the importin/karyopherin-b family. However, import of cargoes that contain a cNLS involves an adaptor protein, importin-a, that serves as an NLS receptor to recognize these cargos in the cytoplasm (4). Importin-a and cNLS cargo interact with importin-b to form a trimeric import complex, which then translocates through the NPC (10). Once in the nucleus, a small GTPase, Ran-GTP (11,12), binds to importin-b causing a con- formational change that results in dissociation of the import complex (13). The importin receptors are then recycled back to the cytoplasm to participate in subse- quent rounds of import (14). In a similar manner, NES- dependent export involves the transport receptor, Crm1 (8,15). Like other export receptors of the importin-b family (16), Crm1 forms a trimeric complex with Ran- GTP and an export cargo (17). The trimeric complex is translocated through the NPC and once it reaches the cytoplasmic face of the NPC, Ran-GTP is hydrolyzed to Ran-GDP resulting in dissociation of the export complex (18). Many proteins contain predicted NLS and NES sequences required for entering and exiting of the nucleus. Often, the function of these predicted sequences is assumed in the absence of supporting experimental data. One example of such a case is the coding sequence of the Saccharomyces cerevisiae control of ploidy protein of 160 kDa, Scp160. Although the exact function of Scp160 is unknown, it is thought to modulate gene expression through interacting with RNA substrates (19). Scp160 contains 14 predicted K-homology (KH) RNA-binding domains and also sequence motifs that have been hypothesized to function as both a cNLS and a classical NES (20,21). Since Scp160 associates with speciﬁc mRNA transcripts (19), it has been hypothesized to shuttle into the nucleus and then exit the nucleus in complexes with mRNA. Despite the presence of a sequence noted as an NLS, the steady-state localization of Scp160 is clearly cytoplas- mic (22–25). This cytoplasmic localization is consistent with at least two models: (i) Scp160 is solely restricted to the cytoplasm; or (ii) Scp160 shuttles into and out of the nucleus but the rate of export exceeds the rate of import. To distinguish between these two possibilities, we exploited yeast mutants that block diﬀerent nuclear export pathways and assessed the localization of Scp160 in these mutant cells under conditions where these speciﬁc nuclear export pathways are blocked. If Scp160 does, in fact, shuttle between the nucleus and the cytoplasm, blocking its export would cause accumulation of the protein within the nucleus. However, if Scp160 does not shuttle, we would expect the localization to remain cytoplasmic despite blocked export pathways. This general approach has been used previously to uncover the nucleocytoplasmic shuttling properties of other proteins. For example, the yeast poly(A)-binding protein 1, Pab1, has a well-characterized function in the cytoplasm where it binds to the poly(A) tail of mRNA transcripts and modulates their stability and translation (26). Consistent with these functions, Pab1 is localized to the cytoplasm at steady state (26). However, recent studies show that Pab1 accumulates within the nucleus of Reference (41) (46) (38) (42) (56) (36) (57) (58) (58) (39) (32) This study This study This study Nucleic Acids Research, 2007, Vol. 35, No. 20 6863 cells that express a conditional mutant of the NES receptor, crm1-1, revealing the surprising ﬁnding that this protein with a well-characterized cytoplasmic function can enter the nucleus (27,28). These studies validate the use of export mutants to test whether Scp160 is also a shuttling protein. The goal of this study was to carry out a functional analysis of the nuclear targeting signals present in the Scp160 protein. To accomplish this goal, we ﬁrst exploited a number of conditional yeast mutants that impact nuclear export. We found that mutations that rendered each of the major export pathways nonfunc- tional did not cause accumulation of Scp160 within the nucleus, suggesting that Scp160 may not enter the nucleus. Second, we generated amino acid substitutions predicted to disrupt the function of the putative NLS of Scp160 and found that these mutations did not signiﬁcantly impair Scp160 function, indicating that the putative NLS is not critical for the key cellular function of Scp160. These data challenge the model that Scp160 performs critical functions in the nucleus, and suggest that Scp160 functions are carried out exclusively in the cytoplasm. MATERIALS AND METHODS Plasmids and yeast strains All yeast manipulations were performed according to standard protocols (29). All yeast strains used in this study are listed and described in Table 1. The wild-type yeast strain used for microscopy, JFY4493, was derived from the haploid parent strain W303 (MAT a ura3-1 leu2-3 his3-11, 15 trp1-1 ade2-1 can1-100 RAD5+, a gift from Dr R. Rothstein, Columbia University, NY, USA) by Table 1. Strains and plasmids used in this study Strains/plasmids Description ACY191 ACY194 ACY372 ACY545 ACY1508 JFY4247 JFY4493 pRS312 pRS315 pAC213 pAC980 JF4470 JF4823 JF4843 mat a rpb1-1, ura2-52, his4-53 mat a rat7-1, ura3-52, leu2 1, his3 200 mat a crm1-3, ade2-1, ura3, leu2 1, his3, trp1 mat a MEX67::KanMX4, ura3, leu2 1, his3 200, [+mex67-5 LEU2] mat a MLP1-RFP::KanMX4, ura3, leu2 1, his3 200, lys2 mat a SCP160::HIS3, EAP1::HIS3, ade2-1, ura3, leu2-3, his3-11, trp1-1, can1-100, [+SCP160-URA3] mat a FLAG-SCP160::SCP160, ade2-1, ura3-1, leu2-3, his3-11, trp1-1, can1-100 TRP1, CEN, AMP LEU2, CEN, AMP SV40 NLS-PK1 NES-GFP, URA3, 2 mM, AMP RGG Nab2-GFP, URA3, CEN, AMP SCP160-GFP, TRP1, CEN, AMP FLAG-SCP160, LEU2, CEN, AMP FLAG-SCP160-NLSmut, LEU2, CEN, AMP