6864 Nucleic Acids Research, 2007, Vol. 35, No. 20 integration of a FLAG epitope onto the N-terminus of the Scp160 open reading frame. All recombinant DNA manipulations were performed according to standard procedures (30) utilizing the XL10-gold (Stratagene) strain of Escherichia coli. All mutagenesis was done using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). Experiments were carried out at 348C unless otherwise noted. Lysate preparation and immunoblot analysis Cells were grown to mid-log phase and harvested by centrifugation (5 min, 4000g at 48C). Following two washes with ddH2O, cells were lyzed by vortex agitation with an equal volume of glass beads in 0.5 ml lysis buﬀer (25 mM Tris pH 7.2, 50 mM KCl, 30 mM MgCl2, 1.5% Triton). Lysates were then transferred to a clean microfuge tube and centrifuged for 5 min at 3000 g at 48C. The supernatant was again transferred to a clean microfuge tube and centrifuged at 12 000 g for 10 min at 48C. Immunoblot analysis was performed essentially as described previously (22). Brieﬂy, samples to be analyzed were mixed with sample buﬀer, boiled and run through a 10% SDS–polyacrylamide gel. The gel was then electro- blotted onto a nitrocellulose membrane (Bio-Rad) and GFP-fusion proteins were detected by incubation of the membrane with a 1:10 000 dilution of an a-GFP polyclonal antibody (31). Direct fluorescence microscopy Imaging was performed as described previously (32). In brief, yeast cells transformed with plasmids expressing GFP-fusion proteins were grown to mid-log phase in media lacking uracil and supplemented with adenine. Cultures were split and half was left at room temperature while the other half was shifted to 378C for 2 h. Direct ﬂuorescence and DIC images were collected using an Olympus BX epiﬂuorescence microscope equipped with a Photometrics Quantix digital camera. IP Lab Spectrum software was used to capture images of cells. Prior to imaging, cells were incubated with 5 mM Hoechst dye to visualize the chromatin within the nucleus. Functional analysis of Scp160 variants in vivo To assess the function of an Scp160 NLS variant (Scp160-NLSmut) four lysine residues (321, 322, 331 and 333) within the predicted NLS were changed to alanines. We then tested whether this variant of Scp160 could rescue the synthetic lethality of an Dscp160Deap1 mutant using a standard plasmid shuﬄe assay (33). Brieﬂy, Scp160-NLSmut (LEU2) was transformed into an Dscp160Deap1 double mutant containing a wild-type URA3 maintenance plasmid. Cells were inoculated into liquid medium and grown to saturation for 2 days. Cell numbers were equalized, serially diluted 1:10, and then spotted onto control plates of synthetic medium lacking leucine and test plates of synthetic medium lacking leucine and containing the drug 5-ﬂuororotic acid (5-FOA). 5-FOA is toxic to cells containing the URA3 gene product and, therefore, the only cells that grow on 5-FOA plates have lost the URA3 plasmid and retain only the LEU2 test plasmid (34). Test plasmids encoding a second wild-type copy of SCP160 and empty vector (pRS315) were analyzed in parallel as positive and negative controls, respectively. Plates were incubated for two days at 348C. RESULTS Scp160-GFP is localized to the cytoplasm at steady state Figure 1A shows a schematic representation of the domain structure and postulated sequence motifs of Scp160. The predicted NES sequence is contained within the N-terminal portion of the protein, while the predicted NLS is located between KH domains three and four (KH3 and KH4), spanning a few residues into both KH3 and KH4. The amino acid sequence for each predicted transport signal is shown. Figure 1B shows that, as previously reported (23–25), Scp160-GFP is localized to the cytoplasm and excluded from the nucleus. To conﬁrm that the GFP signal reﬂects the localization of full-length Scp160, soluble lysates of wild-type cells expressing Scp160-GFP were subjected to immunoblot analysis with an a-GFP antibody (Figure 1C). Lysate from cells expressing NLS-NES- GFP served as a control for the speciﬁcity of the antibody. FLAG-Scp160-GFP migrated as a single band at 200 kDa while NLS-NES-GFP migrated at 35 kDa. As an additional control, we tested whether the GFP tag impacts the function of Scp160 by assaying the ability of Scp160-GFP to replace wild-type Scp160 in vivo using a plasmid shuﬄe assay. A TRP1 plasmid containing SCP160-GFP was transformed into Dscp160Deap1 yeast containing a wild-type URA3 maintenance plasmid of SCP160. Although Scp160 is not essential for cell viability (23), Scp160 becomes essential when the spindle pole body protein, Eap1 (35), is also deleted (36). Thus, we can employ this double mutant background where Scp160 is essential to rapidly assess Scp160 function. Transformants containing both the wild-type URA3 plasmid and the TRP1 test plasmid were streaked onto synthetic complete plates lacking uracil and tryptophan (control) and 5-FOA plates lacking tryptophan. No signiﬁcant growth defect was observed in the Scp160-GFP versus the untagged Scp160 (data not shown). As expected, cells containing vector alone failed to grow, conﬁrming the requirement for Scp160 in this genetic background. Scp160 does not accumulate in the nucleus in a NES export mutant To begin to assess whether Scp160 can enter the nucleus, we took advantage of the observation that Scp160 contains a predicted classical NES (20). If this NES mediates nuclear export of Scp160, then blocking the NES-dependent export pathway should cause nuclear accumulation of Scp160. To block this pathway, we utilized a conditional mutant of the NES receptor, Crm1 (37). crm1-3 cells show defects in export of NES- cargo (38). Plasmids encoding Scp160-GFP or control cargoes either containing a classical NES, NLS-NES-GFP (39), or lacking an NES, RGG-Nab2-GFP (32),