A secretory defect causes specific and significant transcriptional repression of both ribosomal protein and rRNA genes (K. Mizuta and J. R. Warner, Mol. Cell. Biol. 14:2493-2502, 1994), suggesting the coupling of plasma membrane and ribosome syntheses. In order to elucidate the molecular mechanism of the signaling pathway, we isolated a cold-sensitive mutant with a mutation in a gene termed RRS1 (regulator of ribosome synthesis), which appeared to be defective in the signaling pathway. The rrs1-1 mutation greatly reduced transcriptional repression of both rRNA and ribosomal protein genes that is caused by a secretory defect. RRS1 is a novel, essential gene encoding a nuclear protein of 203 amino acid residues that is conserved in eukaryotes. A conditional rrs1-null mutant was constructed by placing RRS1 under the control of the GAL1 promoter. Rrs1p depletion caused defects in processing of pre-rRNA and assembly of ribosomal subunits.Balanced synthesis of cellular components is required for normal cell growth. A temperature-sensitive mutation in SLY1, whose gene product is involved in endoplasmic reticulum-toGolgi trafficking (26), causes the transcriptional repression of both ribosomal protein and rRNA genes in Saccharomyces cerevisiae (20). Further examination using various sec mutants showed that a defect anywhere in the secretory pathway, from a step prior to insertion of the nascent peptide into the endoplasmic reticulum to a step involved in the formation of the plasma membrane, prevents the continued synthesis of the components of the ribosome. Similar results were obtained following treatment of wild-type cells with the secretory inhibitors tunicamycin and brefeldin A (20). Furthermore, many temperature-sensitive mutants in which transcription of ribosomal protein genes is temperature sensitive appear to be defective in the secretory pathway (17). As the membrane is the end product of much of the secretory pathway, these results suggest an important coupling of plasma membrane and ribosome biosynthesis. We proposed the existence of a signal transduction pathway from the plasma membrane to the nucleus. According to this model, a signal generated by the defect in de novo synthesis of membrane should be transmitted to the nucleus and cause specific and significant transcriptional repression of ribosomal genes. It was recently suggested that stress in the plasma membrane is monitored by Pkc1, which initiates a signal transduction pathway that leads to the repression (24). In order to elucidate the molecular mechanism of the signal transduction pathway, we have screened for mutants defective in the response to a secretory defect.Here we describe the isolation and molecular characterization of RRS1, encoding an essential nuclear protein of 203 amino acids. In the rrs1-1 mutant, a secretory defect fails to cause transcriptional repression of either rRNA or ribosomal protein genes. The mutant gene, rrs1-1, had a single nucleotide difference within codon 114, resulting in a stop codon. The amino acid sequence of R...

Background: A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response.

A secretory defect leads to transcriptional repression of both ribosomal protein and rRNA genes in yeast. To elucidate the mechanism of the signaling, we previously isolated rrs mutants that were unable to respond to a secretory defect, and we cloned RRS1 encoding a nuclear protein that was required for ribosome biogenesis (Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T., and Mizuta, K. (2000) Mol. Cell. Biol. 20, 2066 -2074). We identified duplicated genes encoding ribosomal protein L11, RPL11B as a wild-type allele complementing the rrs2 mutation, and RPL11A in two-hybrid screening using RRS1 as bait. Rpl11p was copurified with Rrs1p in immunoprecipitation analysis. Ultracentrifugation analysis revealed that Rrs1p associated fairly tightly with 60 S preribosomal subunits. These results suggest that signaling in response to a secretory defect requires the normal assembly of 60 S ribosomal subunits including Rrs1p and Rpl11p.

We have previously shown that a functional secretory pathway is essential for continued ribosome synthesis in Saccharomyces cerevisiae. When a temperature-sensitive mutant defective in the secretory pathway is transferred to the non-permissive temperature, transcription of both rRNA genes and ribosomal protein genes is nearly abolished. In order to define the cis -acting element(s) of ribosomal protein genes sensitive to a defect in the secretory pathway, we have constructed a series of fusion genes containing the CYH2 promoter region, with various deletions, fused to lacZ. Each fusion gene for which transcription is detected is subject to the repression. Rap1p is the transcriptional activator for most ribosomal protein genes, as well as having an important role in silencing in the vicinity of telomeres and at the silent mating-type loci. To assess its role in the repression of transcription by the defect in the secretory pathway, we have introduced rap1 mutations. The replacement of wild-type Rap1p by Rap1p truncated at the C-terminal region caused substantial attenuation of the repression. Furthermore, we have demonstrated that the Rap1p-truncation affects the repression of TCM1 , encoding ribosomal protein L3, which has no Rap1p-binding site in its upstream regulatory region. These results suggest that the repression of transcription of ribosomal protein genes by a secretory defect is mediated through Rap1p, but does not require a Rap1p-binding site within the UAS.