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J. Biol. Chem., Vol. 276, Issue 9, 6445-6452, March 2, 2001
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From the Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110
Our previous studies have focused on a family of
Saccharomyces cerevisiae nuclear pore complex (NPC)
proteins that contain domains composed of repetitive
tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We
have previously shown that the GLFG regions of Nup116p and Nup100p
directly bind the karyopherin transport factor Kap95p during nuclear
protein import. In this report, we have further investigated potential
roles for the GLFG region in mRNA export. The subcellular
localizations of green fluorescent protein (GFP)-tagged mRNA
transport factors were individually examined in yeast cells
overexpressing the Nup116-GLFG region. The essential mRNA export
factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus.
In contrast, the localizations of Gle1-GFP and Gle2-GFP remained
predominantly associated with the NPC, as in wild type cells. The
localization of Kap95p was also not perturbed with GLFG overexpression.
Coimmunoprecipitation experiments from yeast cell lysates resulted in
the isolation of a Mex67p-Nup116p complex. Soluble binding assays with
bacterially expressed recombinant proteins confirmed a direct
interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions.
Mtr2p was not required for in vitro binding of Mex67p to
the GLFG region. To map the Nup116-GLFG subregion(s) required for
Kap95p and/or Mex67p association, yeast two-hybrid analysis was used.
Of the 33 Nup116-GLFG repeats that compose the domain, a central
subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the
capacity to bind both karyopherins and an mRNA export factor
simultaneously. Taken together, our in vivo and in
vitro results define an essential role for a direct Mex67p-GLFG
interaction during mRNA export.
The GLFG Regions of Nup116p and Nup100p Serve as Binding
Sites for Both Kap95p and Mex67p at the Nuclear Pore Complex*
*
This work was supported by National Institutes of Health
Grant GM51219 (to S. R. W.).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.
To whom correspondence should be addressed: Dept. of Cell Biology
and Physiology, Washington University School of Medicine, 660 S. Euclid
Ave., St. Louis, MO, 63110. Tel.: 314-362-2713; Fax: 314-747-1259;
E-mail: swente@cellbio.wustl.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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