Résumé :
|
Myotubularin MTM1 is a phosphoinositide 3-phosphatase with specificity towards PtdIns3P (Phosphatidylinositol 3-Phosphate) and PtdIns(3,5)P2 (Phosphatidylinositol 3,5-bisPhosphate). Different mutations in the MTM1 gene cause a severe congenital myopathy, X-linked centronuclear myopathy (XLCNM, also called myotubular myopathy). We investigated the cellular effects resulting from the expression of different MTM1 disease causing mutants in the yeast Saccharomyces cerevisiae. The expression of human MTM1 resulted in the enlargement of the vacuole/lysosome, a consequence of its phosphatase activity. We used vacuolar size as a measure of MTM1 in vivo activity. Microscopic observations allowed a classification of MTM1 forms from the most active to the least active in vivo, MTM1R69C, MTM1, MTM1V49F, MTM1N180K, MTM1R421Q and the inactive synthetic mutant MTM1C375S. Determination of the cellular levels of the different phosphoinositides confirmed the link between vacuolar morphology and MTM1 phosphatase activity, showing that certain disease causing mutants have phosphatase activity towards PtdIns3P and PtdIns( 3,5)P2. Furthermore, the increase in PtdIns5P levels resulting from MTM1 phosphatase activity towards PtdIns(3,5)P2 strongly correlates with the increase in vacuolar size for the different MTM1 mutants, showing that the vacuolar size can be used as a marker for MTM1 phosphatase activity. These results show that different MTM1 mutants responsible for severe forms of myopathy display phosphatase activity in vivo. Therefore, several mutations lead to XLCNM through a mechanism not directly linked to their phosphatase activity.
|