The beginning of the end: how scaffolds nucleate autophagosome biogenesis

Abstract

Autophagy is a conserved mechanism that is essential for cell survival in starvation. Moreover, autophagy maintains cellular health by clearing unneeded or harmful materials from cells. Autophagy proceeds by the engulfment of bulk cytosol and organelles by a cup-shaped double-membrane sheet known as the phagophore. The phagophore closes on itself to form the autophagosome, which delivers its contents to the vacuole or lysosome for degradation. A multiprotein complex comprising the protein kinase autophagy-related protein 1 (Atg1) together with Atg13, Atg17, Atg29, and Atg31 (ULK1, ATG13, FIP200, and ATG101 in humans) has a pivotal role in the earliest steps of this process. This review summarizes recent structural and ultrastructural analysis of the earliest step in autophagosome biogenesis and discusses a model in which the Atg1 complex clusters high-curvature vesicles containing the integral membrane protein Atg9, thereby initiating the phagophore.

Keywords: Atg1; Atg13; Atg9; SNAREs; ULK1; autophagy; membrane bending; vesicle tethering.

Figure 1

Autophagy and the Cvt pathway in yeast. A. Starvation-induced bulk autophagy. Autophagosome biogenesis begins with a pool containing an estimated three Atg9-positive vesicles which may be as small as 20 nm in diameter. These vesicles fuse into a double-membrane sheet, which becomes the phagophore. The phagophore grows by addition of membrane and adopts the shape of a cup. The cup engulfs mitochondria, peroxisomes, ribosomes, inclusions, and bulk cytosol. The opening of the phagocytic cup eventually fuses and the structure is now the autophagosome, which in yeast is 300-900 nm in diameter. The autophagosome then fuses with the vacuole (lysosome in mammalian cells) such that the inner membrane and all of the contents are hydrolyzed. B. The Cvt pathway, a constitutive transport process in yeast with analogies to mammalian selective autophagy. The Cvt pathway begins with the fusion of Atg9 vesicles, as in (A). The main difference in this pathway is that the size and shape of the phagocytic cup are dictated by the size and shape of the substrate being engulfed, which is usually a single entity. Here a particle of the vacuolar peptidase precursor prApe1 is shown.

Figure 2

The preautophagosomal structure. A. Fluorescence micrograph of the PAS. B. Schematic for the genetic hierarchy of Atg protein recruitment to the PAS.

Figure 3

Biogenesis and coalescence of Atg9 vesicles. A. Electron micrograph of Atg9 reservoir (arrow; reproduced by permission from [19]). B. Schematic of the trafficking of Atg9 vesicles from the TGN to the Atg9 reservoir, and onward to the PAS. Atg9 emerges from the TGN in tandem with Atg23 and Atg27, a peripheral and single-crossing integral membrane protein, respectively. Atg9 vesicles emerging from the TGN are sorted to a cytosolic reservoir near the mitochondrion in a process depending on the Rab/RabGEF pair Sec2 and Sec4, the SNAREs Ykt6, Sso1/2, and Sec9, and the exocyst complex. Once at the reservoir, the Atg9 vesicles acquire another Rab protein, Ypt1, and its multimeric GEF, the TRAPPIII complex. C. Coalescence of Atg9 vesicles is highlighted most dramatically adjoining a preApe1 particle in atg13Δ cells (reproduced by permission from [19]). V, vacuole, M, mitochondrion, MVB, multivesicular body. Scale bars are 200 nm.

Figure 4

The Atg1 complex and a model for vesicle tethering. A. Crystal structure of the autoinhibited Atg17-Atg31-Atg29 complex [30]. B-E. A hypothesis for the tethering, and fusion of Atg9 vesicles by the Atg1 complex and SNAREs. For clarity Atg13 is not depicted in the cartoon B. In uninduced conditions, the Atg17-Atg31-Atg29 subcomplex resides at the PAS but does not scaffold because the Atg29-Atg31 module sterically blocks the concave surface of Atg17. C. Following autophagy induction, Atg9 vesicles migrate to the PAS, where the binding of Atg9 to Atg17 competitively displaces Atg29-Atg31. Alternatively, some other activation signal might disinhibit Atg17, leading to the subsequent binding of Atg9 vesicles to the double crescent. In this state, Atg9 vesicles are scaffolded in proximity to one another, but are not tethered tightly enough for membrane fusion. D. Atg13 is recruited to the PAS following its dephosphorylation, with the recruitment of Atg1 occurring simultaneously or subsequently. The EAT domain of Atg1 tightly tethers the two vesicles in conjunction with their preexisting scaffolding by Atg17. In the final step in this speculative model, (E), SNAREs form appropriate pairings across the gap between the two vesicles and drive their homotypic fusion.

Figure 5

From initiation to extension. A speculative model for the possible continuing roles of the initiation machinery during phagophore extension. A. A recap of the initial tethering events depicted in Fig. 4(B-D). B. Following Atg9 vesicle fusion, the phagophore begins as a small membrane sheet, visualized in two-dimensional projection as a tube. C. Atg9 vesicles are estimated to provide less than 1 % of the ultimate membrane content of the autophagosome. The source and physical state of the bulk of the membrane remains a topic of controversy. It is noteworthy that the Atg1 EAT domain has no apparent specificity for different lipid compositions, sensing only high curvature. Moreover, membrane-inserting curvature sensors can bind well to loosely packed neutral lipid mixtures, such as those of the ER [12], which is widely considered the major source of lipid supply in autophagosome biogenesis. D. The Atg1 complex, via Atg13, recruits the PI 3-kinase complex, although not necessarily through a direct interaction [51]. After Atg8 conjugation begins, some of the Atg1 subunit appears to dissociate from the PAS [52] and localize to the phagophore surface via its AIM [35, 53, 54]. E. PI(3)P generated by the PI 3-kinase complex diffuses over the phagophore surface, and together with Atg9, recruits the Atg2-Atg18 complex. The Atg2-Atg18 complex, together with Atg1, somehow mediates recycling of Atg9. F. Upon autophagosome closure, the high curvature rim of the phagophore disappears, remove the binding sites for curvature-sensing proteins such as Atg17. The dissociation of these proteins might be sensed by a checkpoint for autophagosome maturation, signaling readiness for fusion with the vacuole.