Nup358, a Cytoplasmically Exposed Nucleoporin with Peptide Repeats, Ran-GTP Binding Sites, Zinc Fingers, a Cyclophilin A Homologous Domain, and a Leucine-rich Region

doi:10.1074/jbc.270.23.14209

Nup358, a Cytoplasmically Exposed Nucleoporin with Peptide Repeats, Ran-GTP Binding Sites, Zinc Fingers, a Cyclophilin A Homologous Domain, and a Leucine-rich Region (*)

From the (1) Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021

^§Supported by National Institutes of Health Fellowship 1F32GM16758-01. To whom correspondence and reprint requests should be addressed. Tel.: 212-327-8100; Fax: 212-327-7880; E-mail: wuj{at}rockvax.rockefeller.edu.

Abstract

The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.

Footnotes

↵^¶ Supported by American Cancer Society-Amgen Fellowship PF-4195.
↵** Supported by Deutsche Forschungsgemeinschaft Fellowship Kr 1412/1-1 and Fritz Thyssen Stiftung Fellowship Az.2.12.
↵^§§ Supported by a fellowship from the Charles H. Revson Foundation.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L41840.
↵¹ The abbreviations used are:

NPC

nuclear pore complex

Ran

Ras-related nuclear protein

RanBP

Ran binding protein

Nup

nucleoporin

NE

nuclear envelope

NLS

nuclear localization sequence

EM

electron microscopy

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction.
↵²M. J. Matunis and G. Blobel, manuscript in preparation.