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.
Highlights
Cdc42p is a Rho-like GTPase that is ubiquitously expressed in eukaryotes and has been implicated in many cellular processes including regulation of cellular polarity, transcriptional activation, and phagocytosis of bacteria into mammalian cells [1]
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
Nrf1 proteins that contained mutations in the putative transmembrane domains designed to perturb the predicted hydophobicity of this domain no longer conferred a lethal phenotype and were mislocalized, with a decrease in membrane localization and an increase in GFP aggregates, suggesting that these domains were necessary for efficient targeting to membranes
Summary
Cdc42p is a Rho-like GTPase that is ubiquitously expressed in eukaryotes and has been implicated in many cellular processes including regulation of cellular polarity, transcriptional activation, and phagocytosis of bacteria into mammalian cells [1]. A ⌬scd mutant is viable and exhibits a round cell morphology, suggesting that Scd1p plays a role in directed cell growth [3]. Mutations generated in the three putative transmembrane domains affected GFP-Nrf1p localization, consistent with the prediction that these transmembrane domains were essential for membrane anchoring. Studies were conducted to further examine the role of Scd1p in Nrf1p localization to the vacuole These studies led to the discovery that ⌬scd and ⌬nrf mutants had a defect in endocytosis, and this defect could be reversed by expression of activated Cdc42p. Together, these data suggest that Cdc42p-dependent signaling pathways play a role in endocytosis in S. pombe
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