Mendelsohn BA, Li AM, Vargas CA, Riehman K, Watson A, Fridovich-Keil JL

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 ...

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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.

Date: Oct. 15, 2003

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