Abstract
In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.
Highlights
In eukaryotes, macroautophagy is a highly conserved degradation process by which cytoplasmic components—such as organelles—are non-selectively engulfed by double-membrane vesicles called autophagosomes
The amplified cDNA encodes a protein of 159 amino acids with a conserved C-terminal glycine that shares a significant level of sequence similarity with ATG12 proteins of plants, animals that have a conserved glycine at the C-terminus (Fig 1)
Most of the residues involved in non-covalent interactions between ATG12 and ATG5, binding and conjugation of ATG3, or binding of ATG8 in the S. cerevisiae and the human ATG12 homologs are conserved in the S. macrospora ATG12 (Fig 1) [24,27,64,65,66]
Summary
Macroautophagy (hereafter autophagy) is a highly conserved degradation process by which cytoplasmic components—such as organelles—are non-selectively engulfed by double-membrane vesicles called autophagosomes. Autophagy-Associated Protein SmATG12 of Sordaria macrospora autophagosome are released into the lumen of the vacuole. These vesicles or autophagic bodies are degraded by hydrolytic enzymes into building blocks that are recycled and released back into the cytoplasm [1]. The autophagic process can be divided into five steps: induction, nucleation, elongation and closure, fusion with the vacuole, and breakdown of autophagic bodies. This process has been studied intensively in the unicellular yeast Saccharomyces cerevisiae. Eight proteins are involved in two ubiquitin-like (UBL) conjugation systems that are essential for autophagosome formation: the conjugation of the UBL protein ATG8 to the lipid phosphatidylethanolamine (PE) and the conjugation of the UBL protein ATG12 to ATG5 [14]
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