The Ty1 integrase protein can exploit the classical nuclear protein import machinery for entry into the nucleus

Abstract

Like its retroviral relatives, the long terminal repeat retrotransposon Ty1 in the yeast Saccharomyces cerevisiae must traverse a permanently intact nuclear membrane for successful transposition and replication. For retrotransposition to occur, at least a subset of Ty1 proteins, including the Ty1 integrase, must enter the nucleus. Nuclear localization of integrase is dependent upon a C-terminal nuclear targeting sequence. However, the nuclear import machinery that recognizes this nuclear targeting signal has not been defined. We investigated the mechanism by which Ty1 integrase gains access to nuclear DNA as a model for how other retroelements, including retroviruses like HIV, may utilize cellular nuclear transport machinery to import their essential nuclear proteins. We show that Ty1 retrotransposition is significantly impaired in yeast mutants that alter the classical nuclear protein import pathway, including the Ran-GTPase, and the dimeric import receptor, importin-alpha/beta. Although Ty1 proteins are made and processed in these mutant cells, our studies reveal that an integrase reporter is not properly targeted to the nucleus in cells carrying mutations in the classical nuclear import machinery. Furthermore, we demonstrate that integrase coimmunoprecipitates with the importin-alpha transport receptor and directly binds to importin-alpha. Taken together, these data suggest Ty1 integrase can employ the classical nuclear protein transport machinery to enter the nucleus.

Figure 1.

Analysis of retrotransposition in mutants of classical nuclear transport proteins. (A) A schematic of the Ty1 retrotransposition assay (38). Yeast cells are transformed with the URA3-marked pAR100 (His- cells) or pSD600 (His+ cells) test plasmid and patched onto SC ura⁻ glu plates. Patches are then replica plated to SC ura⁻ gal plates for 3 days at 25°C and sequentially replica plated to (1) SC ura⁻ glu; (2) YPD; (3) 5-FOA; and (4) SC his⁻ glu for pAR100 or YEPD + G418 for pSD600. (B) Transposition levels in nuclear protein import mutants. gsp1-1, ntf2-1, srp1-5, rsl1-L63A, and Δsxm1 mutant cells were transformed with the appropriate test plasmids and replica plated to selection medium. The final selection plates are YEPD + G418 (gsp1-1, ntf2-1, rsl1-L63A, and Δsxm1) and SC his⁻ glu (srp1-55). As controls, each plate also contained wildtype cells either expressing (+) or lacking (−) the appropriate test plasmid. (C) Transport mutants carrying the Ty1 test plasmid grow approximately as well as wildtype cells on selective media. gsp1-1, ntf2-1, rsl1-L63A and Δsxm1 cells were transformed with the pSD600 (G418) test plasmid while srp1-55 cells were transformed with the pAR100 (His) test plasmid. Cell numbers were equalized, cultures were serially diluted 10-fold, and spotted onto either YEPD + G418 plates (top) or SC-his (bottom) glu plates. Plates were then grown at 25°C for 3 days. Wildtype cells either carrying the appropriate Ty1 test plasmid (+) or an empty vector (−) served as positive and negative controls, respectively.

Figure 2.

Analysis of Ty1 proteins in nuclear transport mutants defective for transposition. (A) A schematic of the Ty1 lifecycle: (1) Ty1 mRNA is transcribed; (2) the mRNA is processed and exported to the cytoplasm; (3) Ty1 proteins are translated and processed; (4) virus-like particles (VLPs) then form; (5) the mRNA is reverse transcribed into cDNA; (6) Ty1 IN and Ty1 cDNA are imported into the nucleus; and finally, (7) IN functions to integrate the Ty1 genome into the cellular genome. (B) Ty1 proteins are translated and properly processed in nuclear transport mutants that show transposition defects. gsp1-1, ntf2-1, srp1-55, rsl1 L63A and Δsxm1 cells were transformed with test plasmid and induced in media containing galactose to undergo retrotransposition. As a control, wildtype cells were also transformed with the test plasmid (+) and a URA (pRS316) vector (−) and induced for retrotransposition. As a further control, Δsxm1 cells, which show no retrotransposition defect, were also transformed with the test plasmid and induced for retrotransposition. Immunoblot analysis of Ty1 reverse transcriptase (RT) and Gag proteins was performed using antibodies against each Ty1 protein (see ‘Materials and Methods’ section). As a loading control, an antibody directed against Pgk1 (3-PhosphoGlycerate Kinase 1) was used (Molecular Probes).

Figure 3.

