Atg8 is Essential for Macropexophagy in Hansenula polymorpha

Abstract

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogenlimitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.Key words: autophagy, methylotrophic yeast, peroxisome degradation, sequestration Received 28 September 2004, revised and accepted for publication 21 October 2004Proper cellular homeostasis involves the efficient re-use of cell components that have been hydrolyzed by the protein degradation machinery. In yeast, two major protein degradation machineries are known -the proteasome and vacuolar hydrolysis. The vacuole contains various hydrolytic enzymes involved in the degradation of cellular constituents.Degradation processes via the vacuole are termed autophagic processes. In yeast, several autophagic processes have been described. Non-selective autophagy is a process characterized by the bulk turnover of portions of the cytoplasm (cytosol plus organelles, and including peroxisomes) (1). However, degradation of yeast peroxisomes may also occur via a strictly selective autophagic process, termed macropexophagy (2,3). The methylotrophic yeast Hansenula polymorpha is an attractive model organism to study peroxisome degradation because the proliferation and degradation of these organelles can be readily prescribed by manipulation of the growth conditions. In H. polymorpha, macropexophagy is induced by exposing methanol-grown cells to new growth environments in which the organelles are redundant for growth (3). However, when the cells are transferred to nitrogen-starvation conditions, microautophagy is induced in this yeast (4). Molecular studies to unravel the principles of pexophagy in H. polymorpha revealed that both macropexophagy and autophagy share many components with other protein/ organelle sorting pathways to the vacuole, e.g. endocytosis, and the Vps and Cvt pathways (3). To circumvent confusion in protein nomenclature, a unified nomenclature has recently been introduced for genes involved in AuTophaGy-related processes (ATG genes) (5).In this study, we describe the isolation and characterization of the H. polymorpha ATG8 gene (previously designated AUT7). We show that a GFP.Atg8 fusion protein is observed as a peri-vacuolar fluoresc...

Functional Analysis of the ATG8 Homologue Ao atg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

“…S. cerevisiae Atg8 is an essential protein in the autophagic pathway, and orthologues of Atg8 in other organisms have been used as specific markers of autophagy (8,19,27,35). In order to isolate the A. oryzae ATG8 gene homologue, we searched the A. oryzae EST and genome databases using the BLAST algorithm.…”

Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance inSaccharomyces cerevisiae

“…Plasmid pUG34DsRed.SKL enables P MET25 -driven expression of DsRed.SKL gene (Monastyrska et al, 2005). For the construction of pUG34DsRed.SKL, a 720 bp BamHI (blunt ended by Klenow treatment)-SalI fragment containing the DsRed.SKL gene, was isolated from plasmid pHIPZ4-DsRed-T1.SKL and inserted between the XbaI (after Klenow treatment) and SalI sites of pUG34, thereby replacing the GFP gene.…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…501,505 The decrease in the level of endogenous proteins such as alcohol oxidase or Pot1 can be followed by western blotting, 412,506-509 TEM, 510 fluorescence microscopy 412,511,512 or laser confocal scanning microscopy of GFP-labeled peroxisomes. 513,514 In yeast, nonspecific autophagy can be induced by nitrogen starvation conditions, whereas degradative types of selective autophagy generally require a carbon source change or ER stress for efficient induction. For example, to induce a substantial level of mitophagy, cells need to be precultured in a nonfermentable carbon source such as lactate or glycerol to stimulate the proliferation of mitochondria.…”