Cvt9/Gsa9 Functions in Sequestering Selective Cytosolic Cargo Destined for the Vacuole

Abstract

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

Macroautophagy versus mitochondrial autophagy: a question of fate?

“…34 A number of genes involved in autophagy and/or the cvt pathway, including Atg1, Atg11 and Atg17, have been implicated in regulation of pexophagy in H. polymorpha and in Saccharomyces cerevisiae, suggesting that there is overlap between the three pathways. 28,[33][34][35] In addition to activation of the autophagy machinery, a key determinant of pexophagy appears to be localized on the peroxisome membrane. Pex14, which is conserved from yeast to humans, has been shown to be a prime target for macropexophagy in H. polymorpha.…”

Atg27 Is Required for Autophagy-dependent Cycling of Atg9

“…The protein extracts were resolved by SDS-PAGE and probed with anti-GFP mAb (Covance Research Products, Berkeley, CA). The alkaline phosphatase assay to measure Ph 8⌬60 activity, Pex14-GFP processing to monitor pexophagy, the protease protection assay, and the subcellular fractionation have been described previously (Harding et al, 1995;Noda et al, 1995;Kim et al, 2001b;Scott et al, 2001;Tucker et al, 2003;Reggiori et al, 2005a). The sucrose density gradient used to fractionate Atg27 was generated as described previously (Reggiori et al, 2005b) with minor modifications (1 ml each of 18, 22, 26, 30, 34, 38, 42, 46, 50, and 54% sucrose).…”

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

“…Using this approach, we have isolated R2, R12, R13, and R22 mutants. The R2 (his4 Ppatg18 -1::zeo R ) and R13 (his4 Ppatg11-1::zeo R ) mutants have been described elsewhere Kim et al, 2001;). The R12 mutants had the pREMI-Z inserted into the PpATG1 gene loci.…”

“…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.…”