Crosstalk between the secretory pathway and ribosome synthesis in Saccharomyces cerevisiae

Abstract

A screen was designed to identify temperature sensitive mutants of Saccharomyces cerevisiae in which the levels of ribosomal protein mRNAs are specifically down-regulated at 37{dollar}\sp\circ{dollar}C. The gene from one such mutant was cloned. The predicted protein is {dollar}\sim{dollar}70% identical to hRAB6, and was named YPT6. The mutant strain is defective in the secretion of invertase, in the maturation of invertase and the vacuolar protein CPY, which accumulate as core-glycosylated forms, suggesting that Ypt6p is involved in an early step of the secretory pathway. Electron microscopic analysis shows accumulation of vesicles and an enlarged vacuole in the mutant. Cloning of suppressors of ypt6 ts revealed two additional proteins influencing protein transport: Ssd1p and Imh1p. The ypt6 ts can be suppressed by YPT1, SLY1-20, or vSNARE genes on a 2 {dollar}\mu{dollar} plasmid, suggesting that Ypt6p is involved in vesicle targeting by activating vSNAREs. Four other mutants selected from the screen were found to have defects in secretion, emphasizing the finding that a functional secretory pathway is essential for ribosome synthesis.;The level of a given mRNA depends on its rate of transcription and its rate of degradation. By testing transcription of a fused gene ACT1-RPL32 (between the ACT1 promoter and the transcript of RPL32), we conclude that the down-regulation occurs mainly at the level of transcription, and to a less extent, at the level of mRNA turnover. I then determined the cis-elements that mediate the repression induced by secretion defects. The promoters of ribosomal protein genes have two Rap1p binding sites, a T rich region and the down-stream region containing the putative TATA box. By replacing (i) the UAS of ACT1 with Rap1p sites in construct ACT1-RPL32; (ii) different parts of RPL32 promoter with Ga14p binding sites which drives GFP, I demonstrated that both Rap1p sites and the cis-element between the T rich regions and transcription initiation site play a role in the repression. We also demonstrated that the SIR proteins are not involved in this repression.