Identification of a Yeast Karyopherin Heterodimer That Targets Import Substrate to Mammalian Nuclear Pore Complexes

doi:10.1074/jbc.270.28.16499

Identification of a Yeast Karyopherin Heterodimer That Targets Import Substrate to Mammalian Nuclear Pore Complexes (*)

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

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Abstract

Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin. Yeast contain a homologue of karyopherin α named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I. To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin α homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera. Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose. Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII. Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin β. The yeast α and β karyopherin subunits were expressed in Escherichia coli and were purified. When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells. We propose that all karyopherins function as α/β heterodimers.

Footnotes

↵§ Supported by Research Fellowship En301/1-1 from the Deutsche Forschungsgemeinschaft.
↵** Supported by the Jane Coffin Childs Memorial Fund for Medical Research.
↵* 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:

NLS

nuclear localization sequence

NPC

nuclear pore complex

GST

glutathione S-transferase

IgG Sepharose

immunoglobulin G-Sepharose

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

PMSF

phenylmethylsulfonyl fluoride.
↵² M. Rout, G. Blobel, and J. Aitchison, unpublished results.