Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Abstract

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration. INTRODUCTIONMacroautophagy (hereafter autophagy) is a conservative eukaryotic pathway for the degradation of intracellular material in the lysosome/vacuole via its sequestration by doublemembrane vesicles called autophagosomes. Autophagy has been implicated in many physiological processes and human diseases (Huang and Klionsky, 2007;Rubinsztein et al., 2007;Mizushima et al., 2008). It is mainly a nonselective process sequestering a random portion of cytoplasm under starvation conditions. However, under certain conditions protein complexes, organelles and intracellular pathogens are selectively removed from the cytosol by specialized autophagy-related pathways. For example, the cytoplasm-tovacuole targeting (Cvt) pathway selectively delivers certain vacuolar resident hydrolases under vegetative conditions (Stromhaug and Klionsky, 2003;Farre et al., 2007), macropexophagy (hereafter pexophagy) ensures selective turnover of peroxisomes under peroxisome repression conditions (Dunn et al., 2005;Monastyrska and Klionsky, 2006;Sakai et al., 2006), and xenophagy selectively disposes microbes that invade cells during infection (Levine and Deretic, 2007;Schmid and Munz, 2007).Irrespective of the material being degraded by autophagyrelated pathways, multiple steps are required to sequester it from the cytosol in double-membrane vesicles. These include induction of the particular pathway, cargo selection and packaging, nucleation of vesicle formation, vesicle expansion and completion, retrieval of autophagic machinery components, fusion of completed vesicles with the lysosome/vacuole, breakdown of the single-membrane vesicle and its cargo, and transport of liberated amino acids and lipids to the cytosol for reuse (Klionsky, 2005;. To date, 32 autophagy-related (Atg) protein...

The machinery of macroautophagy

“…Atg36 and PpAtg30 are receptors for pexophagy in S. cerevisiae and P. pastoris, respectively [143,144]. In both yeast and mammalian cells Pex3 and Pex14 function as cargo ligands [145,146].…”

The role of ALFY in selective autophagy

“…The best described adaptor protein is yeast Atg11, involved in the Cvt pathway, 43 mitophagy 28,44 and pexophagy. 45 The Cvt pathway is a biosynthetic process involving transport of some vacuolar hydrolases (e.g. the precursor aminopeptidase 1 (prApe1) complex) from the cytosol to the vacuole.…”

“…46 Interestingly, increased Atg11 expression facilitates recruitment of Atg8 and Atg9 to the PAS and formation of more Cvt vesicles, suggesting that Atg11 can regulate the rate of selective autophagy. 10 Atg11 interacts also with the autophagy receptors Atg30 (pexophagy) 45 and Atg32 (mitophagy) 28,44 and likely has a similar role in selective autophagy of these cargo, but it is not required for non-selective starvation-induced autophagy.…”

Atg35, a micropexophagy-specific protein that regulates micropexophagic apparatus formation inPichia pastoris

“…Recently, we reported that yeasts from the order Saccharomycetales have either one large Atg28 protein or two smaller proteins, Atg29 and Atg31. 12 Here, we analyzed the genomes of the same eight yeasts and found that all contain a protein with the N-terminal RING and PHD fingers, either a longer Atg35 or a shorter Asr1 (see Fig. S1 in Sup.…”