Overexpression of Kap114 or eIF-5A suppresses the temperature sensitivity of TBP mutant strains. (A) YSB66, a strain bearing the temperature-sensitive mutation Y94C in TBP, was transformed with a 2μ plasmid containing the KAP114/TDS2 gene (YEpTDS2a), with the 2μ vector YEp24 alone (vector) or with a plasmid containing the wild-type TBP gene (pRS316-IID). Individual transformants were streaked onto selective media and were grown at 30°C (not shown) and 37°C for 3 days. (B) YSB67, a temperature-sensitive strain bearing the mutations K133L, K138L in TBP (9, 15), was transformed with a 2μ plasmid containing the TDS3 clone, YEp24 alone (vector), a 2μ TIF51A (eIF-5A) plasmid (YEp352T-5A) (16), or a 2μ plasmid encoding a nonhypusinated mutant of TIF51A [YEp352T-5A(K51R)] (16). Individual transformants were streaked onto selective media and were grown at 30°C and 38°C for 3 days.

The importin Kap114 is most similar to the yeast protein Cse1. The amino acid sequence of Kap114 (S. cerevisiae ORF YGL241W) was aligned with its most similar protein, the importin Cse1, by using the Clustal method (DNAstar, Madison, WI). Identical amino acids are shaded black, and similar amino acids are shaded gray.

TBP is partially mislocalized to the cytoplasm in cells lacking Kap114. (A) A centromeric plasmid encoding GFP-TBP (pDP15GFPTBP) (2) was transformed into a Δkap114 strain (PSY1784) and the isogenic wild-type strain (PSY1785). A centromeric plasmid encoding ³HA-TBP (³HA-TBP/YCplac111) was transformed into a Δkap114 strain (YSB103) and the isogenic wild-type strain (YSB105). Whole cell lysates were prepared from cells grown at 25°C in selective media. Equal amounts of protein were resolved by SDS/PAGE and then were analyzed by immunoblot using α-GFP antibodies (lanes 1–3) or 12CA5 α-HA antibodies (lanes 4–6). Lanes: 1, Δkap114 cells + GFP-TBP; 2, wild-type cells + GFP-TBP; 3, Δkap114 cells; 4, Δkap114 + ³HA-TBP; 5, wild-type cells + ³HA-TBP; 6, wild-type cells + vector (YCplac111). (B) The strains expressing GFP-TBP described in A also were transformed with either vector alone (YEp24) or with a 2μ plasmid expressing Kap114 (YEpTDS2a). Cells were grown to mid-log phase at 25°C in selective media and then were analyzed by fluorescence microscopy. Cells were visualized by using a GFP filter set (Left) and by Nomarski optics (Right). (Top, wild-type + vector) The localization of GFP-TBP in wild-type cells (PSY1785) that have been transformed with pDP15GFPTBP and YEp24 vector. (Middle, Δkap114 + vector) The localization of GFP-TBP in Δkap114 cells that have been transformed with pDP15GFPTBP and YEPp24 vector. (Bottom, Δkap114 + KAP114) The localization of GFP-TBP in Δkap114 cells that have been transformed with pDP15GFPTBP and the 2μ KAP114/TDS2 plasmid YEpTDS2. (C) The strains expressing ³HA-TBP in A were grown to mid-log phase at 25°C in selective media and were fixed, and immunofluorescence was performed by using 12CA5 α-HA antibodies and FITC-conjugated secondary antibodies. Cells were visualized by using a FITC filter set (Left), a DAPI filter set (Center), and Nomarski optics (Right). (Upper) The localization of ³HA-TBP in wild-type cells (YSB105). (Lower) The localization of ³HA-TBP in Δkap114 cells (YSB103).

Overexpression of Kap114 increases TBP levels. Lysates of the TBP temperature-sensitive (ts) strain YSB66 and the isogenic wild-type strain (FY23) bearing either an empty vector (pRS425) or a high copy plasmid encoding Kap114 (pRS425-TDS2) were prepared from cells grown to mid-log phase. Equal amounts of protein were resolved by SDS/PAGE and were immunoblotted using α-TBP antibodies. Lanes: 1 and 2, wild-type cells; 3 and 4, temperature-sensitive TBP cells; 1 and 3, + vector; 2 and 4, +high copy Kap114.

Kap114-GFP localizes primarily to the nucleus. (A) Lysates of wild-type cells (FY23) and cells bearing an integrated Kap114-GFP gene (PSY1783) were prepared from cells grown to mid-log phase. Equal amounts of protein were resolved by SDS/PAGE and were immunoblotted using α-GFP antibodies. (B) Kap114-GFP cells (PSY1783) were grown in selective media at 25°C to mid-log and were analyzed by fluorescence microscopy. (Left) Cells under a GFP filter set. (Right) The corresponding cells by Nomarski optics.

Kap114 binds to TBP. (A) Whole cell lysates were prepared from cells bearing GFP fusions to either Kap95 (PSY1786), Sxm1 (PSY1267) (11), Kap114 (PYS1783), or Cse1 (PSY1262) (19). Lysates were incubated overnight with GST-TBP or GST bound to glutathione-agarose beads. Pellets (bound) and ≈10 μg of supernatants (unbound) were resolved by SDS/PAGE and were immunoblotted by using α-GFP antibodies. (B) The TBP/Kap114 interaction is disrupted by a nonhydrolyzable GTP analogue. Lysates were incubated in the presence and absence of 1 mM GTPγS or 1 mM GDPβS before binding as in A. (C) The blots in A also were probed with α-Srp1 antibodies (Kap114 and Kap95 lanes shown).