Human myotubularin expression in yeast <i>S. cerevisiae</i>.

<p>(A) Representation of the MTM1 protein with its domains, the position of the mutations analyzed and the severity of the resulting myopathy phenotype. MTM1 displays different domains, PH-GRAM (pleckstrin homology-glucosyltransferase, Rab-like GTPase activator and myotubularin), RID (Rac-induced recruitment domain), catalytic phosphatase domain and SID (SET-protein interaction domain). (B) Anti-MTM1 Western-blot on yeast protein extracts. The <i>MTM1</i> gene was placed under the control of the tetracycline-repressible <i>tetO</i> promoter in the low copy number centromeric plasmid (CEN, pVV204, 1 to 3 copies per cell). In the high copy number 2 µ plasmid (2 µ, pVV200, 20 to 50 copies per cell), the <i>MTM1</i> gene was under the control of the strong yeast <i>PGK1</i> promoter. These different plasmids were transformed into the <i>ymr1Δ</i> yeast mutant. Protein extracts of <i>ymr1Δ</i> cells transformed with pVV204 (CEN) or pVV200 (2 µ) empty plasmids (Control) or bearing the different MTM1 forms were analyzed by Western-blot. MTM1 production was detected with the mouse monoclonal 1G6 anti-MTM1 antibody. The different human MTM1 proteins were produced at the expected molecular weight (70 kDa) as compared to the <i>ymr1Δ</i> cells transformed with empty control plasmids that displayed no signal. Protein loading was evaluated by immunodetection of the yeast endogenous 3-phosphoglycerate kinase Pgk1 protein.