Genetic and biochemical interactions between SCP160 and EAP1 in yeast

Abstract

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.

The polysome-associated proteins Scp160 and Bfr1 prevent P body formation under normal growth conditions

“…This role could be regulated by phosphorylation because Scp160 is phosphorylated upon inhibition of the TOR complex by rapamycin, mimicking amino acid starvation (Soulard et al, 2010). In addition, Scp160 is part of a complex that contains Eap1, a negative regulator of translation (Cosentino et al, 2000;Mendelsohn et al, 2003). In fact, the Smy2, Eap1, Scp160, Asc1 (SESA) complex has been shown to act as a translational repressor of the POM34 mRNA in response to spindle pole body (SPB) duplication defects (Sezen et al, 2009).…”

Phosphatidylinositol 4-Phosphate Is Required for Translation Initiation in Saccharomyces cerevisiae

“…We found that in response to rapamycin treatment, isogenic wild-type and eap1⌬ cells both displayed a similar decrease in the P/M ratio (data not shown), which argues in fact against a major role of TORC1 in regulating translation initiation via Eap1p. Interestingly, however, Eap1p is predominantly associated with membranes and interacts genetically and biochemically with the ERassociated polyribosomal protein Scp160p (72). Moreover, it is known that Eap1p inhibits translation initiation following a membrane stress imposed by either application of the amphiphilic drug chlorpromazine or a block (in sec mutant cells) of the membrane transport to the plasma membrane (4,6).…”

The SESA network links duplication of the yeast centrosome with the protein translation machinery

“…We analyzed about 40 genes with roles in translation control, the UPR pathway (Sidrauski and Walter 1997), and nuclear envelope function (Supplemental Table 1) for their requirement in the viability of mps2D 2mm-SMY2 cells. This analysis identified the genes coding for the polysomeassociated, mRNA-binding protein Scp160 that has been shown to exist in a complex with Eap1 (Mendelsohn et al 2003), the Scp160-interacting protein Asc1 (essential for ER localization of Scp160) (Baum et al 2004), and the brefeldin A resistance protein Bfr1 (a component of polyribosome-associated mRNP complexes) (Lang et al 2001), as being essential in the mps2D 2mm-SMY2 background (Figs. 2E,F, 3A; Supplemental Table 1).…”