The Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic trafficking in the fission yeast Schizosaccharomyces pombe

Nrf1p was first identified in a screen for negative regulators of the Cdc42 p GTPase. Overexpression of Nrf1p resulted in dose-dependent lethality, wit h cells exhibiting an ellipsoidal morphology and abnormal vacuolar phenotyp es including an increase in vacuolar fusion. Green fluorescent protein (GFP )-Cdc42p and GFP-Nrf1p colocalized to vacuolar membranes and GFP-Nrf1p vacu olar localization depended on Scd1p, the Schizosaccharomyces pombe homolog of the Cdc24p guanine nucleotide exchange factor. In this study, site-direc ted mutagenesis was conducted on Nrf1p to determine its functional domains. Mutations in the three putative transmembrane domains resulted in mislocal ization of GFP-Nrf1p and an inability to induce lethality, suggesting a los s of function. Mutations in the second extramembranous loop of Nrf1p also r esulted in a loss of function and altered the ability of GFP-Nrf1p to local ize to vacuolar membranes. Analysis of Delta nrf1 and Delta scd1 mutants re vealed defects in endocytosis. In addition, overexpression of constitutivel y active Cdc42(GI2V)p re suited in an increase in endocytosis and an abilit y to rescue the endocytic defects in Delta nrf1 and Delta scd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient end ocytosis, possibly through the regulation of Cdc42p.