Different Binding Domains for Ran-GTP and Ran-GDP/RanBP1 on Nuclear Import Factor p97

doi:10.1074/jbc.272.10.6818

Different Binding Domains for Ran-GTP and Ran-GDP/RanBP1 on Nuclear Import Factor p97*

From the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611

^§ To whom correspondence and reprint requests should be addressed:
Dept. of Cell and Molecular Biology, Northwestern University Medical School, W129, 303 East Chicago Ave., Chicago, IL 60611.
Tel.: 312-503-7525; Fax: 312-503-7912; E-mail: s-adam{at}nwu.edu

Abstract

Several proteins are required for the transport of nuclear proteins from the cytoplasm to the nucleus, including the nuclear location sequence receptor (NLS receptor), p97, the small nuclear GTPase Ran/TC4, and several nucleoporins. The interaction of Ran with p97 is thought to regulate the interaction of these transport components. Ran-GTP alone binds p97, but Ran-GDP binds p97 only in conjunction with RanBP1. Using site-directed mutagenesis and deletion analysis, we have identified two distinct but overlapping binding domains for Ran-GTP and Ran-GDP/RanBP1 on p97. A short acidic sequence in p97 is part of the Ran-GDP/RanBP1 binding domain, possibly functioning in a similar manner as the C-terminal acidic sequence in Ran. A conserved cysteine residue in p97, Cys-158, is required for binding Ran-GDP/RanBP1, but not for binding of Ran-GTP to p97. In a permeabilized cell protein import assay, a mutant p97 with alanine substituted for Cys-158 is unable to support import in the presence of NLS receptor and Ran. These results support a direct active role for Ran-GDP in the receptor complex and provide evidence that the activity of downstream effectors of small GTPases may be regulated by both GTP- and GDP-bound forms of the protein.

Footnotes

↵^‡ Supported by a National Institutes of Health Training Grant.
↵* This work was supported in part by National Institutes of Health Grant 2RO1GM47866 and Training Grant 5-T32-GM08152-10 (to S. A. A.) and by American Cancer Society Grant JFRA-433. 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.
↵¹ The abbreviations used are:

NLS

nuclear location sequence

NEM

N-ethylmaleimide

APC

allophycocyanin.
↵²N. C. Chi and S. A. Adam, manuscript in preparation.
- Received September 26, 1996.
- Revision received November 8, 1996.