The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substrate

doi:10.1074/jbc.270.32.19017

The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substrate (*)

From theLaboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021

^¶ To whom correspondence should be addressed:
Laboratory of Cell Biology, Howard Hughes Medical Inst., The Rockefeller University, New York, NY 10021.
Tel.: 212-327-8096; Fax: 212-327-7880.

Abstract

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound S-labeled mammalian karyopherin β, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin β), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.

Footnotes

↵^§ Supported by a fellowship from the Deutsche Forschungsgemeinschaft (Kr 1412/1-1) and by funds from Fritz Thyssen Stiftung (Az.2.12).
↵* 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.
↵¹ The abbreviations used are:

NPC

nuclear pore complex

NE

nuclear envelope

PAGE

polyacrylamide gel electrophoresis

ORF

open reading frame

PCR

polymerase chain reaction

mAb

monoclonal antibody.
↵²Strambio-de-Castillia, C., Blobel, G., and Rout, M. P. (1995) J. Cell Biol., in press.
- Received May 9, 1995.