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Quantitiative phosphoproteomic analysis of SKS1 signaling network involved in yeast filamentous growth by LC-MS in combination with SILAC

ABSTRACT: Yeast pseudohyphal growth(filamentous growth) is regulated by signaling pathways responsive to nitrogen stress and glucose deprivation, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. To identify the Sks1p signaling network involved in filamentous growth, we applied mass spectrometry-based quantitative phosphoproteomics in combination with SILAC. We investigated differential protein expressions and changes in phosphorylation levels in SKS1 kinase dead mutant yeast cells (SKS1-KD, Lys-4 and Arg-6 labeled) under filamentous growth condition. Twelve SCX fractions of SILAC paired peptides derived from SKS1-KD and wild type yeast cells were subjected to phosphopeptide enrichment by using ZrO2, prior to LC-MSMS by an LTQ-Orbitrap. MaxQunat (ver 1.0.13.8) package incorporating Mascot search was used for identification and quantification of peptides and proteins. RAW files were searched against a composite target-decoy database containing yeast ORFs from SGD allowing variable modifications of phospho (STY) and oxi (M) and a fixed modification of carbamidomethyl(C) within 7 ppm (MS) and 0.5 Da (MSMS) mass tolerances. Peptide and protein identifications were maintained within 5% FDR.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Hye Kyong Kweon

PROVIDER: PXD000414 | Pride | 2014-04-16

REPOSITORIES: Pride

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The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

Johnson Cole C Kweon Hye Kyong HK Sheidy Daniel D Shively Christian A CA Mellacheruvu Dattatreya D Nesvizhskii Alexey I AI Andrews Philip C PC Kumar Anuj A

PLoS genetics 20140306 3

The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as ...[more]

PMID: 24603354

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Project description:Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, the protein complement at the yeast cell periphery plays a critical and tightly regulated role in enabling pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By iTRAQ chemistry and two-dimensional LC-MS/MS, we profiled 2,463 peptides and 356 proteins, from which we identified eleven differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1 (MCC). Pun1p abundance is increased two-fold under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1D mutants are non-invasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1D mutants misregulate amino acid biosynthetic genes during nitrogen deprivation. By chromatin immunoprecipitation and RT-PCR, we find that the filamentous growth factor Mss11p directly binds to the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of protein abundance during pseudohyphal growth, identifying a previously uncharacterized MCC protein required for wild-type nitrogen signaling and filamentous growth. For this study, we constructed a homozygous diploid strain in the filamentous ?1278b background deleted for PUN1; a wild-type diploid strain of the same background served as the control. Both strains were grown under conditions of low nitrogen, and RNA was extracted from three biological replicates of each strain after identical culturing. The RNA samples were analyzed using affymerix DNA microarrays.

Project description:Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes. For each time-course sample, cDNA was coupled to Cy5 and a reference cDNA pool was made by combining RNA from t = 0 and all late time course samples, which was coupled to Cy3. For end point microarray experiments (i.e., established yeast samples compared to established filamentous samples), G217B yeast cDNA was coupled to Cy5 and filament cDNA was coupled to Cy3.

			Action	DRS
	F004716.dat	Other
	F004717.dat	Other
	F004718.dat	Other
	F004719.dat	Other
	MQ_report_SKS1-KD_HK.zip	Other