A B NES 52L P S L K D L P S L61 321 K K L A S N L D E E K V K 333 NLS KH1 KH2 KH3 KH4 KH5 KH6 KH7 KH8 KH9 KH10 KH11 KH12 KH13 KH14 Nucleic Acids Research, 2007, Vol. 35, No. 20 6865 C 250 37 MW kDa control Scp160-GFP Scp160-GFP NLS-NES-GFP Scp160-GFP Mlp1-RFP Hoescht MERGE DIC Figure 1. Scp160 is localized to the cytoplasm at steady state. (A) Schematic representation of Scp160 domain structure and location of putative nuclear targeting motifs. The predicted NLS is indicated by the ﬁlled diamond and the predicted NES is indicated by the open diamond. The sequences for the predicted NLS and NES motifs as well as their positions within the open reading frame are indicated. Numbered boxes represent the 14 KH domains. (B) The Scp160-GFP protein was expressed in wild-type cells that express a red-ﬂuorescent protein-tagged nuclear rim protein, Mlp1 (56) to mark the position of the nucleus. Fluorescent protein localization was examined by direct ﬂuorescence microscopy. Cells were also stained with Hoechst dye to mark the position of chromatin within the nucleus. A merged ﬂuorescence image is shown as well as the corresponding DIC image. (C) Immunoblot analysis of GFP in protein lysate from wild-type cells transformed with plasmids encoding FLAG-Scp160-GFP (right lane) or a control NLS-NES-GFP (left lane). Migration of protein standards is indicated to the left of the image. The position of the bands corresponding to FLAG-Scp160-GFP and NLS-NES-GFP is indicated. were transformed into wild type and crm1-3 cells. Protein localization was assessed by direct ﬂuorescence micro- scopy. Scp160 remained strictly localized to the cytoplasm in crm1-3 cells at the non-permissive temperature with no apparent nuclear concentration (Figure 2). In contrast, the control NLS-NES-GFP accumulated in the nucleus of crm1-3 cells. The localization of a control non-NES cargo protein, RGG-Nab2-GFP, was not altered in the crm1-3 cells compared to wild-type cells. These results indicate that either Scp160 does not enter the nucleus or its export is not dependent on the Crm1 export pathway. Scp160 does not accumulate in the nucleus when poly(A) RNA export is blocked Since Scp160 associates with mRNA (19), we hypothe- sized that Scp160 localization could depend on ongoing mRNA synthesis and mRNA export. A number of mRNA-binding proteins that undergo nucleocytoplasmic shuttling depend both on ongoing transcription and mRNA export (32,40). Therefore, we examined the steady-state localization of Scp160 in both rpb1-1 cells where RNA polymerase II transcription is decreased (41) and mex67-5 mutant cells where poly(A) RNA export from the nucleus is blocked (42,43). As controls we also examined the localization of NLS-NES-GFP, which is not aﬀected by mRNA export (44), and RGG-Nab2-GFP, which depends on ongoing mRNA export and synthesis for export from the nucleus (44). No nuclear accumulation of Scp160 was observed in either the mex67-5 or rpb1-1 cells (Figure 3). As expected, the control mRNA binding protein, RGG-Nab2-GFP, accumulated in the nucleus of both mex67-5 and rpb1-1 cells following a shift to the non-permissive temperature. No change in the localization of NLS-NES-GFP was observed. These results indicate that either Scp160 does not enter the nucleus or its export does not depend on ongoing mRNA synthesis or mRNA export. Scp160 does not accumulate in the nucleus of cells with an export-deficient NPC As a ﬁnal test of whether Scp160 enters the nucleus, we exploited a nuclear pore mutant that blocks all known nuclear export pathways, rat7-1 (32,45–47). We hypothe- sized that if Scp160 enters the nucleus it should accumulate in the nucleus of rat7-1 cells. Although RAT7 (NUP159) is essential, the rat7-1 allele is a temperature-sensitive allele that can be shifted to the non-permissive temperature to inactivate Rat7/Nup159 and block export pathways (46). We examined the localization of Scp160-GFP and control proteins, NLS- NES GFP and RGG-Nab2-GFP, in rat7-1 cells. Following the shift to the non-permissive temperature, Scp160-GFP remained cytoplasmic in rat7-1 cells similar to wild-type cells (Figure 4). In contrast, NLS-NES-GFP