Trs85 (Gsg1), a Component of the TRAPP Complexes, Is Required for the Organization of the Preautophagosomal Structure during Selective Autophagy via the Cvt Pathway

Abstract

Autophagosomes and Cvt vesicles are limited by two membrane layers. The biogenesis of these unconventional vesicles and the origin of their membranes are hardly understood. Here we identify in Saccharomyces cerevisiae Trs85, a nonessential component of the TRAPP complexes, to be required for the biogenesis of Cvt vesicles. The TRAPP complexes function in endoplasmic reticulum-toGolgi and Golgi trafficking. Growing trs85⌬ cells show a defect in the organization of the preautophagosomal structure. Although proaminopeptidase I is normally recruited to the preautophagosomal structure, the recruitment of green fluorescent protein-Atg8 depends on Trs85. Autophagy proceeds in the absence of Trs85, albeit at a reduced rate. Our electron microscopic analysis demonstrated that the reduced autophagic rate of trs85⌬ cells does not result from a reduced size of the autophagosomes. Growing and starved cells lacking Trs85 did not show defects in vacuolar biogenesis; mature vacuolar proteinase B and carboxypeptidase Y were present. Also vacuolar acidification was normal in these cells. It is known that mutations impairing the integrity of the ER or Golgi block both autophagy and the Cvt pathway. But the phenotypes of trs85⌬ cells show striking differences to those seen in mutants with defects in the early secretory pathway. This suggests that Trs85 might play a direct role in the Cvt pathway and autophagy.Starvation-induced autophagy is an unselective, degradative pathway that delivers cytosolic material to the lysosome (vacuole) (1-3). It is well conserved between eukaryotes such as fungi, plants, and mammals. During the last decade work initially using the model eukaryote Saccharomyces cerevisiae led to the identification of a set of more than 20 ATG genes essential for the autophagic process (4 -7). Studies on the mammalian counterparts of the yeast ATG genes uncovered the importance of autophagy in the development of severe diseases such as cancer, cardiomyopathy, Huntington and Parkinson disease (8). Autophagy also plays an important role in the removal of intracellular pathogens (9), and increasing evidence points to a relationship between autophagy and aging (10,11).Autophagy starts at the preautophagosomal structure (PAS), 2 a perivacuolar organelle where numerous Atg proteins colocalize (12, 13). Out of the PAS double membrane-layered transport vesicles, the autophagosomes are formed (14, 15). The outer membrane of the autophagosome then fuses with the vacuolar membrane, and the inner part of the vesicle is released as a still membrane-limited autophagic body into the vacuole. Within the vacuole the autophagic bodies are lysed dependent on the putative lipase Atg15 (16), and the cytosolic material is degraded by the various vacuolar hydrolases. The use of transport vesicles limited by two membrane layers distinguishes autophagy from other transport pathways. Consistently, the molecular mechanisms used for the biogenesis of these vesicles are also unconventional. For example, the homotypic membrane fusion event during t...

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

“…The finding that Trs85 is required for autophagy in yeast (15,16) raised the possibility that there may be a separate pool of Trs85 that functions in autophagy-related processes. To begin to address this possibility, we fractionated cytosol on a Superdex-200 column and blotted the column fractions for Trs85, Trs65, and Trs33.…”

“…Previous studies demonstrate that Trs85 is required for the cytoplasm to vacuole targeting (Cvt) pathway, a specific type of autophagy, and macroautophagy, the nonspecific autophagy of cytoplasm (15,16). These earlier studies did not resolve if a free pool of Trs85 or TRAPP was required for the Cvt pathway and autophagy.…”

Identification of autophagy genes participating in zinc-induced necrotic cell death inSaccharomyces cerevisiae

“…32 One of the enzymes brought there in this way is aminopeptidase I, the product of the gene APE1. We transformed yeast with plasmids encoding an Ape1-RFP fusion protein 33 and used it to monitor ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

“…The plasmids used in this paper are listed in Table 2. The plasmid carrying the GFP-ATG8 fused open reading frame was a generous gift from Dr. Y. Ohsumi, 59 APE1-RFP was kindly provided by Dr. M. Thumm, 33 and ROSELLA constructs were graciously donated by Dr. R. Devenish. 29 Each was transformed into appropriate yeast strains using standard protocols.…”

Mitochondria Autophagy in Yeast

“…Notably, > 40% of the genes identified from both screens are membrane trafficking-related genes (20 among the 45 total genes). The requirement of most of these genes for macroautophagy and/or the Cvt pathway has been previously reported, and it is widely thought that defects in membrane trafficking pathways affect the lipid supply that is needed for extension of the phagophore, the initial sequestering compartment that generates the autophagosome (21,31,49,50,57,59,66,75,81,94,95,97,100). Accordingly, it is reasonable-and partly a verification of the success of the screens-that both groups identified a certain number of membrane traffickingrelated genes.…”