INFORMATIONS The myotubularin - amphiphysin 2 complex in membrane tubulation and centronuclear myopathies

The constructs pEGFP BIN1 full length (isoform 8, 454 aa) and pEGFP BAR+PI (aa 1-281) were obtained from P. De Camilli (Yale University, New York). cDNA corresponding to MTM1 sequence (603 aa) was cloned into pEGFP-C1 (Clontech, N-ter GFP fusion) by PCR amplification followed by BamH1 restriction and into pCS2+ vector ([1], untagged). cDNA corresponding to the full length, BAR+PI, BAR (aa 1-255) and SH3 (aa 380-454) sequences of BIN1, and to the full length sequence of MTM1 were cloned into pENTR1A Gateway entry vector (Invitrogen) and recombined into several destination vectors: pEGFPgateway (N-ter GFP fusion), pSG5EYFP (N-ter YFP fusion), pSG5B10 (N-ter B10 fusion = 14aa epitope of the estrogen receptor alpha) and pDEST15 (N-ter GST fusion). Mutations were introduced in pEGFP BIN1 full length, in pCS2+ MTM1 and in the different pENTR1A constructs by primer-directed PCR mutagenesis. The full length open reading frames for human MTM1 (GenBank U46024), MTM1 GRAM (aa 1-162), MTM1 ΔGRAM (aa 146-603), MTM1 Cter (aa 545-603) and MTMR1, MTMR2 MTMR10 and MTMR12 were subcloned into pENTR Gateway entry vector and recombined into destination vector pGex4T3 (N-ter GST fusion) and pSG5B10 [2]. All the constructs were verified by Sanger sequencing.