Isolation and characterization of Nrf1p, a novel negative regulator of thecdc42p GTPase in Schizosaccharomyces pombe

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction path ways in eukaryotic cells leading to actin rearrangements. A cross-species g enetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24(ls) mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One c DNA, nrf1(+), encoded an similar to 15 kD protein with three potential tran smembrane domains and 78% amino-acid identity to a S. cerevisiae gene, desi gnated NRF1. A S. pombe Delta nrf1 mutant was viable but overexpression of nrf1(+) in S. pombe resulted in dose-dependent lethality, with cells exhibi ting 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 intrace llular membranes, including vacuolar membranes, and to sites of septum form ation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S . pombe Cdc24p homolog Scd1p. Taken together, these data are consistent wit h Nmp functioning as a negative regulator of Cdc42p within the cell polarit y pathway.