Autophagy-deficient Schizosaccharomyces pombe mutants undergo partial sporulation during nitrogen starvation

Abstract

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome-vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagydefective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1D and atg12D mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation. INTRODUCTIONAutophagy is a degradative pathway conserved among eukaryotic cells, and is responsible for turnover of damaged organelles and long-lived proteins. When living organisms are exposed to radical environmental changes such as nutrient starvation, differentiation or development, autophagy is rapidly induced and inner cell components are reorganized. In the budding yeast Saccharomyces cerevisiae, autophagy is triggered by starvation for nitrogen or carbon, after which endogenous proteins and organelles are enclosed within isolated membranes called autophagosomes. The outer membrane of the autophagosome then fuses with the vacuole, allowing the contents of the autophagosome, designated autophagic bodies, to be released and degraded in the vacuole. The molecular mechanisms involved in the process of autophagy have been subjected to detailed genetic analysis and more than 20 AuTophaGy-related (ATG) genes essential for autophagy have been identified in Sac. cerevisiae Kamada et al., 2000;Mizushima et al., 1998).One of the most striking findings with respect to function of the Atg proteins was the discovery of two ubiquitin-like conjugation systems involving Atg12p and Atg8p (Ohsumi, 2001). Atg12p, which has no apparent homology to ubiquitin, covalently attaches to Atg5p in a manner analogous to ubiquitination, and then forms a complex with Atg16p. Atg8p also lacks similarity to ubiquitin, but Abbreviations: DAPI, 49,6-diamidino-2-phenylindole; DIC, differential interference contrast; FSM, forespore membrane.Two supplementary tables a...

Fission yeast Ryh1 GTPase activates TOR Complex 2 in response to glucose

“…1B, bottom). 46,47 These results strongly suggest that TORC2 remains active in fission yeast cells starved of nitrogen, consistent with the observation that the activity of TORC2 and Gad8 is essential for the cell cycle arrest and sexual development after nitrogen starvation. [14][15][16]19,48 The TORC2-Gad8 pathway is responsive to glucose It has been reported that the mitotic control under low glucose conditions is altered in a strain carrying the tor1-L2045D mutation of the TORC2 catalytic subunit.…”

“…46 Briefly, the GFP-Atg8 fusion protein, which was expressed exogenously under the control of the thiamine-repressible nmt1 promoter of pREP41 66 in the absence of thiamine, was examined in immunoblotting with antibodies against GFP for its autophagy-induced cleavage. Psk1 phosphorylation levels were analyzed as described before.…”

“…Crude cell lysate was prepared with TCA and immunoblotted with antibodies against phospho-Gad8 (P-Gad8), Gad8, phospho-human S6K (P-Psk1), and GFP. 46 are starved and stop dividing with high viability. 49 Consistently, when cells grown in the presence of 3% glucose were transferred to the same medium with 0.2% glucose, the Gad8 phosphorylation was unaltered (Fig.…”

Atg20- and Atg24-family proteins promote organelle autophagy in fission yeast

“…To examine whether organelle autophagy occurs in wild-type cells, we applied nitrogen starvation treatment, which is a trigger of general autophagy in S. pombe (Kohda et al, 2007;Mukaiyama et al, 2009;Sun et al, 2013), and monitored the subcellular localization of two organelle markers, an ER integral membrane protein Ost4 and a mitochondrial matrix protein Sdh2. We used Cpy1 as a vacuole lumen marker (Tabuchi et al, 1997).…”

Special issue: Physiology and Systems Biology of the Fungal Cell

“…The special collection of research papers found in this issue spans a range of topics and organisms. Topics covered include primary metabolism (Heyland et al, 2009;Domin et al, 2009;Dawe et al, 2009) and its regulation (Teves et al, 2009;Vongsangnak et al, 2009;Wong et al, 2009), as well as the cellular and systems biology of key processes that underlie fungal growth (Bormann & Tudzynski, 2009;Hutchison et al, 2009;Etxebeste et al, 2009;Janus et al, 2009;Eisenman et al, 2009;Gale et al, 2009;Avanzo et al, 2009;Mukaiyama et al, 2009;Zara et al, 2009). Organisms include well-known model systems (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, Aspergillus nidulans), human pathogens (Candida albicans, Cryptococcus neoformans), plant pathogens (Claviceps purpurea, Cryphonectria parasitica, Fusarium solani) and industrial producers (Penicllium chrysogenum).…”