在酿酒酵母孢子中,存在糖酵解产物3-磷酸甘油醛(3-glyceraldehyde phosphate, GAP)参与的抑制萌发的负调节机制,但是目前的调控途径并不完善。该研究目的是发现GAP对萌发孢子的影响,并深入了解其负调控机制。重点分析以GAP为底物的糖酵解酶——三磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)在调控机制中的作用。该文采用富营养培养基和热处理等多种条件诱导酵母孢子萌发,并通过荧光增白剂染色来测定萌发率。分别在有无GAP的条件下诱导萌发,研究GAPDH的表达水平。接着利用qPCR的方法检验GAPDH mRNA的表达水平。此外,在酵母中敲除TDH1、TDH2和TDH3这3个GAPDH的结构基因,分析突变体的萌发效率。结果显示当营养物诱导或加热处理促使孢子萌发时,GAPDH的表达水平显著降低,而在孢子萌发的过程中添加GAP反而会增加GAPDH水平。qPCR结果显示孢子萌发时GAPDH的mRNA水平没有降低,故GAPDH表达水平的降低是因为在萌发过程中蛋白被降解。且GAPDH单个结构基因的缺失会增加孢子的萌发率。结果表明GAPDH参与GAP介导的负调控机制。
Spores exit a quiescent state and start mitotic growth when they encounter sufficient nutrients.This process is called germination.In Saccharomyces cerevisiae spores, there is a negative regulatory mechanism which inhibits germination.A glycolytic product 3-glyceraldehyde phosphate (GAP) is known to be involved in the negative regulatory mechanism and spore germination is inhibited in the presence of GAP.However, the entire process of the regulatory mechanism remains elusive.The objectives of this study were to find the effects of GAP on germinating spores and to gain insight into the mechanism of the negative regulatory mechanism.In particular, the role of glyceraldehyde triphosphate dehydrogenase (GAPDH), which was a glycolytic enzyme using GAP as a substrate, in the regulatory mechanism was analysed.Yeast spore germination was induced by various conditions including nutrient-rich media and by heat-treatment.Germination efficiency can be assayed by calcofluor whit staining.Expression levels of GAPDH was investigated when germination was induced in the presence or absence of GAP.In yeast, there are three structural genes of GAPDH, TDH1, TDH2, and TDH3.These genes were disrupted and germination efficiencies of these mutants were analysed.Expression levels of GAPDH were significantly reduced when germination was induced either by nutrients or heat.However, GAPDH levels were increased by the addition of GAP even if spores were incubated in nutrient-rich media.Since levels of GAPDH mRNAs were not decreased, GAPDH proteins were likely degraded during germination.Germination efficiencies were increased when these genes, especially, TDH2 and TDH3, were deleted.GAPDH is known as a multifunction protein and regulates various processes.Results suggest that GAPDH is involved in the negative regulatory mechanism, possibly as a downstream regulator of GAP.
[1] WERNER-WASHBURNE M, BRAUN E, JOHNSTON G C, et al.Stationary phase in the yeast Saccharomyces cerevisiae[J].Microbiological Reviews, 1993, 57(2):383-401.
[2] PLANTE S, MOON K M, LEMIEUX P, et al.Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome[J].PLoS Biology, 2023, 21(4):e3002042.
[3] MOIR A, COOPER G.Spore germination[J].Microbiology Spectrum, 2015, 3(6).
[4] ROZALI S N M, MILANI E A, DEED R C, et al.Bacteria, mould and yeast spore inactivation studies by scanning electron microscope observations[J].International Journal of Food Microbiology, 2017, 263:17-25.
[5] MANTZOURANI I, PLESSAS S, SAXAMI G, et al.Study of kefir grains application in sourdough bread regarding rope spoilage caused by Bacillus spp[J].Food Chemistry, 2014, 143:17-21.
[6] PINTO C A, MOREIRA S A, FIDALGO L G, et al.Effects of high-pressure processing on fungi spores:Factors affecting spore germination and inactivation and impact on ultrastructure[J].Comprehensive Reviews in Food Science and Food Safety, 2020, 19(2):553-573.
[7] DIJKSTERHUIS J.Fungal spores:Highly variable and stress-resistant vehicles for distribution and spoilage[J].Food Microbiology, 2019, 81:2-11.
[8] TSUYUZAKI H, UJIIE R, SATO M.Wake-up alarm:Virtual time-lapse gene expression landscape illuminates mechanisms underlying dormancy breaking of germinating spores[J].Current Genetics, 2021, 67(4):519-534.
[9] AKEKAWATCHAI C, JITRAPAKDEE S.Chapter Twelve Cellular signals integrate cell cycle and metabolic control in cancer[J].Advances in Protein Chemistry and Structural Biology, 2023, 135:397-423.
[10] ALFATAH M, WONG J H, KRISHNAN V G, et al.TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae[J].BMC Biology, 2021, 19(1):95.
[11] WIESE W, BARCZUK J, RACINSKA O, et al.PI3K/Akt/mTOR signaling pathway in blood malignancies-new therapeutic possibilities[J].Cancers, 2023, 15(21):5297.
[12] LIU G Y, YANG Y, YANG G L, et al.Triosephosphate isomerase and its product glyceraldehyde-3-phosphate are involved in the regulatory mechanism that suppresses exit from the quiescent state in yeast cells[J].Microbiology Spectrum, 2022, 10(4):e0089722.
[13] NICHOLLS C, LI H, LIU J P.GAPDH:A common enzyme with uncommon functions[J].Clinical and Experimental Pharmacology & Physiology, 2012, 39(8):674-679.
[14] BUTERA G, MULLAPPILLY N, MASETTO F, et al.Regulation of autophagy by nuclear GAPDH and its aggregates in cancer and neurodegenerative disorders[J].International Journal of Molecular Sciences, 2019, 20(9):2062.
[15] HUO J F, DONG W, XU J K, et al.Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage[J].Experimental Neurology, 2024, 371:114577.
[16] HABER J E.Mating-type genes and MAT switching in Saccharomyces cerevisiae[J].Genetics, 2012, 191(1):33-64.
[17] LONGTINE M S, MCKENZIE A 3rd, DEMARINI D J, et al.Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[J].Yeast, 1998, 14(10):953-961.
[18] ESCORCIA W, FORSBURG S L.Random spore analysis in fission yeast[J].Methods in Molecular Biology, 2018, 1721:189-195.
[19] SHEN C L, LI W J, WANG Y J.Research on the oncogenic role of the house-keeping gene GAPDH in human tumors[J].Translational Cancer Research, 2023, 12(3):525-535.
[20] HUANG T S, NAGY P D.Direct inhibition of tombusvirus plus-strand RNA synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, in yeast and plants[J].Journal of Virology, 2011, 85(17):9090-9102.
[21] VAN LEEMPUTTE F, VANTHIENEN W, WIJNANTS S, et al.Aberrant intracellular pH regulation limiting glyceraldehyde-3-phosphate dehydrogenase activity in the glucose-sensitive yeast tps1Δ mutant[J].mBio, 2020, 11(5):e02199-20.
[22] LINCK A, VU X K, ESSL C, et al.On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae[J].FEMS Yeast Research, 2014, 14(3):389-398.
[23] RANDEZ-GIL F, SÁNCHEZ-ADRIÁ I E, ESTRUCH F, et al.The formation of hybrid complexes between isoenzymes of glyceraldehyde-3-phosphate dehydrogenase regulates its aggregation state, the glycolytic activity and sphingolipid status in Saccharomyces cerevisiae[J].Microbial Biotechnology, 2020, 13(2):562-571.
