Membrane association of myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain and a coiled-coil dimerization module

Abstract

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.

Fig. 1.

Membrane association of Mtmr2. (A) Domain structure of Mtmr2. Disease-associated mutations are indicated below the schematic structure and artificially designed mutations are indicated above. PH-G, PH-GRAM domain; SID, SET-interacting domain; CC, coiled-coil domain; PDZ-B, PDZ-binding motif. (B) A cytosolic localization of Mtmr2 with enrichment in the perinuclear region was observed in unstressed COS cells transfected with a construct expressing wild-type (wt) Mtmr2. (C) Under hypoosmotic (hypo) stress conditions, Mtmr2 binds to the membrane of large vacuoles. The boxed region is shown at higher magnification in D. (E and F) Mtmr2-C417S and Mtmr2-T641X also bind to membranes of the vacuoles. (G–J) Mtmr2-G103E, Mtmr2-Y579X, and Mtmr2-G103E-Y579X do not bind efficiently to these vacuoles. (I) A higher-power view of H.(K) The percentage of cells containing vacuoles with membrane staining. (Scale bars, 20 μm for B, C, E–H, and J; 4 μm for D and I.)

Fig. 2.

Homodimerization of Mtmr2. (A) A gel filtration elution diagram of His-tagged Mtmr2 protein and SDS/PAGE analysis is shown (Lower). The two peaks elute with molecular masses of ≈170 and 70 kDa, respectively. The first peak contains full-length protein (73 kDa) (Mtmr2 full-length), whereas the protein in the second peak is truncated (Mtmr2-ΔC). (B) Western blot analysis with anti-His antibody of peak fractions (lanes I and II) reveals that the N-terminal His-tag is present in both protein species (FL and ΔC). (C) Analytical ultracentrifugation of full-length Mtmr2 indicates a molecular mass of 140 kDa, which is consistent with Mtmr2 forming a homodimer. Sample purity is shown by SDS/PAGE and staining with Coomassie blue (Inset).

Fig. 3.

The Mtmr2 coiled-coil domain is a dimerization module. (A) Size-exclusion chromatography of His₆TRXNCoil, His₆TRXN, and the coiled-coil domain of Mtmr2. The A_{280 nm} trace of the His₆TRXNCoil (solid line) is superimposed on the trace of the individual proteins after TEV cleavage (dashed line) marked His₆TRXN + Coil. Homogeneity of His₆TRXNCoil was confirmed by SDS/PAGE (Inset). The fusion protein elutes at an apparent molecular mass of 37.6 kDa, indicating dimer formation. The TEV cleavage products, His₆TRXN and the coiled coil, elute in one peak with an apparent molecular mass of 11.9 kDa. The presence of both proteins was confirmed by SDS/PAGE, using a 20% acrylamide gel (Inset). His₆TRXN (13.9 kDa) elutes as a monomer, and the coiled-coil domain (monomer size: 4.3 kDa) elutes as a dimer. (B) TEV protease cleavage of His₆TRXNCoil fusion protein. Reaction was complete after 1 h, as shown by SDS/PAGE (lane 1, directly after addition of TEV protease; lane 2, after 1 h incubation). (C) The CD signal (solid line) of the coiled-coil domain shows the spectrum of an α-helix. The coiled-coil domain was purified to homogeneity as confirmed by SDS/PAGE (Inset).

Fig. 4.

The Mtmr2 coiled-coil domain forms a parallel dimer. (A) A model of the Mtmr2 coiled-coil module, comprising 35 amino acids in two orthogonal views is shown. A dimer of two Mtmr2 coiled-coils extends to ≈45 Å.(B) FRET experiments are shown in a schematic representation. Coexpression of isolated BFP and GFP molecules (I) yields a blue color on excitation at 366 nm (blue arrows). Covalent linking of BFP and GFP (II) leads to FRET, resulting in emission of green light by GFP on excitation at 366 nm (green arrow). (III) Coil-GFP and BFP-Coil adopting a parallel arrangement are shown. Green light is not emitted, due to the location of the fluorophores at opposite ends of the coiled-coil dimer. (IV) Coil-GFP and Coil-BFP forming a parallel coiled-coil dimer are shown. The fluorophores are now located at close proximity, and FRET occurs, resulting in a green color. Results illustrated in I–IV are depicted in C, showing bacteria expressing the respective protein, which is plated on IPTG-containing dYT-agar.

Fig. 5.

Protein-lipid overlay assay. (A) Serial dilutions of spotted phospholipids (100–3 pmol in two-fold dilution steps) were incubated with Mtmr2-C417S, a protein without phosphatase activity, to prevent dephosphorylation of the lipids. Mtmr2 binds to PI(4)P, PI(5)P, PI(3,5)P₂, and PI(3,4,5)P₃, but not to PI, PI(3)P, PI(3,4)P₂, or PI(4,5)P₂.(B) Phospholipids were incubated with Mtmr2-E276X, which contains only the N-terminal part with the PH-G domain. The same specificity as with the full-length protein is observed. See Fig. 7, which is published as supporting information on the PNAS web site, www.pnas.org, for additional blots.

Fig. 6.

Proposed models for membrane association mediated by the PH-G and coiled-coil domains. (A) Homodimerization provides two PH-G domains, leading to enhanced affinity. xP, specifically bound phosphoinositides (see text). (B) Heterodimerization with sbf1/MTMR5 or hypothetically sbf2/MTMR13 allows interaction with other as-yet-unidentified phosphoinositides (yP and zP). (C) A hypothetical interaction with a potential membrane-bound interaction partner.