The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.

To explore the regulatory elements that maintain the balanced synthesis of the components of the ribosome, we isolated a temperature-sensitive (ts) mutant of Saccharomyces cerevisiae in which transcription both of rRNA and of ribosomal protein genes is defective at the nonpermissive temperature. Temperature sensitivity for growth is recessive and segregates 2:2. A gene that complements the ts phenotype was cloned from a genomic DNA library. Sequence analysis revealed that this gene is SLY), encoding a protein essential for protein and vesicle transport between the endoplasmic reticulum and the Golgi apparatus. In the strain carrying our ts allele of SLYI, accumulation of the carboxypeptidase Y precursor was detected at the nonpermissive temperature, indicating that the secretory pathway is defective. To ask whether the eflect of the ts allele on ribosome synthesis was specific for sly) or was a general result of the inactivation of the secretion pathway, we assayed the levels of mRNA for several ribosomal proteins in cells carrying ts alleles of secl, sec7, secil, secl4, secl8, sec53, or sec63, representing all stages of secretion. In each case, the mRNA levels were severely depressed, suggesting that this is a common feature in mutants of protein secretion. For the mutants tested, transcription of rRNA was also substantially reduced. Furthermore, treatment of a sensitive strain with brefeldin A at a concentration sufficient to block the secretion pathway also led to a decrease of the level of ribosomal protein mRNA, with kinetics suggesting that the effect of a secretion defect is manifest within 15 to 30 min. We conclude that the continued function of the entire secretion pathway is essential for the maintenance of ribosome synthesis. The apparent coupling of membrane synthesis and ribosome synthesis suggests the existence of a regulatory network that connects the production of the various structural elements of the cell.

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...

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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.