Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe.

Davey, John; Davis, Kevin; Imai, Yoshiyuki; Yamamoto, M.; Matthews, Glenn

doi:10.1002/j.1460-2075.1994.tb06936.x

Cited by 54 publications

(59 citation statements)

References 79 publications

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“…A number of phenotypic defects have been described for kex2 mutants of different yeast species (5,17,20,30). We did not observe obvious aberrant morphologies of cells grown in YPD, nor did we notice any growth arrest at 16°C as reported for S. cerevisiae (30).…”

Section: Resultssupporting

confidence: 71%

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Werten¹,

Wolf²

2005

Appl Environ Microbiol

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Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae ␣-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the ␣-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the ␣-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.

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Section: Resultssupporting

confidence: 71%

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Werten¹,

Wolf²

2005

Appl Environ Microbiol

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“…Deletion of KEX2 abrogates the ability of ␣ cells to mate with a cells, but not the ability of a cells to mate with ␣ cells, 2 suggesting that C. albicans ␣ cells may secrete an ␣-specific mating pheromone processed by Kex2p. Although well-characterized peptide mating pheromones such as the ␣-factor of S. cerevisiae and the P-factor of S. pombe are cleaved by kexins (7,38), our semiautomated annotation of the list of candidate Kex2p cleavage substrates did not describe a protein having a significant degree of sequence similarity to known fungal pheromones. This is not surprising, as fungal pheromones such as ␣-factor and P-factor do not bear much sequence similarity to one another (see Fig.…”

Section: One Of the Candidate Cleavage Substrates Of Kex2p Is Structumentioning

confidence: 91%

Inactivation of Kex2p Diminishes the Virulence of Candida albicans

Newport¹,

Kuo²,

Flattery³

et al. 2003

Journal of Biological Chemistry

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Deletion of the kexin gene (KEX2) in Candida albicanshas a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae ␣-factor.Serine proteinases of the kexin/subtilisin superfamily activate precursor forms of many exported eukaryotic proteins by specific endoproteolytic cleavage adjacent and C-terminal to dibasic residues. Members of the superfamily play a crucial role in a large and varied set of biological processes in animals, plants, and fungi. In metazoan cells, kexins participate in both the constitutive and regulated secretory pathways, with substrates including hormones, serum proteins, neuropeptides, cell surface receptors, components of the extracellular matrix (ECM), 1 and proteinases, which modify the ECM (1). Furin, a member of the mammalian kexin family, processes several ECM metalloproteinases requisite for the dissemination of cancer cells (2). Kexins have also been suborned to mediate the maturation of viral proteins and bacterial toxins (3). In fungi, kexins cycle between the outer face of the Golgi and the prevacuolar compartment where they process proteins involved in maintenance and remodeling of the cell wall (4, 5), proteins associated with the formation of aerial hyphae (6), ␣-type mating prepropheromones (7), killer toxins (7) , zymogens of secreted proteinases (8 -10), lipases (11), polysaccharide-degrading enzymes (12, 13), and themselves (14). Deleting the kexin of the yeast Yarrowia lipolytica abolishes the formation of hyphae (15). kex2-null mutants of the yeast Saccharomyces cerevisiae are viable but exhibit conditional morphological abnormalities (16), defective vacuolar proton-translocating V-ATPase activity (17), cold-sensitive growth (16), and a partial defect in meiosis (genome-www.stanford.edu/Saccharomyces). Genetic data indicate that ...

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“…Soon after the discovery of Kex2, numerous Kex2-like processing systems were identified in other eukaryotic species, including humans (6), plants (7), the fission yeast Schizosacchoromyces pombe (8), and the plant pathogenic fungus Ustilago maydis (9). Characteristic of target proteins of Kex2-like proteases are N-terminal processing sites with dibasic amino acids, in particular Lys-Arg.…”

mentioning

confidence: 99%

Glycosylphosphatidylinositol-anchored Proteases of Candida albicans Target Proteins Necessary for Both Cellular Processes and Host-Pathogen Interactions

Albrecht

Felk

Pichová

et al. 2006

Journal of Biological Chemistry

237

248

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Intracellular and secreted proteases fulfill multiple functions in microorganisms. In pathogenic microorganisms extracellular proteases may be adapted to interactions with host cells. Here we describe two cell surface-associated aspartic proteases, Sap9 and Sap10, which have structural similarities to yapsins of Saccharomyces cerevisiae and are produced by the human pathogenic yeast Candida albicans. Sap9 and Sap10 are glycosylphosphatidylinositol-anchored and located in the cell membrane or the cell wall. Both proteases are glycosylated, cleave at dibasic or basic processing sites similar to yapsins and Kex2-like proteases, and have functions in cell surface integrity and cell separation during budding. Overexpression of SAP9 in mutants lacking KEX2 or SAP10, or of SAP10 in mutants lacking KEX2 or SAP9, only partially restored these phenotypes, suggesting distinct target proteins of fungal origin for each of the three proteases. In addition, deletion of SAP9 and SAP10 modified the adhesion properties of C. albicans to epithelial cells and caused attenuated epithelial cell damage during experimental oral infection suggesting a unique role for these proteases in both cellular processes and host-pathogen interactions.Proteases possess multiple functions in nature ranging from regulation of subtle cellular processes by activating distinct proproteins to nonspecific degradation of proteins for recycling of biomolecules. Several pathogenic microorganisms have adapted this biochemical property to fulfill a number of specialized functions during the infective process. The substrate specificities of these proteases may be very narrow, as in the case of bacterial toxins responsible for botulism or anthrax. In contrast, facultative pathogens may secrete proteases that have more general and much broader effects and play important roles in both saprophytic growth and infection.The yeast Saccharomyces cerevisiae has served as a eukaryotic model organism to study functions of regulatory proteases including the Kex2 protein, a proprotein processing subtilisin-like serine protease of the late Golgi compartment (1-5). Soon after the discovery of Kex2, numerous Kex2-like processing systems were identified in other eukaryotic species, including humans (6), plants (7), the fission yeast Schizosacchoromyces pombe (8), and the plant pathogenic fungus Ustilago maydis (9). Characteristic of target proteins of Kex2-like proteases are N-terminal processing sites with dibasic amino acids, in particular Lys-Arg. Although Kex2 was shown to be the major protease for processing at dibasic residues, alternative pathways exist in S. cerevisiae (10). Gene products of YPS1 (yapsin 1) and YPS2 (yapsin 2), two closely related glycosylphosphatidylinositol (GPI) 2 -anchored aspartic proteases, were able to cleave the ␣-factor pheromone precursor (a well known Kex2 substrate) at Lys-Arg sites (10 -12), and overexpression of YPS1 and YPS2 partially compensated for the loss of Kex2 (10, 13). However, the biological roles of yapsins are unknown.The human...

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Isolation and characterization of krp, a dibasic endopeptidase required for cell viability in the fission yeast Schizosaccharomyces pombe.

Cited by 54 publications

References 79 publications

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a Pichia pastoris kex2 Disruptant

Inactivation of Kex2p Diminishes the Virulence of Candida albicans

Glycosylphosphatidylinositol-anchored Proteases of Candida albicans Target Proteins Necessary for Both Cellular Processes and Host-Pathogen Interactions

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