Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior tode Novo Vesicle Formation

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2002; 277(1):763-73. DOI: 10.1074/jbc.M109134200
Source: PubMed


Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.

Download full-text


Available from: Per E Strømhaug, Jun 29, 2015
  • Source
    • "The PCR fragments were cloned into the pRS416 vector (Sikorski and Hieter, 1989) using HindIII and KpnI before inserting the GAL1 promoter using XhoI and HindIII. The pCuGFPATG8414 and pCuGFPATG8416 plasmids expressing GFP-Atg8 under the control of the CUP1 promoter have been described elsewhere (Kim et al., 2002). To create the integrative pCFPATG8406 plasmid that leads to the expression of the CFP-Atg8 fusion protein from the authentic ATG8 promoter, the backbone of the pRS314 ECFP-AUT7 plasmid (Suzuki et al., 2001) was exchanged for that of the pRS406 vector (Sikorski and Hieter, 1989) using XhoI and SacII. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a β-propeller formed by 7 WD40 repeats, which contains a conserved FRRG motif that binds phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. It is unknown, however, how Atg18 association with these organelles is regulated as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds Atg2 through a specific stretch of amino acids in the β-propeller on the opposite surface from the FRRG motif. As in absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model, where the Atg18 β-propeller provides organelle specificity by binding two determinants on the target membrane.
    Full-text · Article · Dec 2012 · Journal of Cell Science
  • Source
    • "An immunoprecipitation analysis showed that Atg19 physically interacts with the propeptide of prApe1, and the coiled-coil domain of Atg19 mediates this interaction [48]. Atg19 localizes at the PAS with the Ape1 complex [49]; the combination of the Ape1 complex bound to Atg19 is referred to as the Cvt complex. In atg19Δ cells, GFP-Ape1 forms a dodecamer, but it does not localize at the PAS. "
    [Show abstract] [Hide abstract]
    ABSTRACT: From today's perspective, it is obvious that macroautophagy (hereafter autophagy) is an important pathway that is connected to a range of developmental and physiological processes. This viewpoint, however, is relatively recent, coinciding with the molecular identification of autophagy-related (Atg) components that function as the protein machinery that drives the dynamic membrane events of autophagy. It may be difficult, especially for scientists new to this area of research, to appreciate that the field of autophagy long existed as a "backwater" topic that attracted little interest or attention. Paralleling the development of the autophagy field was the identification and analysis of the cytoplasm-to-vacuole targeting (Cvt) pathway, the only characterized biosynthetic route that utilizes the Atg proteins. Here, we relate some of the initial history, including some never-before-revealed facts, of the analysis of the Cvt pathway and the convergence of those studies with autophagy.
    Full-text · Article · Feb 2012 · International Journal of Cell Biology
  • Source
    • "Direct binding to Atg8/LC3 allows the cargos to be selectively enclosed by autophagosomes. In yeast, Ape1, Ams1, and Atg19 are present on the preautophagosomal struc­ ture (PAS), where most Atg proteins gather and autophagosome are generated (Suzuki et al., 2001; Kim et al., 2002; Chang and Huang, 2007). Importantly, localization of the Cvt complex to the PAS requires Atg19 but does not absolutely depend on other Atg proteins including Atg8, suggesting that cargo re­ cruitment can be achieved independently of the Atg8–cargo interaction (Cao et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.
    Full-text · Article · Jan 2011 · The Journal of Cell Biology
Show more