Cvt18/Gsa12 Is Required for Cytoplasm-to-Vacuole Transport, Pexophagy, and Autophagy inSaccharomyces cerevisiaeandPichia pastoris

Abstract

Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro-and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.

PpATG9 Encodes a Novel Membrane Protein That Traffics to Vacuolar Membranes, Which Sequester Peroxisomes during Pexophagy inPichia pastoris

“…However, when R19 cells were adapted to glucose, the sequestering membranes appear to be absent, but instead the vacuole appeared only slightly indented ( Figure 2E), with an occasional short armlike projection ( Figure 2I). Similar vacuole morphology has been reported for Ppatg11, Ppatg18, and Ppvps15 mutants (Stasyk et al, 1999;Guan et al, 2001;Kim et al, 2001). Next, we examined whether the MIPA was forming in R19 cells.…”

“…On homotypic membrane fusion of the arms, the peroxisomes are incorporated into autophagic bodies within the vacuole where they are degraded. We have previously shown that PpAtg2 (Gsa11), PpAtg7 (Gsa7), PpAtg11 (Gsa9), and PpAtg18 (Gsa12) are required for the sequestration events of micropexophagy (Yuan et al, 1999;Guan et al, 2001;Kim et al, 2001;. In this study, we show that PpAtg9 is required for an early sequestration event in micropexophagy, possibly the initial formation of the sequestration membranes that extend from the vacuole.…”

“…However, no loss of AOX was detected in R19 cells over 24 h, suggesting that macropexophagy was suppressed in the cells lacking PpAtg9. Our previous studies have shown that a number of those REMI mutants defective in pexophagy were also defective in nitrogen starvation-enhanced proteolysis (Yuan et al, 1999;Guan et al, 2001;. Therefore, we next quantified protein degradation in R19 starved for amino acids ( Figure 1C).…”

“…Despite their differences, these pathways share a number of common molecular events. For example, Atg7/Gsa7/Apg7, Atg1/Gsa10/Apg1/Aut3, Atg2/Gsa11/Apg2, Atg18/Gsa12/Cvt18, and Vps15 are required for pexophagy, autophagy, and CVT pathways, whereas Atg11/Gsa9/Cvt9 and Vac8 are essential for pexophagy and CVT pathways, but not autophagy (Stasyk et al, 1999;Yuan et al, 1999;Scott et al, 2000;Guan et al, 2001;Kim et al, 2001;Strømhaug and Klionsky, 2001;Wang et al, 2001;Huang and Klionsky, 2002;Abeliovich et al, 2003). Because many autophagy genes and their orthologues have been identified using different yeast models, the nomenclature for these genes had become confusing.…”

“…To better understand the functions of these proteins, we examined the vacuole morphology upon glucose-induced pexophagy. For example, PpVps15 (Paz13), PpAtg11 (Gsa9), and PpAtg18 (Gsa12) appear to act at early sequestration events (Stasyk et al, 1999;Guan et al, 2001;Mukaiyama et al, 2002), PpAtg2 (Gsa11) and PpAtg7 (Gsa7/Paz12) at intermediate sequestration events (Yuan et al, 1999;, and PpAtg1 (Paz1/Gsa10) at late sequestration events (Mukaiyama et al, 2002). Many of these proteins are structurally and functionally homologous to those proteins essential for autophagy and CVT pathways in Saccharomyces cerevisiae (Habibzadegah-Tari and Dunn, 2003;.…”