Two yeast nuclear pore complex proteins involved in mRNA export form a cytoplasmically oriented subcomplex - PubMed (original) (raw)

Two yeast nuclear pore complex proteins involved in mRNA export form a cytoplasmically oriented subcomplex

M E Hurwitz et al. Proc Natl Acad Sci U S A. 1998.

Abstract

We sublocalized the yeast nucleoporin Nup82 to the cytoplasmic side of the nuclear pore complex (NPC) by immunoelectron microscopy. Moreover, by in vitro binding assays we showed that Nup82 interacts with the C-terminal region of Nup159, a yeast nucleoporin that previously was also localized to the cytoplasmic side of the NPC. Hence, the two nucleoporins, Nup82 and Nup159, form a cytoplasmically oriented subcomplex that is likely to be part of the fibers emanating from the cytoplasmic ring of the NPC. Overexpression of Rss1/Gle1, a putative nucleoporin and/or mRNA transport factor, was shown previously to partially rescue depletion of Nup159. We show here that overexpression of Rss1/Gle1 also partially rescued depletion of Nup82. Depletion of either Nup82, Nup159, or Rss1/Gle1 was shown previously to inhibit mRNA export. As was reported previously for depletion of Nup159 or of Rss1/Gle1, we show here that depletion of Nup82 has no detectable effect on classical nuclear localization sequence-mediated nuclear import. In summary, the nucleoporins Nup159 and Nup82 form a cytoplasmically oriented subcomplex of the NPC that is likely associated with Rss1/Gle1; this complex is essential for RNA export, but not for classical nuclear localization sequence-mediated nuclear protein import.

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Figures

Figure 1

Figure 1

Depletion of Nup82 has no significant effect on nuclear protein import. NUP82-wt and NUP82-Δ108 yeast containing pGFP-URA were grown to mid-log phase and then incubated at 30°C or 37°C for 3 hr and tested in the NLS-GFP nuclear import assay.

Figure 2

Figure 2

Immunoelectron microscopic localization of Nup82 to the cytoplasmic side of the NPC in isolated crude nuclear envelopes. (A) Side view. (Bar = 150 nm.) (B) Front view. (Bar = 55 nm.) (C) Quantitation of distance of gold particles from the NPC. The distance measured is a straight line perpendicular to the central plane of the NPC. Forty-eight particles were measured, and the mean and SD were found to be 29.9 nm and 10.9 nm, respectively.

Figure 3

Figure 3

Nup82 binds to the coiled-coil domain of Nup159. (A) Nup159 was expressed in Escherichia coli in five different segments, p159-1, amino acid residues 1–175; p159-2, residues 176–440; p159-3, residues 441–876; p159-4, residues 877-1222; p159-5, residues 1223–1460. The position of the FXFG peptide repeats, coiled coil, and amino acid number are shown [modified from Kraemer et al. (17)]. (B) Fractions containing the expressed proteins were subjected to SDS/PAGE, transferred to nitrocellulose, incubated with GST-Nup82 or GST alone, and then incubated with a polyclonal rabbit anti-GST antibody. The arrowhead shows where p159-5 migrates. The asterisk is at approximately double the molecular weight of p159-5.

Figure 4

Figure 4

Overexpression of Rss1/Gle1 partially rescues the ts phenotype seen in NUP82-Δ108U yeast. The (+) rows contain NUP82-Δ108U yeast harboring pVDP7 (which overexpresses Rss1/Gle1). The (−) rows contain NUP82-Δ108U yeast harboring pRS315 (empty plasmid). Cells were grown in selective medium (-LEU) at 30°C overnight. Equivalent numbers of cells and serial 10-fold dilutions (based on OD600) were plated on either complete medium (YPD) or selective medium (-LEU) and incubated at 30°C or 37°C for 4 days.

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