Fig. 1. Construction of the forced localization cassettes. (a) Cassettes containing two NLS or NES localization motifs separated by a T7 epitope tag were added to the C-terminus of Cln2 and Cln3. (b) Active and inactive versions of both a SV40 nuclear localization signal (NLS) and a PKI nuclear export signal (NES) were created. Inactive versions contain point mutations previously shown to prevent transport activity in S. cerevisiae (Nelson and Silver, 1989) (Murphy and Wente, 1996). (c) The localization cassettes direct GFP to the expected locations. Forced localization cassettes fused to GFP under control of the GAL1,10 promoter were grown to mid-log growth phase. GFP expression was induced by the addition of 0.1% galactose for 1-2 hours, and live cells were subsequently stained, observed and digitally captured as described in Materials and Methods. DIC, differential interference contrast; Hoechst represents DNA staining; –A represents the active/functional transport sequences; –I represents the inactive/nonfunctional transport sequences.
