In vitro analysis of the ubiquitin - like reactions of autophagy

Two ubiquitin-like conjugation events are essential for autophagy in yeast and mammals. The first involves the conjugation of the ubiquitin-like protein Atg12 to Atg5. The Atg12-Atg5 conjugate then associates with autophagosomal membrane as it elongates, perhaps acting as a coat. In the absence of this first conjugate, the second covalent linkage between the ubiquitin-like protein Atg8 and phosphatidylethanolamine (PE) does not efficiently occur, Atg8 is not properly localized, and autophagy is inhibited. The Atg8-PE conjugation reaction has been reconstituted in vitro with only the addition of its cognate E1and E2-like enzymes, ATP, and PE containing liposomes. Although the E1and E2-like enzymes for the Atg12-Atg5 reaction have been identified, it is not known whether additional components such as an E3-like enzyme are required. The Atg8-PE reaction seems to depend on the Atg12-Atg5 conjugate, but the molecular relationship between these two reactions has not been defined. I postulate that the Atg12-Atg5 conjugate plays a direct role in stimulating the Atg8-PE reaction. I also hypothesize that, in analogy to the Atg8-PE conjugation reaction, the E1and E2-like enzymes, Atg7 and Atg10 respectively, are sufficient to catalyze the Atg12-Atg5 reaction. This proposal seeks to reproduce a biologically active Atg12-Atg5 reaction in vitro and to directly test the influence of the Atg12-Atg5 conjugate on the Atg8-PE reaction using the yeast Saccharomyces cerevisiae. Specific Aims 1. Identify the minimal requirements of the Atg12-Atg5 conjugation reaction. 2. Isolate additional factors that affect the Atg12-Atg5 reaction and investigate the influence of the Atg12-Atg5 conjugation system on the Atg8-PE reaction. 3. Examine the biological activity of the Atg12-Atg5 conjugate produced in Aim 1. Background and Significance Eukaryotic cells possess at least two conserved modes to recycle their components. The ubiquitin-proteasome system is generally considered to be used for the degradation of short-lived and aberrantly folded proteins, where as the vacuole/lysosome breaks down long-lived proteins, protein aggregates, and superfluous organelles (Levine and Klionsky, 2004). The vacuole receives material from the secretory pathway, the endocytic pathway, and the cytoplasm. While delivery via the secretory and endocytic routes use vesicular intermediates that bud from donor compartments, the pathway from the cytoplasm requires a unique mechanism since a topologic reversal, from ‘in’ to ‘out’, is required (Figure 1A). This pathway is generally referred to as autophagy, though variations exist. Chaperone-mediated autophagy, which appears to be limited to mammalian cells, involves the Hsp70-mediated lysosomal targeting of certain soluble polypeptides, their translocation directly across the lysosomal membrane and subsequent digestion (Massey et al., 2004). Microautophagy is characterized by the constitutive invagination of the vacuolar membrane, which then buds into the vacuole lumen, thus engulfing cytoplasm (Muller et al., 2000). The budded membrane and its internal components are then degraded by resident hydrolases. Macroautophagy, hereafter referred to as autophagy, involves the sequestration of cytoplasm in a cup-shaped membrane of unknown origin that fuses with itself to form a doublemembraned vesicle, known as the autophagosome (Figure 1). The outer membrane of the autophagosome then fuses with the vacuole, releasing the inner membrane and its enclosed cytoplasmic components into the vacuolar lumen. The components of these vesicles are then degraded as in microautophagy. These events are summarized in Figure 1A. In yeast, autophagy is stimulated by