RESUMO
Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Fosfotransferases/metabolismo , Agregados Proteicos , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/ultraestrutura , Sequência Conservada , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Espaço Intracelular/metabolismo , Mutação/genética , Fosfotransferases/química , Homologia de Sequência de AminoácidosRESUMO
Calmodulin (CaM) performs essential functions in cell proliferation in Saccharomyces cerevisiae. Previously, we isolated fourteen temperature-sensitive Phe-to-Ala mutations of the CaM-encoding gene CMD1. These mutations were classified into four intragenic complementation groups, suggesting that each group represents a loss of CaM interaction with its specific essential target protein. Nuf1p/Spc110p, one of the essential targets, is a spindle pole body component that is required for proper mitosis. We investigated which intragenic complementation group of CaM represents the malfunction of Nuf1p. Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature. The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1. Additionally, these mutants displayed characteristic mitotic defects: an increased ratio of artificial chromosome loss, which could be suppressed by the CaM-independent dominant allele of NUF1, and aberrant microtubule structures. These results indicate that these cmd1 mutants display the temperature-sensitive growth due to the compromised interaction with Nuf1p. However, the interaction was restored in a heterozygous diploid of the two cmd1 alleles, suggesting that intragenic complementation between these cmd1 alleles occurs by a novel mechanism, whereby co-presence of both mutant proteins rescues the interaction with Nuf1p.
Assuntos
Alanina/genética , Calmodulina/genética , Teste de Complementação Genética , Mutação/genética , Fenilalanina/genética , Saccharomyces cerevisiae/genética , Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina , Cromossomos Artificiais de Levedura/genética , Cromossomos Artificiais de Levedura/metabolismo , Proteínas do Citoesqueleto , Imunoprecipitação , Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.
