Both KH and non-KH domain sequences are required for polyribosome association of Scp160p in yeast.
Scp160p is a 160 kDa RNA-binding protein in yeast previously demonstrated to associate with specific messages as an mRNP component of both soluble and membrane-bound polyribosomes. Although the vast majority of Scp160p sequence consists of 14 closely spaced KH domains, comparative sequence analyses also demonstrate the presence of a potential nuclear localization sequence located between KH domains 3 and 4, as well as a 110 amino acid non-KH N-terminal region that includes a potential nuclear export sequence (NES). As a step toward investigating the structure/function relationships of Scp160p, we generated two truncated alleles, FLAG.SCP160DeltaN1, encoding a protein product that lacks the first 74 amino acids, including the potential NES, and FLAG.SCP160DeltaC1, encoding a protein product that lacks the final KH domain (KH14). We report here that the N-truncated protein, expressed as a green fluorescent protein fusion in yeast, remains cytoplasmic, with no apparent nuclear accumulation. Biochemical studies further demonstrate that although the N-truncated protein remains competent to form RNPs, the C-truncated protein does not. Furthermore, polyribosome association is severely compromised for both truncated proteins. Perhaps most important, both truncated alleles appear only marginally functional in vivo, as demonstrated by the inability of each to complement scp160/eap1 synthetic lethality in a tester strain. Together, these data challenge the notion that Scp160p normally shuttles between the nucleus and cytoplasm, and further implicate polyribosome association as an essential component of Scp160p function in vivo. Finally, these data underscore the vital roles of both KH and non-KH domain sequences in Scp160p.
Li
Ai-min
AM
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.
Vargas
Claudia A
CA
Brykailo
Melissa A
MA
Openo
Kimberly K
KK
Corbett
Anita H
AH
Fridovich-Keil
Judith L
JL
D000483:Alleles; D000595:Amino Acid Sequence; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D008565:Membrane Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C097262:SCP160 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins