide region of prApe1 is recognized by its receptor Cvt19, which independently recruits Ams1. This process is termed the cargo selection step for the Cvt pathway. Cvt9 subsequently binds to the complete Cvt complex (Cvt19 bound to cargo) and mediates the tethering step of the Cvt pathway. When the Cvt complex reaches its destination, the perivacuolar preautophagosomal structure (PAS), Cvt19 further interacts with Aut7 accompanied by the release of Cvt9. The Cvt19-Aut7 interaction ensures that the cargo proteins are packaged inside the forming Cvt vesicle.

Figure 6

Two PtdIns 3-kinase complexes. Both complexes contain Vps34, Vps15, and Apg6/Vps30. Complex I also has Apg14 and is used primarily in the Cvt and autophagy pathways. Complex II contains Vps38 and functions in the Vps pathways that target proteins to the vacuole through a portion of the secretory pathway; most resident vacuolar hydrolases transit from the ER to the Golgi complex and are then diverted away from the secretory pathway and targeted to the vacuole, usually via an endosomal intermediate. Modified from Stromhaug and Klionsky (55).

Figure 7

Two conjugation systems and the Apg1 regulatory complex are required for vesicle formation. A: Apg12-Apg5 conjugation. Apg12 is activated by an ubiquitin activating enzyme E1 homologue, Apg7, through the formation of a thioester linkage through a C-terminal glycine of Apg12. The activated Apg12 is then transferred to the E2 enzyme Apg10. Finally, Apg10 transfers Apg12 to Apg5 resulting in the formation of a stable isopeptide bond between Apg12 and an internal lysine residue of Apg5. The Apg12-Apg5 conjugate binds to Apg16. Apg16 undergoes tetramerization resulting in a multi-conjugate complex that is required for autophagosome and Cvt vesicle formation. B: Aut7 lipid conjugation. Prior to Aut7 conjugation, the carboxyl-terminal arginine (R) residue of Aut7 is proteolytically removed by a cysteine protease, Aut2. The exposed glycine (G) residue of Aut7 is activated by the E1-like enzyme Apg7, and subsequently transferred to an E2 enzyme, Aut1. Aut7 is finally conjugated to PE by the formation of an amide bond between PE and Aut7. The resulting Aut7-PE conjugate allows Aut7 to be tightly associated with membrane that is involved in autophagosome and Cvt vesicle formation. A pool of Aut7 is subsequently cleaved from PE by the action of Aut2. C: The Apg1 regulatory complex. The Cvt pathway and autophagy are controlled by nutrient conditions through the Tor pathway. Under nutrient rich conditions, Tor kinase is active, and Apg13 is maintained in a hyperphosphorylated state resulting in a weak association with Apg1 kinase. Apg13 is only required for autophagy and physically associates with Vac8 that is only required for the Cvt pathway. Apg1 kinase also is phosphorylated and physically interacts with the Cvt pathway specific protein Cvt9 and the autophagy specific protein, Apg17. Apg17 associates with 2 additional Cvt specific proteins, Cvt13 and Cvt20, both of which contain PX domains that bind PtdIns(3)P. The phosphorylation state of Cvt13, Cvt20, and Apg17 is not known. Dephosphorylated Apg13 shows enhanced interaction with Apg1 under starvation conditions. Modified from Khalfan and Klionsky (56).

Figure 8

Fusion of Cvt vesicles and autophagosomes with the lysosome/vacuole must be regulated. A: Vesicles that bud from a donor organelle are physically tethered to the organelle and prevented from prematurely fusing with the acceptor compartment. Coat proteins drive the curvature and budding of the vesicle and must be removed prior to the final fusion with the acceptor compartment. B: Cvt vesicles and autophagosomes may not form from a pre-existing organelle and accordingly are free to diffuse in the cytosol. Fusion prior to vesicle completion would not result in delivery of the cargo into the lumen of the acceptor compartment. C: Normally the Cvt vesicle and autophagosome are completed prior to fusion with the lysosome/vacuole. This results in delivery of the cargo into the lumen of the acceptor organelle. It is not known if coat proteins are involved in regulating this process.