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J. Biol. Chem., Vol. 276, Issue 5, 3004-3009, February 2, 2001
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From the Department of Microbiology and Molecular Genetics and the
Markey Center for Molecular Genetics, University of Vermont,
Burlington, Vermont 05405
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
The Cdc42p GTPase and Its Regulators Nrf1p and Scd1p Are
Involved in Endocytic Trafficking in the Fission Yeast
Schizosaccharomyces pombe*
nrf1 and scd1
mutants revealed defects in endocytosis. In addition, overexpression of
constitutively active Cdc42G12Vp resulted in an increase in
endocytosis and an ability to rescue the endocytic defects in
nrf1 and scd1 cells. These data are consistent with Nrf1p and Scd1p being necessary for efficient endocytosis, possibly through the regulation of Cdc42p.
*
This research was supported by American Cancer Society Grant
RPG-89-012-08 and a predoctoral fellowship from the National Science
Foundation-VT EPSCoR (to J. M. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology
and Molecular Genetics, 202B Stafford Hall, University of Vermont,
Burlington, VT 05405. Tel.: 802-656-8203; Fax: 802-656-8749; E-mail:
dijohnso@zoo.uvm.edu.
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