Integrated RNA- and protein profiling of fermentation and respiration in diploid budding yeast provides insight into nutrient control of cell growth and development

Abstract

Diploid budding yeast undergoes rapid mitosis when it ferments glucose, and in the presence of a non-fermentable carbon source and the absence of a nitrogen source it triggers sporulation. Rich medium with acetate is a commonly used pre-sporulation medium, but our understanding of the molecular events underlying the acetate-driven transition from mitosis to meiosis is still incomplete. We identified 263 proteins for which mRNA and protein synthesis are linked or uncoupled in fermenting and respiring cells. Using motif predictions, interaction data and RNA profiling we find among them 28 likely targets for Ume6, a subunit of the conserved Rpd3/Sin3 histone deacetylase-complex regulating genes involved in metabolism, stress response and meiosis. Finally, we identify 14 genes for which both RNA and proteins are detected exclusively in respiring cells but not in fermenting cells in our sample set, including CSM4, SPR1, SPS4 and RIM4, which were thought to be meiosis-specific. Our work reveals intertwined transcriptional and post-transcriptional control mechanisms acting when a MATa/α strain responds to nutritional signals, and provides molecular clues how the carbon source primes yeast cells for entering meiosis. Biological significance Our integrated genomics study provides insight into the interplay between the transcriptome and the proteome in diploid yeast cells undergoing vegetative growth in the presence of glucose (fermentation) or acetate (respiration). Furthermore, it reveals novel target genes involved in these processes for Ume6, the DNA binding subunit of the conserved histone deacetylase Rpd3 and the co-repressor Sin3. We have combined data from an RNA profiling experiment using tiling arrays that cover the entire yeast genome, and a large-scale protein detection analysis based on mass spectrometry in diploid MATa/α cells. This distinguishes our study from most others in the field—which investigate haploid yeast strains—because only diploid cells can undergo meiotic development in the simultaneous absence of a non-fermentable carbon source and nitrogen. Indeed, we report molecular clues how respiration of acetate might prime diploid cells for efficient spore formation, a phenomenon that is well known but poorly understood.

The anti-cancer drug 5-fluorouracil affects cell cycle regulators and potential regulatory long non-coding RNAs in yeast

“…We also observed genes important for meiosis (MEI4, RED1) and spore formation (SPO19, SPO73, SSP2) in C2. This is unsurprising, since our experimental protocol involved arresting diploid cells in G1 via starvation, which primes them for entry into the meiotic differentiation pathway (the full execution of which requires, however, the simultaneous absence of a fermentable carbon source and nitrogen; see methods) [45].…”

The protein expression landscape of mitosis and meiosis in diploid budding yeast

“…Fermenting cells undergoing mitotic growth (YPA) and meiotic cells at two time points (SPII 6h and 8h) were harvested and frozen using liquid nitrogen prior to storage at -80ºC. Total protein extracts were prepared using a tandem buffer system for soluble and membrane bound proteins and fractionated into 30 trypsin-digested samples via SDS-PAGE gel electrophoresis as described [10,11].…”

“…In previous work, we used Direct Iterative Protein Profiling (DIPP) to determine the proteome of rapidly dividing diploid SK1 MATa/ yeast cells cultured in rich medium (YPD), and respiring cells cultured in rich medium with acetate as the sole carbon source (YPA), which is often used as pre-sporulation medium [10,11]. Here, we compared the proteomes in dividing versus differentiating yeast cells using acetate as a carbon source, by analysing samples cultured in YPA (mitosis) and SPII at 6h and 8h (broadly corresponding to meiosis I and II) with the quantitative label-free T3PQ method.…”

“…Yeast genes are exceptionally well defined, since a large number of genomes from different strain backgrounds have been sequenced [1]. The transcriptome and proteome in important phases of yeast's haploid and diploid life cycle were analysed, which revealed complex and often uncorrelated mRNA/protein expression patterns [2][3][4][5][6][7][8][9][10][11]. These profiling data were complemented by a variety of functional genomics studies that yielded insight into the roles of nearly all currently known protein-coding genes during mitotic growth, stress response, gametogenesis (sporulation) and spore germination [12][13][14][15][16].…”

Similar environments but diverse fates: Responses of budding yeast to nutrient deprivation

“…For example, energy is required to express hundreds of gene products required for meiosis and spore wall formation including many, such as spore wall proteins, that are produced to very high levels 4445707172. In addition, many of the cellular processes required in sporulation have additional energy requirements - e.g.…”