RESUMEN
Vph2 is a putative V-ATPase assembly factor. Our previous study has characterized its roles in localization of V-ATPase subunit, cell wall composition, hyphal development and virulence. In this study, our results further demonstrated that Vph2 was localized around the nucleus and in patches close to the periphery of the cell, indicating that Vph2 was located to the endoplasmic reticulum (ER), which was consistent with that in Saccharomyces cerevisiae. Disruption of VPH2 led to hypersensitivity to reducing stresses induced by dithiothreitol (DTT) and ß-mercaptoethanol (ß-ME), and displayed increased GSH content and up-regulation of unfolded protein response (UPR)-related genes, such as PRB1 and PMT4. However, the induced UPR and growth defect on ß-ME plates of vph2Δ/Δ mutant could be partly alleviated by the GSH-specific scavenger 1-chloro-2, 4-dinitrobenzene (CDNB). These results indicated that loss of VPH2 led to an increase in GSH levels, which induced the UPR and caused the defective growth on reductive stress induced by ß-ME. In summary, Vph2 is necessary to maintain resistance against reductive stresses.
Asunto(s)
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Candidiasis/microbiología , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/análisis , Humanos , Oxidación-Reducción , Respuesta de Proteína Desplegada , ATPasas de Translocación de Protón Vacuolares/análisisRESUMEN
The vacuolar-type H+-ATPase (V-ATPase) is known to be associated with various cellular processes. Several V-ATPase subunits have been identified in C. albicans. However, there are still a few V-ATPase subunits and assembly factors that remain uncharacterized. In this study, we identified one of putative V-ATPase assembly factors, Vph2, and V0 subunit, Vma6, and explored their potential functions in C. albicans. Our results revealed that Vph2 and Vma6 were required for the correct distribution of V0 subunit Vph1 and V1 subunit Tfp1. Furthermore, Vph2 and Vma6 played an important role in endocytosis and vacuolar acidification. Disruption of VPH2 or VMA6 affected cell wall stress resistance and composition, accompanying induction of cell wall integrity (CWI) pathway. Besides, deletion of VPH2 or VMA6 led to weakened hyphal development in Spider medium that was not dependent on Hog1 activation. Moreover, the vph2Δ/Δ and vma6Δ/Δ mutants displayed attenuated virulence in a mouse model of systemic candidiasis. Taken together, our data indicated that Vph2 and Vma6 were essential for the proper localization of V-ATPase subunits, cell wall functions, filamentous growth and C. albicans pathogenesis, and provided the potential to better exploit V-ATPase-related proteins as antifungal targets.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Candida albicans/genética , Pared Celular/metabolismo , Endocitosis , Femenino , Proteínas Fúngicas/genética , Hifa/crecimiento & desarrollo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos ICR , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Vacuolas/metabolismoRESUMEN
Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.