The NTF2 Gene Encodes an Essential, Highly Conserved Protein That Functions in Nuclear Transport in Vivo

doi:10.1074/jbc.271.31.18477

The NTF2 Gene Encodes an Essential, Highly Conserved Protein That Functions in Nuclear Transport in Vivo*

From the Division of Cellular and Molecular Biology, Dana Farber Cancer Institute and the Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115

^‡ To whom correspondence should be addressed:
849 Mayer Bldg., Dana Farber Cancer Institute, 44 Binney St., Boston, MA 02115.
Tel.: 617-632-5102; Fax: 617-632-5103.

Abstract

The small protein p10/Ntf2p has been implicated in protein import in vitro (Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10212-10216; Paschal, B. M., and Gerace, L. (1995) J. Cell Biol. 129, 925-937). Here we present the first evidence that demonstrates an essential in vivo role for the NTF2 gene product in nuclear transport. The NTF2 locus was identified in a screen for temperature-sensitive Saccharomyces cerevisiae mutants defective in the localization of nuclear proteins. Genetic analysis demonstrates that the NTF2 gene is essential for viability in budding yeast. Two temperature-sensitive mutants, ntf2-1 and ntf2-2, that each contain single point mutations in highly conserved amino acid residues show defects in the localization of nuclear proteins but not in the export of poly(A)⁺ RNA following a shift to the nonpermissive temperature. An epitope-tagged version of Ntf2p was used to show that the protein is concentrated at the nuclear envelope. Finally, the human gene under the control of the yeast promoter fully substitutes for the deleted yeast gene. Taken together, these results demonstrate the exquisite functional conservation of this protein throughout evolution and indicate a critical in vivo role in nuclear transport.

Footnotes

↵* This work was funded by grants from the National Institutes of Health (to P. A. S.) and by a National Institutes of Health postdoctoral fellowship (to A. H. C.). 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:

GAP

GTPase activating protein

bp

base pair

DAPI

4′,6-diamidino-2-phenylindole

FITC

fluorescein isothiocyanate

FOA

5′-fluoroorotic acid

GFP

green fluorescent protein

GST

glutathione S-transferase

NLS

nuclear localization signal

NTF2

nuclear transport factor 2

PSL1

profilin synthetic lethal 1

YEPD

yeast extract plus dextrose

PCR

polymerase chain reaction.
↵² A. H. Corbett, B. K. Haarer, and P. A. Silver, unpublished observations.
- Received April 12, 1996.
- Revision received May 15, 1996.