The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae

Abstract

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.

FIG. 1.

Complementation analysis of Rfa-GFP fusions. (A) Full-length and truncated Rfa1-, Rfa2-, and Rfa3-GFP chimeras were generated with GFP fused to the C-terminus of each. The numbers shown indicate the amino acid present at the N- and C-termini of each Rfa fusion encoded in each construct. (B) Diploid yeast heterozygous for an rfa1Δ deletion were transformed with full-length RFA1-GFP, rfa1Δ353-GFP, or rfa1[1–337,344–621]-GFP expressed under the control of the GAL1 promoter. Diploid transformants were sporulated and resulting tetrads dissected on selective media containing glucose (left) or galactose (right) as a carbon source. The growth of colonies from a single representative tetrad from each plate is depicted. (C) Haploid yeast containing the rfa2-210 temperature-sensitive mutation (Maniar et al., 1997) were transformed with plasmids expressing wild-type RFA2 (RFA2), empty vector (GFP), full-length RFA2 fused to GFP (RFA2-GFP), and two C-terminal rfa2 truncations fused with GFP (rfa2Δ169-GFP and rfa2Δ247-GFP). Transformants were streaked on selective media at permissive and restrictive temperatures assayed for growth. Images were taken 3 and 2 days after streaking to 24°C and 37°C, respectively. (D) Haploid cells containing rfa3-313^ts were transformed with plasmids expressing wild-type RFA3 (RFA3), GFP alone (GFP), and full-length (RFA3-GFP) and truncated (rfa3Δ46-GFP) alleles of RFA3. Complementation was determined by assaying for growth at 24°C and 37°C after 48–72 h.

FIG. 2.

Functional alleles of RPA localize to the nucleus, whereas nonfunctional truncations do not. (A) RFA1-GFP fusions under control of their endogenous promoter were expressed in wild-type cells and localized by fluorescence microscopy (GFP) and DIC microscopy of live cells. Rfa1-GFP localized predominantly to the nucleus, whereas the truncations remained in the cytosol. (B) Full-length RFA1-GFP and rfa1 deletion mutants under control of the GAL1 promoter were induced for 2 h with 2% galactose and examined for intracellular localization. RFA1-GFP is primarily in the nucleus, whereas deletion mutants lacking residues 338–343 or 1–329 are retained within the cytoplasm. (C, D) Full-length and truncated RFA2-GFP and RFA3-GFP fusions were expressed in live cells and examined by live cell microscopy. For Rfa2, the full-length protein and the functional rfa2Δ247-GFP fusion localized to the nucleus, whereas the nonfunctional rfa2Δ169-GFP did not. Rfa3-GFP was efficiently imported into the nucleus, whereas the fusion encoded by the rfa3Δ46 truncation remained primarily cytosolic. DIC, differential interference contrast; RPA, replication protein A.

FIG. 3.

Kap95 is important for efficient RPA import, whereas Msn5 is not essential. (A) RFA1-GFP was expressed in wild-type, msn5 deletion (msn5Δ), and kap95 temperature-sensitive cells (rsl1-1 and rsl1-4 alleles of kap95^ts) and grown at 24°C. Cells were retained at 24°C or shifted to 37°C for 2 h and observed for localization of Rfa1-GFP. Wild-type and msn5Δ cells contain Rfa1-GFP predominantly in the nucleus at both temperatures. kap95^ts cells exhibit an increased level of cytoplasmic fluorescence at both temperatures and nearly equivalent nuclear and cytoplasmic fluorescence in the rsl1-4/kap95^ts allele at 37°C. (B) The same experiment was performed with Rfa3-GFP expressed in wild-type, msn5Δ, and kap95^ts cells. Rfa3-GFP remained apparent in the nucleus in all three strains, with increased cytoplasmic fluorescence in the kap95^ts mutant.

FIG. 4.

Ybr137w is not essential for import of Rfa1 or Rfa2. Full-length Rfa1-GFP and Rfa2-GFP fusions were expressed in cells lacking the RPA-binding protein Ybr137w (ybr137wΔ) and in wild-type cells. Both Rfa1-GFP and Rfa2-GFP are localized to the nucleus with no change in localization in the absence of Ybr137w.

FIG. 5.

Truncated Rfa-GFP fusions that fail to enter the nucleus also fail to bind other members of the RPA complex. Whole cell protein extracts containing one Rfa protein fused to protein A (PrA) and another linked to GFP were immunoprecipitated using IgG-sepharose beads and western blotted using anti-GFP antibodies. For each panel, the top blot contains proteins isolated by immunoprecipitation (I.P.) and the bottom blot contains total soluble protein (total extract). (A) Full-length Rfa1 precipitates functional Rfa2- and Rfa3-GFP fusions (Rfa2-GFP, Rfa2Δ247-GFP, and Rfa3-GFP) but does not associate with nonfunctional C-terminal truncations (Rfa2Δ169-GFP and Rfa3Δ46-GFP). (B) Only full-length Rfa3-GFP was precipitated by Rfa2-PrA. (C) Rfa3-PrA binds functional Rfa1- and Rfa2-GFP fusions (Rfa1-GFP, Rfa2-GFP, and Rfa2Δ247-GFP) but not inviable truncations (Rfa1Δ353-GFP, Rfa1Δ330-GFP, and GFP).

FIG. 6.

Rfa1-GFP is mislocalized to the cytoplasm in rfa2^ts and rfa3^ts mutant cells. Plasmids expressing Rfa1-GFP or cNLS-GFP were transformed into wild-type, rfa2-215^ts, and rfa3-313^ts cells. Cells were grown overnight in media lacking galactose, and then induced for Rfa1-GFP expression by addition of galactose 3 h before microscopy. Rfa1-GFP is detectable in the nucleus of wild-type cells, but remains in the cytoplasm in rfa2^ts and rfa3^ts cells. The cNLS-GFP control is nuclear in all three strains. cNLS, classical nuclear localization signal.