6862–6869 Nucleic Acids Research, 2007, Vol. 35, No. 20 doi:10.1093/nar/gkm776 Published online 11 October 2007 Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160 Melissa A. Brykailo1, Laura M. McLane 2 , Judith Fridovich-Keil1 and Anita H. Corbett2,* 1Department of Human Genetics and 2Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA Received June 27, 2007; Revised September 11, 2007; Accepted September 17, 2007 ABSTRACT Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady- state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were com- plemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence. INTRODUCTION Eukaryotic cells are characterized by the separation of the genome and transcriptional machinery in the nucleus from the translational machinery in the cytoplasm. This separa- tion is mediated by the double membrane of the nuclear envelope, which necessitates a system for transport between these discrete compartments. All macromolecular trans- port between the nucleus and the cytoplasm occurs via nuclear pore complexes (NPCs) embedded in the nuclear envelope (1,2). NPCs are massive multi-protein structures each composed of 30 proteins, which are collectively referred to as nucleoporins (3). Although passive diﬀusion of molecules 440 kDa through NPCs can occur, most macromolecules require speciﬁc transport signal sequences to enter and exit the nucleus via the NPC (4). Nuclear targeting signals mark proteins for transport into and out of the nucleus (4). The best-characterized nuclear targeting signal is the classical nuclear localization signal (cNLS), which consists of a monopartite or bipartite signal (5,6). The monopartite NLS contains a single cluster of four to six basic amino acids, while a bipartite NLS contains two stretches of basic amino acids separated by a stretch of 8–10 spacer residues (6,7). Nuclear export signals (NES) have also been identiﬁed. These targeting signals consist of a weak consensus of three to four hydrophobic residues (8). These classical targeting sequences can be, and often are, identiﬁed by computer searches; however, in many instances the function of these sequences is not experimentally veriﬁed. Targeting signals are recognized by soluble nuclear transport receptors that shepherd cargo proteins through the nuclear pore complex (9). These receptors are termed karyopherins or importins/exportins. Many transport pathways involve direct cargo binding to receptors of *To whom correspondence should be addressed. Tel: +1 404 727 4546; Fax: +1 404 727 3452; Email: acorbe2@emory.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.