FIGURE 2.

Yar1 is a shuttling protein. A, Yar1 has a cytoplasmic steady-state localization. A strain containing a chromosomal YAR1-GFP fusion was grown to mid-log phase and the localization of the fusion protein was inspected by fluorescence microscopy. Note that Yar1-GFP is predominantly localized in the cytoplasm, while only a faint nuclear staining is visible. B, Yar1 transiently enters the nucleus. A leptomycin sensitive crm1 strain deleted for YAR1 and containing YAR1-GFP on a centromeric plasmid was grown at 30 °C in SDC-medium to logarithmic growth phase. Cells were inspected by fluorescence microscopy with or without treatment with 200 ng/ml leptomycin B (LMB) for 60 or 120 min. The crm1 strain transformed with plasmids expressing Rps3-GFP and Rpl25-GFP served as positive control for export inhibition of pre-40S and pre-60S subunits. C, Rps3 contains a monopartite classical NLS. Multiple Sequence Alignment of the 15 N-terminal amino acids of Rps3 from Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Homo sapiens (Hs). D, N-terminal 15 amino acids of Rps3 target a 3xyEGFP reporter to the nucleus. Amino acids 1–15 of Rps3 were fused to a triple yEGFP reporter and the fusion protein was expressed from a plasmid under the control of the ADH1 promoter in cells expressing the nucleolar marker protein Nop58-RedStar2. As controls, 3xyEGFP and SV40NLS-3xyEGFP were also localized.