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First published online 19 April 2005
doi: 10.1242/jcs.02325

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Analysis of phosphoinositide binding domain properties within the myotubularin-related protein MTMR3

Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK

View larger version (9K): [in a new window]   Fig. 1. Domain structure of MTMR3. MTMR3 contains, from the N-terminus, PH-Gram (PH-G), phosphatase (PTP), coiled-coil and FYVE domains. Deletion and point mutations used in this study are indicated.

View larger version (41K): [in a new window]   Fig. 2. MTMR3 shows lipid binding associated with its PH-GRAM, but not its FYVE domain. (A) Lipid overlay assay. The isolated GST-MTMR3-FYVE domain does not present specific binding. As a control, equimolar GST-EEA1-FYVE domain demonstrates specific binding to Ptdins3P. (B) Binding of 3H-labelled liposomes to immobilised GST-FYVE domains. In contrast to GST-EEA1-FYVE, the GST-MTMR3-FYVE domain does not show PtdIns3P-dependent binding to liposomes. Black bars correspond to +1% PtdIns3P; white bars, no PtdIns3P. Results are the mean of duplicate points from a representative experiment. (C) The MTMR3 PH-G domain binds phosphoinositide lipids. Full length GST-MTMR3 (top panel), GST-MTMR3 (PH-G) (centre) or GST-2 xPH-G (bottom) were tested for lipid binding in an overlay assay. (D) Liposome binding assay. Liposomes containing 0.5 or 0.25 mol% of the indicated phosphoinositides were tested for binding to GST-2 xPH-G. Blots were developed using anti-GST HRP-coupled antibody. MTMR3-PH-G domain showed increased selectivity for PtdIns5P phosphoinositide at reduced mole fractions (pellets, lower panels).

View larger version (28K): [in a new window]   Fig. 3. PH-G domain of MTMR3 binds PtdIns5P. HeLa cells were transfected with HA-MTMR3 (A and B), HA-MTMR3-PH-G (C) or RFP-2 xPH-G (D,E). In addition, some cells were cotransfected with EGFP-IpgD phosphatase (B-D), EGFP-IpgD staining is shown for each in the corresponding lower panel (F-H). IpgD expression results in MTMR3 translocation to the plasma membrane (B). Deletion of the PH-G domain (PH-G) from MTMR3 abolishes translocation (C), whereas RFP-2 xPH-G redistributes similarly to wild-type protein (D). Bar, 10 µm.

View larger version (17K): [in a new window]   Fig. 4. Requirement of MTMR3 domains for enzymatic activity. (A) Deletion of PH-G domain abolishes MTMR3 phosphatase activity. GST-MTMR3 (white bars) and GST-MTMR3 (PH-G) (black bars) were tested for lipid phosphatase activity using a colorimetric assay as described in Materials and Methods. (B) The FYVE domain is not essential for MTMR3 activity. The FYVE domain mutant (His6-MTMR3 (C1174S); black bars) shows similar phosphatase activity towards PtdIns3P and PtdIns3,5P2 compared with wild-type His6-MTMR3 (white bars). Graphs show the mean±s.d. of 4 separate experiments.

View larger version (50K): [in a new window]   Fig. 5. Mutations in MTMR3 alter subcellular localisation. HeLa cells were transfected with HA-MTMR3 constructs and stained with anti-HA antibodies (A-E,G) or GRASP55 antibodies (F,H). Images of HA-MTMR3 constructs, PH-G (A), C1174S (B), C413S (C), C413/1174S (D), PH-G/C413S (E,F) and PH-G/C413/1174S (G,H) were obtained by confocal microscopy. Bar, 10 µm.

View larger version (73K): [in a new window]   Fig. 6. Golgi organisation is lost in cells expressing HA-MTMR3 (C413S). HeLa cells were transfected with wild-type HA-MTMR3 (A,B) or HA-MTMR3 (C413S) (C,D) and stained with anti-HA antibodies (A,C) and GRASP55 antibodies (B,D). Images were then obtained by confocal microscopy. Bar, 10 µm.