(A) Schematic diagram of human MTMR4, based on [33]. Protein domains predicted with SMART (http://smart.embl-heidelberg.de/) and PFSCAN (http://hits.isb-sib.ch/cgi-bin/PFSCAN) include GRAM (glucosyltransferase, Rab-like GTPase activator of myotubularins) domain, RID (Rac-induced recruitment domain), PTP/DSP domain, SID (SET-interacting domain), PY motif (XPPXY), CC (coiled-coil) domain and FYVE domain. (B) Western blot of hMTMR4, Nedd4 and tubulin from transfected (MTM tx) or untransfected (untx) HEK-293 lysate immunoprecipitated with anti-MTMR4 antibodies (MTM I.P.). Equal amounts of whole lysate were run as a control. (C) Lysates (lysate) and immunoprecipitates using anti-Nedd4 antibodies (Nedd4 I.P.) or anti-(p70 S6 kinase) antibodies (p70 S6 I.P.) as negative control, of HeLa cells transfected with HA–hMTMR4 wild-type (MTM WT), HA–hMTMR4 PY mutant (MTM PY), pcDNA 3.1 vector alone (vec) or untransfected (untx). Immunoblotting (IB) was performed with anti-HA (monoclonal) antibodies followed by anti-Nedd4 (polyclonal) antibodies and blots were stripped and re-probed with anti-tubulin antibodies as control. Molecular masses are indicated in kDa.

GST-fusion proteins of the area encompassing the PY motif of hMTMR4 (GST-hMTMR4 PY) and of a non-PY-containing proline-rich region of hMTMR4 (GST-hMTMR4 PRO) as control were generated in bacteria as described in the Experimental section. Samples of 0.3 μg of soluble and GST–PY and GST–Pro were incubated with 0.5 μg of His₆-tagged hNedd4–WWI, –WWII, –WWIII or –WWIV immobilized on Ni²⁺-nitrilotriacetate beads (His-hNedd4 WW domain I–IV) or His₆–beads alone (His). Following thorough washes of the beads, bound proteins were eluted with sample buffer, separated by SDS/PAGE, and Western blotting was performed with anti-GST and anti-His antibodies. Molecular masses are indicated in kDa.

(A) HeLa cell lysate transfected with HA-tagged hMTMR4 or HA-tagged hMTMR4 PY motif mutant, together with hNedd4 or untransfected lysate (untx) were subjected to immunoprecipitation (I.P.) with anti-MTMR4 antibodies. Beads were washed and separated by SDS/PAGE with total lysate and then subjected to Western blotting with anti-ubiquitin (Ub) antibodies to detect ubiquitinated MTMR4. Blots were re-probed with anti-HA antibodies to detect transfected hMTMR4 and then stripped and re-probed with anti-Nedd4 antibodies to detect transfected Nedd4. Aliquots of whole-cell lysate were subjected to immunoblotting with anti-HA and anti-Nedd4 antibodies. Blots were stripped and re-probed with anti-α-tubulin antibodies as a loading control. Ubiquitinated MTMR4 is seen as a high-molecular-mass smear (top-left-hand panel) in the hMTMR4 transfected lysate only. Molecular masses are indicated in kDa.

(A) Immunofluorescence of HeLa cells transfected with HA-tagged hMTMR4 (red) and GFP–hNedd4 (green). Overlay shows merging of the two images. (B) Co-localization of HA–hMTMR4 with markers of late endosomes (Rab7) and recycling endosomes (transferrin receptor). HA–hMTMR4 does not co-localize with markers of early endosomes (Rab5). (C) Triple labelling of HeLa cells transfected with HA–hMTMR4 (blue), GFP–Nedd4 (green) and PX–mCherry (red) to represent PtdIns3P localization. Arrowheads point to examples of co-localization. Mander's co-localization coefficients, r (degrees of freedom), are shown for each set of co-staining. Scale bars, 11 μm.

(A) Representative Western blot of MTMR4, Nedd4 and HPRT (as loading control) from gastrocnemius muscle insoluble protein fractions from denervated (Den) and contralateral control (Con) limbs. MTM tx is positive control lysate from MTMR4 transfected or untransfected (untx) HEK-293 cells. Molecular masses are indicated in kDa. (B) Quantification of the chemiluminescent signal from Western blots performed using Bio-Rad Fluor-S Max acquisition system and Quantity One software. Each bar represents the results from eight muscles (mean+S.E.M.). P<0.05 denotes statistically significant differences between mean values. CNT, counts.