Abstract
ABSTRACTAutophagy is a conserved cellular degradation pathway wherein double-membrane vesicles called autophagosomes capture long-lived proteins, and damaged or superfluous organelles, and deliver them to the lysosome for degradation. Septins are conserved GTP-binding proteins involved in many cellular processes, including phagocytosis and the autophagy of intracellular bacteria, but no role in general autophagy was known. In budding yeast, septins polymerize into ring-shaped arrays of filaments required for cytokinesis. In an unbiased genetic screen and in subsequent targeted analysis, we found autophagy defects in septin mutants. Upon autophagy induction, pre-assembled septin complexes relocalized to the pre-autophagosomal structure (PAS) where they formed non-canonical septin rings at PAS. Septins also colocalized with autophagosomes, where they physically interacted with the autophagy proteins Atg8 and Atg9. When autophagosome degradation was blocked in septin-mutant cells, fewer autophagic structures accumulated, and an autophagy mutant defective in early stages of autophagosome biogenesis (atg1Δ), displayed decreased septin localization to the PAS. Our findings support a role for septins in the early stages of budding yeast autophagy, during autophagosome formation.This article has an associated First Person interview with the first author of the paper.
Highlights
Macroautophagy is an evolutionarily conserved intracellular waste disposal and recycling process that is critical for normal cellular and organismal homeostasis
Autophagy defects in septin mutants To identify autophagy defects in viable mutant yeast strains, we introduced into a collection of temperature-sensitive (Ts−) mutants in a POT1-GFP strain, which expresses a marker of pexophagy (Kondo-Okamoto et al, 2012), a specialized form of autophagy in which peroxisomes are degraded (Oku and Sakai, 2016)
Unlike in wild-type (WT) cells, where free GFP accumulated at both 22°C and 37°C, in cells expressing any of several Ts− mutant alleles of the septin CDC10 (G100E or P3S G44D) or CDC11 (G29E, G34D or S31F S100P) more free GFP was detected at 22°C, compared to what was seen at 37°C, and the Pot1–GFP fusion remained intact at 37°C (Fig. 1A; Fig. S1A)
Summary
Macroautophagy ( autophagy) is an evolutionarily conserved intracellular waste disposal and recycling process that is critical for normal cellular and organismal homeostasis. Autophagy involves the formation of double-membrane vesicles called autophagosomes that engulf intracellular material destined for degradation. The site of autophagosome formation is known as the pre-autophagosomal structure (PAS) and is perivacuolarly located. Recent work has shown that the PAS is tethered to endoplasmic reticulum (ER) exit sites where multiple autophagy proteins colocalize in a hierarchical sequence (Graef et al, 2013; Suzuki et al, 2007). The membrane source for the developing autophagosome is contributed by the trafficking of Atg along with its transport complex (Atg1–Atg11–Atg13–Atg23– Atg27–Atg2–Atg18–TRAPIII) to help build the initial cup-shaped structure, the phagophore (Legakis et al, 2007; Reggiori et al, 2004; Tucker et al, 2003). Additional recruitment of the Atg5–Atg12– Atg complex as well as Atg allows the completion of the autophagosome (Feng et al, 2014)
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