FIGURE 8.

Association of Lsb1 with nuclear-ER rim. A–G, representative images of live cells are shown. At least 100 cells were analyzed for each experiment. Wild type GFP-Lsb1 and its mutant derivatives were expressed from the copper-inducible promoter, P_CUP1. A, GFP-Lsb1 forms a rim and small dots at the cell periphery in 40–50% of cells growing at 25 °C. At 30 °C, GFP-Lsb1 is not present in the rim but is distributed over the cytoplasm and forms small dots in 96% of cells. B, GFP-Lsb1 rim, formed at 25 °C, is colocalized with the nuclear-ER rim. Live cells expressing GFP-Lsb1 from P_CUP1 were stained with Hoechst to indicate the positions of the nuclei. C, Lsb1 colocalizes with ER marker protein Sbh1. Proteins were expressed from plasmid promoters P_CUP1- mCherry-Lsb1 and P_GAL-GFP-Sbh1 for 18 h. Colocalization was detected in all cells. D, GFP-Lsb1 expressed from the P_CUP1 promoter colocalizes with nuclear envelope protein Mlp1-RFP expressed from endogenous promoter. Colocalization was detected in all cells. E, GFP-Lsb1 mutant deficient in processing (Y182A,Y183A) is present in the nuclear nuclear-ER rim in 40–50% of cells, whereas the truncated version (1–183 amino acids) corresponding to the processed form of Lsb1 is observed only in the cytoplasm in all cells growing at 25 °C. F, ubiquitination affects localization of Lsb1 to the nuclear-ER rim. GFP-Lsb1 WT and mutants not deficient in ubiquitination (K41R and W90S) do not associate with the nuclear-ER rim in almost all cells growing at 30 °C. However, Lsb1 mutants deficient in ubiquitination (K79R, and K41R,K79R, and P135A,P136A) associate with the nuclear-ER rim in 60–80% of cells growing at 30 °C. The W90S mutant, defective in interaction with Las17, does not form small dots as has been reported previously. G, deletion of get2Δ abolishes localization of wild type and ubiquitination-deficient (P135A,P136A) mutant GFP-Lsb1 to nuclear-ER rim at 25 °C.