Abstract
Nrf1p was first identified in a screen for negative regulators of the Cdc42p GTPase. Overexpression of Nrf1p resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotypes including an increase in vacuolar fusion. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacuolar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor. In this study, site-directed mutagenesis was conducted on Nrf1p to determine its functional domains. Mutations in the three putative transmembrane domains resulted in mislocalization of GFP-Nrf1p and an inability to induce lethality, suggesting a loss of function. Mutations in the second extramembranous loop of Nrf1p also resulted in a loss of function and altered the ability of GFP-Nrf1p to localize to vacuolar membranes. Analysis of Deltanrf1 and Deltascd1 mutants revealed defects in endocytosis. In addition, overexpression of constitutively active Cdc42(G12V)p resulted in an increase in endocytosis and an ability to rescue the endocytic defects in Deltanrf1 and Deltascd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Cell Cycle Proteins / physiology*
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / physiology*
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Endocytosis / physiology*
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Fungal Proteins*
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GTP Phosphohydrolases / physiology*
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Guanine Nucleotide Exchange Factors*
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Membrane Fusion / physiology
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Membrane Proteins / physiology
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Mitogen-Activated Protein Kinases / metabolism
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Nuclear Respiratory Factors
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Osmotic Pressure
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Protein Structure, Tertiary
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Proto-Oncogene Proteins / physiology*
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Saccharomyces cerevisiae Proteins*
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Schizosaccharomyces / physiology*
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Schizosaccharomyces pombe Proteins*
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Trans-Activators / chemistry
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Trans-Activators / genetics
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Trans-Activators / physiology*
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cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / physiology*
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rab GTP-Binding Proteins / metabolism
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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Fungal Proteins
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Guanine Nucleotide Exchange Factors
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Membrane Proteins
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Nuclear Respiratory Factors
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Proto-Oncogene Proteins
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces pombe Proteins
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Trans-Activators
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scd1 protein, S pombe
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Mitogen-Activated Protein Kinases
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Pmk1 protein, Magnaporthe grisea
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GTP Phosphohydrolases
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YPT7 protein, S cerevisiae
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cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
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rab GTP-Binding Proteins