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Identification and characterization of multiple novel Rab-myosin Va interactions.

Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B - Mol. Biol. Cell (2013)

Bottom Line: Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems.Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes.We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork, Ireland Centre de Recherche, Molecular Mechanisms of Intracellular Transport, Institut Curie, CNRS UMR144, F-75248 Paris, France School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland Structure and Membrane Compartments, Institut Curie, CNRS UMR144, F-75248 Paris, France Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, CNRS UMR144, F-75248 Paris, France.

ABSTRACT
Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A', 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

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Calcium is required for membrane binding of myosin Va. GFP-MyoVaFL(F)WT (A) or GFP-MyoVaFL(D)WT (B) and their Y1203A or Q1753R mutants were treated with 5 μM ionomycin, or solvent alone, for 4 min at 37°C. The cells were then fixed immediately and processed for fluorescence microscopy. (C) Percentage of cells displaying vesicular labeling of the GFP–myosin Va fusion proteins for each condition (mean ± SEM; n = 150 cells from three independent experiments).
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Figure 5: Calcium is required for membrane binding of myosin Va. GFP-MyoVaFL(F)WT (A) or GFP-MyoVaFL(D)WT (B) and their Y1203A or Q1753R mutants were treated with 5 μM ionomycin, or solvent alone, for 4 min at 37°C. The cells were then fixed immediately and processed for fluorescence microscopy. (C) Percentage of cells displaying vesicular labeling of the GFP–myosin Va fusion proteins for each condition (mean ± SEM; n = 150 cells from three independent experiments).

Mentions: Many of the identified Rab partners of myosin Va have been the focus of considerable research efforts and reported to function on overlapping transport pathways, largely in the endocytic recycling and post-Golgi secretory systems (Chen et al., 1998; Darchen and Goud, 2000; Junutula et al., 2004; Larance et al., 2005; Miserey-Lenkei et al., 2007; Stenmark, 2009; Grigoriev et al., 2011; Hutagalung and Novick, 2011). Rab39B, which is less well characterized, is neuronal specific and has been reported to localize to the Golgi complex (Giannandrea et al., 2010). To evaluate the degree of overlap between myosin Va and its Rab partners, we established stable cell lines expressing the GFP-fused D and F isoforms of full-length myosin Va in A431 cells. Under basal conditions, both isoforms display a predominantly cytosolic distribution, likely due to the fusion protein adopting the closed, “inactive” conformation (see later discussion of Figure 5, A and B). Micromolar levels of calcium and/or cargo binding induce myosin Va to undergo a conformational switch to its open, active conformation (Liu et al., 2006; Thirumurugan et al., 2006). Indeed, subcellular fractionation experiments demonstrated that the majority of endogenous myosin Va partitioned in the membrane fraction when A431 cell lysates were prepared with CaCl2, whereas when calcium was chelated by ethylene glycol tetraacetic acid (EGTA), the major pool of myosin Va was found in the cytosolic fraction (Figure 2A). To rule out the possibility that calcium results in myosin Va becoming insoluble, thus accounting for its recovery in the high-speed pellet, we pretreated A431 lysates with 1% TX-100 to solubilize the membranes before centrifugation. Under these conditions the majority of myosin Va was found in the high-speed supernatant (Figure 2A). This indicates that calcium allows myosin Va to adopt a conformation that is capable of binding to membranes.


Identification and characterization of multiple novel Rab-myosin Va interactions.

Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B - Mol. Biol. Cell (2013)

Calcium is required for membrane binding of myosin Va. GFP-MyoVaFL(F)WT (A) or GFP-MyoVaFL(D)WT (B) and their Y1203A or Q1753R mutants were treated with 5 μM ionomycin, or solvent alone, for 4 min at 37°C. The cells were then fixed immediately and processed for fluorescence microscopy. (C) Percentage of cells displaying vesicular labeling of the GFP–myosin Va fusion proteins for each condition (mean ± SEM; n = 150 cells from three independent experiments).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814135&req=5

Figure 5: Calcium is required for membrane binding of myosin Va. GFP-MyoVaFL(F)WT (A) or GFP-MyoVaFL(D)WT (B) and their Y1203A or Q1753R mutants were treated with 5 μM ionomycin, or solvent alone, for 4 min at 37°C. The cells were then fixed immediately and processed for fluorescence microscopy. (C) Percentage of cells displaying vesicular labeling of the GFP–myosin Va fusion proteins for each condition (mean ± SEM; n = 150 cells from three independent experiments).
Mentions: Many of the identified Rab partners of myosin Va have been the focus of considerable research efforts and reported to function on overlapping transport pathways, largely in the endocytic recycling and post-Golgi secretory systems (Chen et al., 1998; Darchen and Goud, 2000; Junutula et al., 2004; Larance et al., 2005; Miserey-Lenkei et al., 2007; Stenmark, 2009; Grigoriev et al., 2011; Hutagalung and Novick, 2011). Rab39B, which is less well characterized, is neuronal specific and has been reported to localize to the Golgi complex (Giannandrea et al., 2010). To evaluate the degree of overlap between myosin Va and its Rab partners, we established stable cell lines expressing the GFP-fused D and F isoforms of full-length myosin Va in A431 cells. Under basal conditions, both isoforms display a predominantly cytosolic distribution, likely due to the fusion protein adopting the closed, “inactive” conformation (see later discussion of Figure 5, A and B). Micromolar levels of calcium and/or cargo binding induce myosin Va to undergo a conformational switch to its open, active conformation (Liu et al., 2006; Thirumurugan et al., 2006). Indeed, subcellular fractionation experiments demonstrated that the majority of endogenous myosin Va partitioned in the membrane fraction when A431 cell lysates were prepared with CaCl2, whereas when calcium was chelated by ethylene glycol tetraacetic acid (EGTA), the major pool of myosin Va was found in the cytosolic fraction (Figure 2A). To rule out the possibility that calcium results in myosin Va becoming insoluble, thus accounting for its recovery in the high-speed pellet, we pretreated A431 lysates with 1% TX-100 to solubilize the membranes before centrifugation. Under these conditions the majority of myosin Va was found in the high-speed supernatant (Figure 2A). This indicates that calcium allows myosin Va to adopt a conformation that is capable of binding to membranes.

Bottom Line: Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems.Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes.We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork, Ireland Centre de Recherche, Molecular Mechanisms of Intracellular Transport, Institut Curie, CNRS UMR144, F-75248 Paris, France School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland Structure and Membrane Compartments, Institut Curie, CNRS UMR144, F-75248 Paris, France Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, CNRS UMR144, F-75248 Paris, France.

ABSTRACT
Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A', 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

Show MeSH
Related in: MedlinePlus