Analysis of a predicted nuclear localization signal: implications for the intracellular localization and function of the Saccharomyces cerevisiae RNA-binding protein Scp160.
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 complemented 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.
Brykailo
Melissa A
MA
Department of Human Genetics and Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
McLane
Laura M
LM
Fridovich-Keil
Judith
J
Corbett
Anita H
AH
D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000032:analysis*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C097262:SCP160 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins