Fig. 5. Effect of protein domains and XLMTM mutations on the subcellular localization of myotubularin. (A) Schematic representation of myotubularin showing protein domains. GRAM, glucosyltransferase, Rab-like GTPase activator and myotubularin common domain (Doerks et al., 2000) (aa 29-97); RID, Rac1-induced localization to membrane ruffles (around aa 233-237); PTP, tyrosine phosphatase-like signature implicated in the lipid phosphatase activity (aa 371-385) with catalytic residues D278, C375 and R381; SID, SET-interacting domain (aa 435-486); PEST, (aa 581-598) with a significant PESTfind score of +8.23; PDZ-BS, putative PDZ-binding site functional in the hMTMR1 homolog (Fabre et al., 2000) (aa 599-603). Highly conserved regions through evolution correspond to a high frequency of missense mutations in XLMTM patients and are also indicated (aa 170-330, 45% identity with the S. cerevisiae protein; and aa 370-490, 55% identity with the S. cerevisiae protein). Below are indicated the subcellular localization and the Rac1-induced localization to membrane ruffles for some constructs. The subcellular localization of the depicted constructs were obtained from immunofluorescence experiments with either the N-terminal 1G6 or the C-terminal 1D10 anti-myotubularin antibodies on transfected COS and HeLa cells. Other constructs produced unstable proteins (aggregates in the cytoplasm and near the nucleus probably in the Golgi and very low myotubularin levels on western blot): del(1-95), del(97-122), del(183-245), del(224-245), del(308-325), del(396-406), del(437-469), del(482-494) and amino acid changes G378R, D394A, G402A, E404K, E410A, D443A, C444Y and H469P. Mutation of the conserved aspartate at position 257 (D257A) did not affect the localization of myotubularin. Note that missense mutations affecting R241 (mild phenotype) impaired the in vitro enzymatic activity toward PtdIns3P (Taylor et al., 2000) and lead to a decrease in protein level in a patient cell line (Laporte et al., 2001b), while mutations G378R (severe phenotype) impaired the in vitro enzymatic activity and G402A (probably severe) leads to a decrease in protein level in a patient cell line. (B) Confocal microscopy analysis of a truncated myotubularin shows nuclear localization (C-ter construct). Deletion of the SET-interacting domain from this construct abolished the nuclear localization. (C) Co-localization of the C-ter construct with PML in more than 50% of co-transfected cells (confocal microscopy). In the same experiment, other co-transfected cells showed no obvious co-localization.

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