yeast-containing vector alone (pRS315) were unable to grow on 5-FOA medium. These results suggest that the predicted NLS does not aﬀect the global in vivo function of Scp160. DISCUSSION This study was designed to test whether the yeast RNA-binding protein, Scp160, is a nucleocytoplasmic shuttling protein and hence to examine the function of the predicted nuclear targeting signals within Scp160. Previous studies have revealed that Scp160 is localized to the cytoplasm at steady state (22–25) and also provided evidence that Scp160 associates with speciﬁc mRNAs (19). Given the presence of sequences reminiscent of both an NLS and an NES motif (21), one possible mechanism for Scp160 function, consistent with other shuttling RNA binding proteins (26,32), is that Scp160 could enter the nucleus, associate with RNA substrates, and then accompany these RNA transcripts to the cytoplasm. However, results of our analysis indicate that Scp160 is not likely to enter the nucleus. Furthermore, the predicted NLS is not required for Scp160 function. Taken together, these experiments argue for an alternative model for Scp160 function. More generally, this study underlines the importance of experimental analysis to support the identiﬁcation of putative nuclear transport signals in a protein of interest. In this study, we localized Scp160 in a number of conditional mutants to test the hypothesis that Scp160 is able to move into the nucleus and accompany transcripts to the cytoplasm by virtue of its predicted NLS and NES signals. We localized Scp160 in mutants blocked in well-characterized export pathways, including NES-protein export (crm1-3), mRNA biosynthesis (rbp1-1), mRNA export (mex67-5) and all known export pathways (rat7-1). Despite its putative targeting sequences, Scp160 did not accumulate in the cell nucleus when any of these pathways were blocked. Furthermore, we have shown that amino acid changes within the predicted NLS of Scp160 do not severely aﬀect Scp160 function in vivo. Due to the slight growth defect we observed when four lysines within the NLS were changed to alanines, we cannot eliminate the possibility that these residues are important for some other function of Scp160, however, taken together our data suggest that Scp160 does not enter the nucleus. We cannot, however, eliminate the possibility that Scp160 enters the nucleus under speciﬁc growth conditions not tested here. There is also the possibility that Scp160 export could depend on a pathway not examined here, but the nuclear pore mutant, which blocks all known export pathways, makes this interpretation of our results unlikely. As a whole, these data suggest that Scp160 functions exclusively in the cytoplasm where it most likely associates with mRNA. A further extension of these ﬁndings is that the pleiotropic eﬀects observed in Dscp160 cells (20,23,48), including increased DNA content per cell, missegregation of nutritional markers through meiosis and abnormal cell morphology, are likely due to indirect Nucleic Acids Research, 2007, Vol. 35, No. 20 6867 eﬀects, perhaps caused by changes in expression of genes required for these pathways. Thus, deregulation of mRNA transcripts that bind to Scp160 may cause some of the phenotypes observed in Dscp160 cells. Alternatively, some phenotypes may be due to loss of direct interaction of Scp160 with other proteins within the cell resulting in incompletely formed mRNP complexes. Taken with the localization studies described here, we conclude that the predicted nuclear targeting sequences in Scp160 are non-functional. Scp160 is most closely related to the vigilin class of proteins, which, like Scp160, contain a large array of 14 to 15 repeated KH domains (21). Vigilins control gene expression through multiple mechanisms including reg- ulating mRNA stability in the cytoplasm (49,50) and modulating heterochromatin formation in the nucleus (51,52). One piece of data that links Scp160 function with related proteins from higher eukaryotes is the ﬁnding that the Drosophila vigilin protein, Ddp1, can comple- ment one phenotype associated with deletion of the SCP160 gene in S. cerevisiae (48). Speciﬁcally, this study demonstrated that expression of Ddp1 in yeast cells lacking Scp160 could complement the ploidy defect observed when Scp160 is absent. However, this study did not test whether the Ddp1 entered the yeast nucleus and thus the mechanism underlying the phenotypic suppression remains unclear. Furthermore, given the vast diﬀerences in centromere sequence and organization in S. cerevisiae and Drosophila (53,54), it seems extremely unlikely that Ddp1 could complement the Dscp160 yeast mutant by interacting with yeast centromeres. Thus, Ddp1 may share other evolutionarily conserved functions with Scp160 that have yet to be deﬁned. Taken together, these ﬁndings suggest that the vigilin family of proteins controls gene expression through multiple mechanisms and that those functions that are located within the nucleus may be present only in higher eukaryotes. It is interesting that the role of vigilins in modulating hetero- chromatin is related to the RNAi pathway and that this pathway is absent from S. cerevisiae (55). Perhaps the nuclear function of vigilins arose in concert with the RNAi pathways. Beyond the implications for Scp160 and vigilin func- tion, this study highlights the importance of experimen- tally testing predicted targeting signals. Many proteins contain putative classical nuclear transport signals pre- sumed to contribute to protein function in the absence of corroborating experimental evidence. Thus, caution must be used when incorporating in silico predictions into models for protein function. ACKNOWLEDGEMENTS We gratefully acknowledge Allison Lange for her helpful discussions about site-directed mutagenesis and nuclear transport; Sara Leung for microscopy reagents, protocols and advice, and all other members of the Corbett and Fridovich-Keil laboratories for their many helpful discussions and contributions. This work was supported in part by funds from the NSF (J.F.K.), funds from