Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy

Abstract

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.utophagy is a catabolic process in which damaged or superfluous cytoplasmic components are degraded in response to stress conditions; it is evolutionarily conserved in eukaryotes and is integrally involved in development and physiology (1, 2). The morphological hallmark of autophagy is the formation of doublemembrane cytosolic vesicles, autophagosomes, which sequester cytoplasm. The autophagosomes then fuse with the lysosome, resulting in the degradation of the cargo. The mechanism of autophagosome formation is distinct from that used for vesicle formation in the secretory or endocytic pathways and is said to be de novo in that it does not occur by direct budding from a preexisting organelle. Instead, a nucleating structure, the phagophore, appears to expand by the addition of membrane possibly through vesicular fusion. One consequence of this mechanism is that it allows the sequestration of essentially any sized cargo, including intact organelles or invasive microbes, and this capability is critical to autophagic function. When autophagy is induced there is a substantial demand for membrane, and a major question in the field concerns the membrane origin; nearly every organelle has been implicated in this role (3). The early secretory pathway is likely one such membrane source for autophagy (4, 5).Rab GTPases are key regulators of membrane traffic that mediate multiple events including vesicle tethering and membrane fusion. These molecular switches cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The yeast Rab Ypt1, which is essential for ER-Golgi and Golgi traffic (6), is activated by the multimeric guanine nucleotide exchange factor (GEF) called TRAPP (7,8). Two forms of the TRAPP complexes have been identified (9). These two complexes share several subunits, including four (Bet3, Bet5, Trs23, and Trs31) that are essential to activate Ypt1. How each of th...

Regulation of selective autophagy onset by a Ypt/Rab GTPase module

“…Therefore, overexpression of Ypt1 is expected to suppress the cargo-processing defect in cells in which its upstream regulator, Trs85, is deleted but not in cells in which its downstream effector, Atg11, is deleted. Suppression of the Ape1-processing phenotype of trs85Δ was shown previously when the GTP-restricted form, but not wild-type Ypt1, was expressed from the GAL1 promoter (17). We observed that overexpression of Ypt1 from its own promoter suppresses the Ape1-processing defect of ypt1-1 and trs85Δ but not that of atg11Δ (Fig.…”

“…GFP-Ypt1 localizes to multiple puncta per cell (26). Previously published studies of intracellular localization of Trs85 tagged with GFP or 3×GFP were inconclusive (17,27). We tagged endogenous Trs85 with yeast-optimized EGFP and demonstrated that it is functional and localizes to multiple puncta per cell (Fig.…”

“…Cells carrying the ypt1-1 mutation, T40K, in the effector-binding domain of Ypt1 are defective in autophagy (15,17). Therefore, we tested whether the Ypt1-1 mutant protein is defective in the interaction with Atg11 using the three interaction assays mentioned above: yeast-two hybrid, coprecipitation with Atg11-HA from yeast lysates and coprecipitation with bacterially expressed His-Atg11-CC2-3 ( Fig.…”

“…Ypt1 and its mammalian homolog Rab1 play a role in autophagy (15,16), and Trs85, in the context of the TRAPP III complex, can act as a Ypt1 GEF in this process (17). However, the molecular mechanism by which Ypt1 and Rab1 regulate autophagy is unknown, and it is not clear whether it is dependent on their well-documented function in ER-toGolgi transport.…”

A current perspective of autophagosome biogenesis

“…Immunoelectron microscopy analysis in another study detected Atg9 in clusters of small vesicles and tubules, and it was proposed that the isolation membrane may be initiated by the fusion of these clustered vesicles/tubules [89]. The Atg9 vesicle fusion mechanism may involve the yeast Rab protein Ypt1 and its GEF complex (the TRAPP III complex), which are required for autophagy and were identified in purified Atg9 vesicles [90,91]. In addition, SNARE proteins required for autophagosome formation in yeast have also been implicated in Atg9 vesicle fusion [92].…”