Localization and expression of GFP-IN, IN NLS and IN NLS_mut in wildtype cells. (A) A schematic of the GFP-LacZ-Ty1 IN (32) and GFP₂-IN NLS constructs is shown. Full-length IN is under control of the GAL1 promoter (DSM1); IN NLS and IN NLS_mut are under control of the MET25 promoter (pAC1804 and pAC2431, respectively). Full-length IN contains three domains: an N-terminal zinc-finger domain, a central catalytic domain, and a C-terminal domain containing the IN NLS, consisting of basic regions 1 and 2 (BR1 and BR2). A GFP-fused truncated form of IN containing the last 54 amino acids (aa 582–636) (including BR1 and BR2 indicated in bold lettering—⁵⁹⁵SKKRSLEDNETEIKVSRDTWNTKNMRSLEPPRSKKRI⁶³¹) was used in most experiments and is referred to as GFP₂-IN NLS. Previously defined key lysine residues are highlighted in bold. (B) GFP-fused IN and GFP₂-IN NLS are targeted to the nucleus while GFP₂-IN NLS_mut is not. Cells expressing either full-length IN, GFP₂-IN NLS, or GFP₂-IN NLS_mut (⁵⁹⁶KKR⁵⁹⁸ → AAA and ⁶²⁸KKR⁶³⁰ → AAA) were analyzed using direct fluorescence microscopy (GFP). Cells were stained with Hoechst to indicate the location of the nucleus and corresponding DIC images are shown.

Figure 4.

GFP₂-IN NLS localization in classical nuclear protein import mutants. (A) GFP₂-IN NLS is mislocalized to the cytoplasm in temperature-sensitive mutants of Ran and its regulatory proteins. Wildtype, gsp1-1, rna1-1 and prp20-1 cells expressing GFP₂-IN NLS were grown to log phase at the permissive temperature and then shifted to 37°C for 3 h and analyzed by direct fluorescence microscopy. Hoechst dye was used to visualize the location of the nucleus. Corresponding DIC images are shown. (B) GFP₂-IN NLS is mislocalized to the cytoplasm in a temperature-sensitive mutant of importin-α and β. Wildtype, srp1-55 (importin-α mutant), or rsll L63A (importin-β mutant), cells expressing either GFP₂-IN NLS (top) or a control Nab2-GFP protein (bottom) were shifted to 37°C for 3 h and analyzed by direct fluorescence microscopy. Hoechst dye was used to visualize the location of the nucleus. Corresponding DIC images are shown.

Figure 5.

The initial rate of GFP₂-IN NLS import is reduced in classical nuclear protein import mutants. The initial rate of nuclear import of GFP₂-IN NLS is dramatically reduced in gsp1-1, rna1-1, prp20-1, srp1-55 and rsl1 L63A cells relative to wildtype cells. Wildtype, gsp1-1, rna1-1, prp20-1, srp1-55 and rsll L63A cells containing GFP₂-IN NLS were analyzed using a semi-quantitative kinetic import assay as described in the ‘Materials and Methods’ section (43). Import in wildtype cells was analyzed at both 37 and 18°C. Data obtained was identical at both temperatures and results are shown only for the 37°C sample. The percentage of cells with GFP₂-IN NLS nuclear accumulation was plotted versus time to obtain an approximation of initial import rates.

Figure 6.

Ty1 IN interacts with importin-α. (A) IN NLS coimmunoprecipitates with importin-α from whole cell yeast lysate. Yeast cells expressing importin-α-myc were transformed with plasmids expressing GFP (control), GFP₂-IN NLS, or GFP₂-IN NLS_mut. Cell lysates were prepared from induced cultures and epitope-tagged importin-α protein was purified using anti-myc beads as described in the ‘Materials and Methods’ section. GFP proteins present in the bound (B) and unbound (UB) fractions were detected with an anti-GFP antibody. An anti-myc antibody was used to ensure enrichment of importin-α-myc in the bound fraction. (B) Importin-α binds directly to IN NLS in vitro. Purified ΔIBB-importin-α was incubated with GST-IN NLS, or as controls, GST alone or GST-IN NLS_mut. GST-fusion proteins and any associated proteins were purified on glutathione sepharose beads as described in the ‘Materials and Methods’ section. Bound fractions were collected and resolved on a 10% SDS-PAGE gel. As a control, 4 μg of purified ΔIBB-importin-α was loaded in the first lane (Input). ΔIBB-importin-α migrates at approximately 70 kDa; GST migrates at approximately 25 kDa; and GST-IN NLS migrates at approximately 35 kDa.