Functional analysis of the yeast Ran exchange factor Prp20p: in vivo evidence for the RanGTP gradient model.
Numerous cellular processes rely on the movement of macromolecules into and out of the nucleus. The primary regulator of this movement is the small GTPase Ran. Like other small GTPases, the nucleotide-bound state of Ran is regulated by effectors that enhance the rate of nucleotide exchange or hydrolysis. Current models for vectorial nuclear transport suggest that it is the strict compartmentalization of these Ran effector molecules that generates a gradient of RanGTP between the nucleus and the cytoplasm to impart directionality to the transport process. Here we investigate the mechanism by which the Ran exchange factor is targeted to the nucleus, and test the impact of disrupting this nuclear compartmentalization on nucleocytoplasmic transport in vivo. Our results indicate that in Saccharomycces cerevisiae the nucleotide exchange factor Prp20p can be targeted to the nucleus via a classical nuclear localization sequence. This transport mechanism is dependent both on Ran and the receptor that recognizes the nuclear localization sequence, importin alpha. Mutations in the evolutionarily conserved nuclear localization sequence only partially inhibit nuclear import of Prp20p, suggesting the existence of a secondary mechanism for this critical nuclear targeting. In an in vivo test of the RanGTP gradient model, we demonstrate that overexpression of a functional cytoplasmic exchange factor inhibits cell growth and blocks both protein import and RNA export in wild-type cells that contain the endogenous nuclear Prp20 protein. Taken together, our results provide in vivo evidence for the idea that the compartmentalization of the exchange factor serves as a mechanism for establishing directional nuclear transport.
Akhtar
N
N
Department of Biochemistry, Emory University School of Medicine, 4117 Rollins Research Center, Atlanta, GA 30322, USA.
Hagan
H
H
Lopilato
J E
JE
Corbett
A H
AH
D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D020662:Guanine Nucleotide Exchange Factors; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D020931:ran GTP-Binding Protein / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C067050:SRM1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D020931:ran GTP-Binding Protein