FIG 2.

Vps75 import depends on a classical nuclear localization signal and is mediated by Kap60. (A) Schematic of Vps75 with amino acid numbers, domains and NLS labeled. (B) The indicated GFP fusion proteins were expressed in vps75Δ, induced for 1 hour and observed by fluorescent imaging. KG,KG represents Vps75(K260G, K261G). DIC image is also shown (right panel). (C) Vps75(243–264)-GFP₂ and GFP₂ were observed in vps75Δ by fluorescent imaging after 1 hour induction. DIC image is also shown (right panel). (D) Recombinant MBP-Vps75 wild type, MBP-Vps75 truncated forms (indicated by V followed by amino acid numbers), MBP-Vps75(K260G, K261G) (abbreviated V(KG,KG)) and MBP-LacZ (negative control) were incubated with immobilized GST, GST-Kap60 and GST-Rtt109. The bound material from binding assays or 25% input samples were separated by SDS-PAGE and western blotted with anti-MBP antiserum (middle and right panels). 25% input samples of GST proteins were stained with Coomassie Brilliant Blue (CBB) (Left panel). (E) Vps75-GFP₂, Vps75(243–264)-GFP₂ or Yra1-GFP₂ was expressed in the indicated strains, which were grown at room temperature (RT) or 30°C or shifted to 37°C for 4 hours including 2h induction in -met and observed by fluorescent imaging. (F) Vps75-GFP₂ expression was induced for 2h in the strains indicated.