Dataset Information

Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa.

ABSTRACT: Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast, Candida maltosa (T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030-5036, 1997). To understand the mechanism of this transition, we screened the gene library of C. maltosa for sequences which could suppress this morphological change. As a result, we isolated the 5' end of a new gene, EPD1 (for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epdlp was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae, and Phr1p and Phr2p of Candida albicans. The expression of EPD1 was moderately regulated by environmental pH. A homozygous EPD1 null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble beta-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by n-hexadecane at pH 4 or pH 7, suggesting that EPD1 is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the GAL promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length EPD1 did not. This result and the initial isolation of the 5' end of EPD1 as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.

SUBMITTER: Nakazawa T

PROVIDER: S-EPMC107133 | biostudies-literature | 1998 Apr

REPOSITORIES: biostudies-literature

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Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa.

Nakazawa T T Horiuchi H H Ohta A A Takagi M M

Journal of bacteriology 19980401 8

Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast, Candida maltosa (T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030-5036, 1997). To understand the mechanism of this transition, we screened the gene library of C. maltosa for sequences which could suppress this morphological change. As a result, we isolated the 5' end of a new gene, EPD1 (for essential for pseudohyphal development), and then cloned the entire ...[more]

PMID: 9555889

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Project description:Pseudohyphal growth is a nutrient-regulated program in which budding yeast form multicellular filaments of elongated and connected cells. Filamentous growth is required for virulence in pathogenic fungi and provides an informative model of stress-responsive signaling. The genetics and regulatory networks modulating pseudohyphal growth have been studied extensively, but little is known regarding the changes in metabolites that enable pseudohyphal filament formation. Inositol signaling molecules are an important class of metabolite messengers encompassing highly phosphorylated and diffusible inositol polyphosphates (InsPs). We report here that the InsP biosynthesis pathway is required for wild-type pseudohyphal growth. Under nitrogen-limiting conditions that can induce filamentation, InsPs exhibit characteristic profiles, distinguishing the InsP7 pyrophosphate isoforms 1PP-InsP5 and 5PP-InsP5. Deletion and overexpression analyses of InsP kinases identify elevated levels of 5PP-InsP5 relative to 1PP-InsP5 in mutants exhibiting hyper-filamentous growth. Overexpression of KCS1, which promotes formation of inositol pyrophosphates, is sufficient to drive pseudohyphal filamentation on medium with normal nitrogen levels. We find that the kinases Snf1p (AMPK), Kss1p, and Fus3p (MAPKs), required for wild-type pseudohyphal growth, are also required for wild-type InsP levels. Deletion analyses of the corresponding kinase genes indicate elevated InsP3 levels and an absence of exaggerated 5PP-InsP5 peaks in trace profiles from snf1Δ/Δ and kss1Δ/Δ mutants exhibiting decreased pseudohyphal filamentation. Elevated 5PP-InsP5:1PP-InsP5 ratios are present in the hyperfilamentous fus3 deletion mutant. Collectively, the data identify the presence of elevated 5PP-InsP5 levels relative to other inositol pyrophosphates as an in vivo marker of hyper-filamentous growth, while providing initial evidence for the regulation of InsP signaling by pseudohyphal growth kinases.

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, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in 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 isobaric tags for relative and absolute quantification chemistry and two-dimensional liquid chromatography-tandem mass spectrometry, we profiled 2463 peptides and 356 proteins, identifying 11 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. Pun1p abundance is doubled under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1Delta mutants are noninvasive, 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 pun1Delta mutants misregulate amino acid biosynthetic genes during nitrogen stress. By chromatin immunoprecipitation and reverse transcription-PCR, we find that the filamentous growth factor Mss11p directly binds the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of differential protein abundance during pseudohyphal growth, identifying a previously uncharacterized membrane compartment of Can1 protein required for wild-type nitrogen signaling and filamentous growth.

Dataset Information

Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa.

Publications

Isolation and characterization of EPD1, an essential gene for pseudohyphal growth of a dimorphic yeast, Candida maltosa.

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