6866 Nucleic Acids Research, 2007, Vol. 35, No. 20 GFP DIC GFP DIC GFP DIC GFP DIC Wildtype crm1-3 Wildtype rat7-1 NLS- NES- GFP ∆ RGG- Nab2- GFP Scp160- GFP NLS-NES- GFP ∆ RGG- Nab2-GFP Scp160- GFP Figure 2. Localization of Scp160-GFP in crm1-3 and wild-type cells. The localization of Scp160-GFP was examined in crm1-3 cells following a 2 h shift to 378C where NES-dependent nuclear export is blocked. Scp160 is localized to the cytoplasm in both wild-type and crm1-1 cells. As controls, we also localized NLS-NES-GFP and RGG-Nab2-GFP. As expected, NLS-NES-GFP accumulates in the nucleus of crm1-1 cells, but RGG-Nab2-GFP, which is exported via an mRNA export-dependent pathway, does not. Corresponding DIC images are shown. GFP DIC GFP DIC GFP DIC NLS- NES- GFP ∆ RGG- Nab2- GFP Scp160- GFP A B Figure 4. Localization of Scp160-GFP in rat7-1 and wild-type cells. The localization of Scp160-GFP was examined in rat7-1 cells following a 30 min shift to 378C where all known transport pathways are blocked. As shown, Scp160 is localized to the cytoplasm in rat7-1 cells. As controls, we also visualized NLS-NES-GFP and RGG-Nab2- GFP. As expected both control proteins accumulate in the nuclei of rat7-1 cells. Scp160 K* K*LASNLDEE-- K*V K* Nucleoplasmin KRPAATKKAGQAKKKKLD p53 KRALPNNTSSSPQPKKK vector Scp160 Wildtype rpb1-1 mex67-5 Scp160-NLSmut Figure 3. Localization of Scp160-GFP in rpb1-1, mex67-5 and wild- type cells. The localization of Scp160-GFP was examined in wild-type, rpb1-1 and mex67-5 cells following a 1 h shift to 378C. Results indicates that Scp160 is localized to the cytoplasm in both rpb1-1 and mex67-5 cells. As controls, we also localized NLS-NES-GFP and RGG-Nab2- GFP. As expected, RGG-Nab2-GFP, which is exported in an mRNA export-dependent manner, accumulates in the nuclei of both rpb1-1 and mex67-5 cells, but NLS-NES-GFP does not. and RGG-Nab2-GFP both accumulated in the nuclei of rat7-1 cells. These results taken together with localization of Scp160 in other export mutants suggest that Scp160 does not enter the nucleus, at least under normal growth conditions. In vivo function of an Scp160 NLS mutant Based on its primary amino acid sequence, Scp160 has been predicted to contain a bipartite NLS (20). Like other previously characterized bipartite NLS sequences, Scp160 contains two stretches of basic amino acids separated by a short spacer region (Figure 5A). However, results of our localization studies suggest that the predicted NLS within Scp160 does not target the protein to the nucleus and, therefore, may not be functional. If this assessment is correct, amino acid substitutions within the key NLS Control 5-FOA Figure 5. Functional analysis of Scp160-NLSmut. (A) An alignment of the predicted NLS sequence from Scp160 with functional NLS sequences from nucleoplasmin and p53 is shown. The lysine residues that were changed to alanines to create Scp160-NLSmut are indicated by asterisks. (B) To assess the functional importance of the predicted NLS within Scp160, we tested whether an Scp160 variant with a mutant NLS (Scp160-NLSmut) could replace wild-type Scp160 in Dscp160Deap1 cells where Scp160 is required for viability (36). Cultures were grown to log phase and equal numbers of cells were serially diluted by orders of magnitude and spotted onto control plates lacking uracil and leucine (left) or test plates lacking leucine but containing 5-FOA (right). The top row shows cells carrying LEU2 vector (pRS315); the middle row shows cells containing a wild-type copy of Scp160; the bottom row shows cells that express Scp160-NLSmut as the sole copy of Scp160. residues in the sequence should not signiﬁcantly impact the function of Scp160. To test this hypothesis, four basic residues within the predicted NLS of Scp160 were changed to alanines to create Scp160-NLSmut (residues changed are indicated by asterisks in Figure 5A). This Scp160 variant was then assayed for its ability to rescue the synthetic lethality of the Dscp160Deap1 cells. Growth of Dscp160Deap1 cells expressing Scp160-NLSmut was not signiﬁcantly aﬀected compared to growth of these cells expressing wild-type Scp160 (Figure 5B). In contrast,