Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module.
Mutations in the myotubularin (MTM)-related protein 2 (MTMR2) gene are responsible for the severe autosomal recessive neuropathy Charcot-Marie-Tooth disease type 4B1. MTMR2 belongs to the MTM family of dual-specific phosphatases that use phosphatidylinositol (PI) 3,5-bisphosphate [PI(3,5)P2] and PI 3-phosphate [PI(3)P] as their substrate. Because these substrates are localized in the membrane bilayer, membrane targeting of Mtmr2 is an important regulatory mechanism. In hypoosmotically stressed COS cells with increased levels of PI(3,5)P2, Mtmr2 is bound to the membrane of vacuoles formed under these conditions. Using several mutant forms of Mtmr2, we identified two domains that are necessary for membrane association: (i) A pleckstrin homology-GRAM domain; and (ii) a coiled-coil module. Protein-lipid overlay assays show that the pleckstrin homology-GRAM domain binds to PI(3,5)P2 and PI(5)P, a substrate and a product of the Mtmr2 enzyme, respectively. We also demonstrate that Mtmr2 forms a dimer and that the C-terminal coiled-coil is responsible for homodimerization, in addition to membrane association. Our data indicate that phosphoinositide-protein interactions, as well as protein-protein interactions, are necessary for the correct regulation of MTMR2.
Berger
Philipp
P
Institute of Cell Biology and Institute for Molecular Biology and Biophysics, Department of Biology, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule-Hönggerberg, CH-8093 Zürich, Switzerland.
Schaffitzel
Christiane
C
Berger
Imre
I
Ban
Nenad
N
Suter
Ueli
U
D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000737:chemistry; D019556:COS Cells; D002607:Charcot-Marie-Tooth Disease / Q000201:enzymology / Q000235:genetics; D004247:DNA / Q000235:genetics; D019281:Dimerization; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001798:Blood Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011994:Recombinant Proteins; C039077:platelet protein P47; D004247:DNA; C408986:MTMR2 protein, human; C498717:Mtmr2 protein, mouse; D017027:Protein Tyrosine Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor