Genetic and biochemical interactions between SCP160 and EAP1 in yeast.
Scp160p is a multiple KH-domain RNA-binding protein in yeast known to associate with polyribosomes as an mRNP component, although its biological role remains unclear. As a genetic approach to examine Scp160p function, we applied an ethyl methanesulfonate (EMS) screen for loci synthetically lethal with scp160 loss, and identified a single candidate gene, EAP1, whose protein product functions in translation as an eIF4E-binding protein, with additional uncharacterized spindle pole body functions. To reconfirm scp160/eap1 synthetic lethality, we constructed a strain null for both genes, supported by an SCP160 maintenance plasmid. We used this strain to establish a quantitative assay for both Scp160p and Eap1p functions in vivo, and applied this assay to demonstrate that Y109A EAP1, a previously described allele of EAP1 that cannot bind eIF4E, is markedly impaired with regard to its SCP160-related activity. In addition, we explored the possibility of physical interaction between Eap1p and Scp160p, and discovered that Eap1p associates with Scp160p-containing complexes in an RNA-dependent manner. Finally, we probed the impact of EAP1 loss on Scp160p, and vice versa, and found that loss of each gene resulted in a significant change in either the complex associations or subcellular distribution of the other protein. These results clearly support the hypothesis that Scp160p plays a role in translation, demonstrate that the interaction of SCP160 and EAP1 is biologically significant, and provide important tools for future studies of the in vivo functions of both genes.
Mendelsohn
Bryce A
BA
Department of Biology, Emory College, Emory University School of Medicine, Room 325.2 Whitehead Building, 615 Michael Street, Atlanta, GA 30322, USA.
Li
Ai-Min
AM
Vargas
Claudia A
CA
Riehman
Kristen
K
Watson
Alice
A
Fridovich-Keil
Judith L
JL
D000483:Alleles; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D005804:Genes, Lethal / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D039561:Eukaryotic Initiation Factor-4E; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; C097262:SCP160 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C410795:eIF4E-associated protein Eap1; D005020:Ethyl Methanesulfonate