FIGURE 4:

Nuclear targeting of Sis1 is dependent on nuclear localization sequences in Btn2 and Cur1 and requires the α-importin Srp1. (A) W303 yeast expressing GFP-tagged Btn2 or Cur1 from a low-copy plasmid were grown at 23 or 37°C or in the presence of the proteasome inhibitor MG132. Left, the GFP channel. Right, an overlay with an mCherry-tagged nuclear marker. (B) W303 yeast cells expressing GFP-tagged Btn2, Cur1, Btn2ΔNLS, or Cur1ΔNLS were subjected to fluorescence microscopy at 30°C. (C) BY4741 yeast cells carrying a GFP-tagged chromosomal copy of SIS1 were transformed with low-copy plasmids for Btn2, Cur1, Btn2ΔNLS, or Cur1ΔNLS. The resulting transformants were grown at 25°C and subjected to fluorescence microscopy. (D) Wild-type yeast (WT) or yeast carrying a temperature-sensitive mutation in SRP1 (srp1-31) were cotransformed with expression plasmids for Sis1-GFP and Orange (control), Btn2, or Cur1. The cells were subjected to fluorescence microscopy after a shift to the nonpermissive temperature for 1 h. The relative nuclear:cytosolic GFP pixel intensity of the strains was determined. *p = 2.7 × 10⁻⁸; **p = 0.0027; ***p = 2.9 × 10⁻⁸.