Isolation and characterization of Nrf1p, a novel negative regulator of the Cdc42p GTPase in Schizosaccharomyces pombe.
The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ts) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1(+), encoded an approximately 15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe Deltanrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1(+) also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.
Murray
J M
JM
Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
Johnson
D I
DI
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000378:metabolism*
D004271:DNA, Fungal; D005656:Fungal Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C499587:VTC1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C499586:nrf1 protein, S pombe; D020558:GTP Phosphohydrolases; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae