The functionally conserved nucleoporins Nup124p from fission yeast and the human Nup153 mediate nuclear import and activity of the Tf1 retrotransposon and HIV-1 Vpr

Abstract

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.

Figure 1.

Nup124 is required for translocation of Tf1-Gag from the NPC into the nucleus. An inducible YFP-tagged Tf1-Gag construct was expressed in multicopy plasmids in WT (YNB540, top) and Δnup124 (YNB541, bottom) host strains. The nuclear envelope and Tf1-Gag:YFP were visualized by indirect immunofluorescence using MAb414 and GFP antibodies that react with FXFG-containing nucleoporins and YFP, respectively (GFP polyclonal antibody recognizes YFP). Nuclear DNA was stained with DAPI. Strains were grown overnight to saturation (stationary phase) in the absence of thiamine to induce nmt1-Tf1 expression. Images were acquired through a 100× objective lens using a Zeiss LSM 510 confocal microscope. Cells were imaged in Z-series and deconvolved. MAb414 (red) staining was used to fix the upper and lower limits of the Z-series. Figure depicts a single plane Z-section passing through the center of the Z-series.

Figure 2.

Functional domains of Nup124. (A) Multicopy plasmids containing full-length and mutant versions of Nup124 whose transcription was under the control of its native promoter were transformed into a Δnup124 mutant strain YNB19 along with the Tf1 reporter plasmid pHL 449-1. (B) Previously described genetic assays (Balasundaram et al., 1999) measured the ability of strains containing epigenetically expressed WT (YNB58) and mutant versions of nup124 (nup124^{ΔAA111–331}, YNB244 and nup124^{ΔAA571–1111}, YNB230) to reinstate levels of Tf1 cDNA recombination and transposition in a Δnup124 mutant (YNB8) to that of endogenous or epigenetically expressed WT levels. PROT^fs (YNB10) contains a Tf1-reporter plasmid with a protease frameshift that blocks the expression of (Tf1) protease, reverse transcriptase, and integrase and serves as a negative control for the assay. Because protease is involved in the processing of the Tf1 genome, all the activities of the retrotransposon are blocked as a result of the frameshift mutation. IN^fs (YNB11) contains a Tf1-reporter plasmid with a frameshift in the integrase so Tf1 transposition is decreased but not Tf1 cDNA recombination. (C) Cellular localization of Nup124p. The Δnup124 mutant expressing the full-length (3HA:Nup124, YNB58) or lacking the N-terminal domain (3HA: nup124^{ΔAA111–331}, YNB244) or FXFG-repeat domain (3HA: nup124^{ΔAA571–1150}, YNB230) were prepared for immunofluorescence microscopy as given in Materials and Methods. The red signal in the panels on the left are specific for the HA-tagged proteins by virtue of their immunoreaction with the anti-HA MAb, 12CA5.

Figure 3.

HIV-1 Vpr activity in S. pombe is Nup124p-dependent. (A) Absence of Nup124p enables cells to be more resistant to Vpr-induced cell killing activity. WT (YNB560) and Δnup124 (YNB562) cells expressing HIV-1 Vpr were grown overnight in medium containing 15 mM thiamine (Vpr expression-off) and washed twice in medium lacking thiamine. “Washed cells” were used as the source to be streaked for single colonies on plates with (Vpr expression-off) or without (Vpr expression-On) 15 μM thiamine. The top sectors of plates contained WT and Δnup124 strains with control plasmids lacking Vpr (YNB563 and YNB557, respectively). (B) To ascertain whether Δnup124 cells had a growth advantage over WT expressing HIV-1 Vpr, the washed cells described above were spread on plates at the indicated cell densities without thiamine to induce for expression of Vpr. Plates were photographed after 5 d incubation at 32°C.

Figure 4.

Localization of HIV-1 Vpr in the presence and absence of Nup124p. WT (YNB560, top) and Δnup124 (YNB562, bottom) strains expressing HIV-1 CFP:Vpr under the control of an nmt1 promoter were grown in the absence of thiamine and prepared for immunofluorescence as described in Materials and Methods. The nuclear envelope and HIV-1 CFP:Vpr were visualized by indirect immunofluorescence with MAb414 antibody that specifically reacts with FXFG-containing nucleoporins and αnti-GFP antibody, respectively. Nuclear DNA was stained with DAPI. Arrows indicate nuclear blebs or cytoplasmic projections. Experimental and image-capture conditions were identical to those detailed in Figure 1. Figure shown depicts a single plane Z-section passing through the center of the series.

Figure 5.

Coimmunoprecipitation of Nup124p with Tf1-Gag and HIV-1 Vpr. (A) Proteins were transcribed and translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine. (B) Translation products were mixed in various combinations as indicated. Coimmunoprecipitation assays were performed as described in Materials and Methods with the appropriate antibodies as indicated. The immunoprecipitates were resolved on 8–16% SDS-PAGE and detected by fluorography. The relative mobilities of full-length/+tag proteins are indicated by arrows.

Figure 6.

Schematic representation of ScNsp1p, ScNup1p (from S. cerevisiae), SpNup124p (from S. pombe), huNup153 (from human), and sequence of the conserved domain(s) in SpNup124p and HuNup153. (A) Schematic diagram showing the relative positions of the FXFG-repeat and other unique and conserved domains of Nup153 and Nup124p (see text for details). (N) denotes the number of the FXFG-repeats within the domain. (B–C) The Nup124p amino acid sequence from the N-terminal (AA 272–454) region that is similar to the N-terminal region (AA 448–634) of huNup153. Shaded stretches of amino acids are numbered boxes 1–4 and were aligned using the ClustalW program. Identical residues are underlined and in boldface type, and similar residues are boxed in dark gray. (D) The alignment of the C-terminal (FXFG) domain of the indicated proteins. Asterisk (*), identical residues; colon (:), highly conserved residues; period (.), less conserved residues. Amino acid ranges are indicated at the start and finish of each peptide sequence.

Figure 7.

Coimmunoprecipitation of Nup153 with Tf1-Gag and HIV-1 Vpr. (A) Indicated proteins were transcribed and translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine as depicted in Figure 5A. Translated products as shown were mixed in equimolar amounts in the indicated combinations. Coimmunoprecipitation and all other experimental conditions were identical to those for Figure 5. (B) Coimmunoprecipitation of Kap95 with Tf1-Gag and HIV-1 Vpr. ³⁵S-labeled proteins (lanes 3–5) were transcribed and translated in rabbit reticulocyte lysates as depicted in Figure 5A. Translated products were mixed in equimolar amounts in the indicated combinations. Coimmunoprecipitation and all other experimental conditions were identical to those for Figure 5.

Figure 8.

Rescue of Tf1 transposition in a nup124 mutant by a unique domain of Nup153. (A) Genetic assays to test the efficacy of Tf1 cDNA recombination and transposition were conducted exactly as described earlier in Figure 2 so as to measure the ability of epigenetically expressed mutants (3–6) to reinstate levels of Tf1 cDNA recombination and transposition in a Δnup124 mutant (1) compared with epigenetically expressed Nup124 (2) and the null strain (1). (B) Strains expressing full-length versions of Nup153^AA1–1475 (YNB964–965), Nup124p^AA1–1159 (YNB895), and Δnup124 (YNB893) were assayed for Tf1 activity as described above.

Figure 9.

Overexpression of unique domains of Nup124p and Nup153 inhibit Tf1 activity without significantly affecting growth. (A) Multicopy plasmids containing fragments encoding the indicated amino acids from Nup124p and Nup153 whose transcription was under the control of either the nmt81 (weak) and nmt1 (strong) promoter were tested in a WT strain for Tf1 activity. (1) Null mutant containing an empty vector served as a negative control (YNB893). (2) WT strain expressing genomic Nup124 and served as a positive control (YNB932). All other strains used are as indicated. (3–4) Multicopy plasmid with Nup124 expressed from its native promoter in a null mutant background. (5–6, YNB) Multicopy plasmid with Nup124 expressed from an nmt81 promoter in a null mutant background. (7–8, YNB909–10), (11–12, YNB933–34), and (9–10, YNB911–12), (13–14, YNB935–36) represent Nup124p and Nup153 fragments, respectively, being overexpressed from the indicated promoters. (B) Western blots of nup124^AA264–454p and nup153^AA449–634p without (Off) and with (On) induction of nmt81 and nmt1 promoters as indicated. Equal volumes of crude extracts from 30 ODs of cells were subjected to PAGE on 8–16% gels. Arrow indicates the protein species recognized by a 1:1000 dilution of anti-V5 HRP (R-961-25, Invitrogen, Carlsbad, CA). (C) The indicated strains were streaked for single colonies onto EMM plates-ura-leu with (Tf1-Off) and without (Tf1-On) thiamine and incubated at 32°C.