Résumé :
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The human myotubularin gene (hMTM1) that is mutated in a severe congenital neuromuscular disease, X-linked myotubular myopathy (XLMTM), encodes a phosphatase specific for phosphatidylinositol 3-phosphate (PtdIns(3)P) and PtdIns(3,5)P2. In Saccharomyces cerevisiae, Ymr1p is the hMTM1 orthologue and the unique member of the myotubularin family. In order to study myotubularin intracellular functions as well as the impact of disease-causing mutations, hMTM1 wild-type and mutants constructs were introduced in yeast. Heterologous expression of hMTM1 wild type led to an enlarged vacuole phenotype, while a phosphatase inactive mutant (C375S) had no effect. The results obtained suggest that dephosphorylation of PtdIns(3)P and PtdIns(3,5)P2 by human MTM1 in yeast impairs membrane trafficking, in particular the trafficking of carboxypeptidase Y (CPY). Depending on the mutation carried there is a difference in the severity of the vacuole phenotype in both wt and ymr1? strains. Mutation N180K responsible for a very mild form of XLMTM provoked a phenotype similar to that of the wild type myotubularin, Mutation V49F responsible for severe XLMTM triggered an increase in vacuole size, although less pronounced, and mutation R421Q (severe XLMTM) did not have an effect when compared to the C375S phosphatase inactive mutant. Surprisingly, mutation R69C (intermediate form of XLMTM) caused an enlargement of the vacuole that was more pronounced than the wild-type construct, suggesting a possible dominant negative effect of the mutation in this system. Apart from establishing a suitable model for studying the function of human myotubularin, the present work sheds light on the possible causes of X-linked myotubular myopathy such as defects in membrane trafficking. Similar studies for other members of the myotubularin family such as hMTMR2, mutated in Charcot-Marie-Tooth peripheral neuropathy, could give information about the pathological processes related to other neuro-muscular diseases.
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