RESUMO
Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12-Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.
Assuntos
Autofagia/genética , Proteínas de Membrana/genética , Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Relacionadas à Autofagia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Genoma Fúngico , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Saccharomyces cerevisiae , Deleção de Sequência , VacúolosRESUMO
Macroautophagy (autophagy) is driven by the coordinated actions of core autophagy-related (Atg) proteins. Atg8, the core Atg protein generally considered acting most downstream, has recently been shown to interact with other core Atg proteins via their Atg8-family-interacting motifs (AIMs). However, the extent, functional consequence, and evolutionary conservation of such interactions remain inadequately understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg38, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex I, interacts with Atg8 via an AIM, which is highly conserved in Atg38 proteins of fission yeast species, but not conserved in Atg38 proteins of other species. This interaction recruits Atg38 to Atg8 on the phagophore assembly site (PAS) and consequently enhances PAS accumulation of the PtdIns3K complex I and Atg proteins acting downstream of the PtdIns3K complex I, including Atg8. The disruption of the Atg38-Atg8 interaction leads to the reduction of autophagosome size and autophagic flux. Remarkably, the loss of this interaction can be compensated by an artificial Atg14-Atg8 interaction. Our findings demonstrate that the Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop between Atg8 and the PtdIns3K complex I to promote efficient autophagosome formation, underscore the prevalence and diversity of AIM-mediated connections within the autophagic machinery, and reveal unforeseen flexibility of such connections. Abbreviations: AIM: Atg8-family-interacting motif; AP-MS: affinity purification coupled with mass spectrometry; Atg: autophagy-related; FLIP: fluorescence loss in photobleaching; PAS: phagophore assembly site; PB: piggyBac; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/fisiologia , Fagossomos/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , LevedurasRESUMO
Spore killers in fungi are selfish genetic elements that distort Mendelian segregation in their favor. It remains unclear how many species harbor them and how diverse their mechanisms are. Here, we discover two spore killers from a natural isolate of the fission yeast Schizosaccharomyces pombe. Both killers belong to the previously uncharacterized wtf gene family with 25 members in the reference genome. These two killers act in strain-background-independent and genome-location-independent manners to perturb the maturation of spores not inheriting them. Spores carrying one killer are protected from its killing effect but not that of the other killer. The killing and protecting activities can be uncoupled by mutation. The numbers and sequences of wtf genes vary considerably between S. pombe isolates, indicating rapid divergence. We propose that wtf genes contribute to the extensive intraspecific reproductive isolation in S. pombe, and represent ideal models for understanding how segregation-distorting elements act and evolve.