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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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Myosin Va interacts with multiple Rab GTPases. (A) Schematic diagram depicting the extended “active” structure of myosin Va. The location of the alternatively spliced exons B, D, and F are indicated. GTD, globular-tail domain; MD, motor domain. (B) HeLa cells expressing dominant-active (DA) or dominant-negative (DN) mutants of the indicated Rab GTPases fused to GFP were lysed, and the fusion proteins were immunoprecipitated with an anti-GFP antibody. The ability of myosin Va to form a complex with these Rabs was tested by Western blot analysis using anti-myosin Va antibody. Input represents 10% of the starting material used in all conditions. (C) Regions of the myosin Va tail tested using the yeast two-hybrid technique. The constitutively active mutant of each Rab GTPase was used. CC, coiled coil; GTD, globular tail domain. (D) Identification of a critical amino acid for binding to Rab6 and Rab14. ClustalW alignment of the Rab6/Rab14-binding regions of human myosin Va and myosin Vb. Arrows indicate the conserved amino acids that were mutated to alanine, and the red arrow indicates tyrosine 1203, which, when mutated, abolishes the Rab6 and Rab14 interaction. The table indicates the strength of interaction of each mutant with constitutively active Rab6A, Rab11A, and Rab14 using the yeast two-hybrid HIS3 and LacZ reporter assays. (E) Identification of critical amino acids that mediate binding to Rab3A, Rab11A, and Rab39B. The amino acids in myosin Va that correspond to those identified by Lipatova et al. (2008) that mediate the binding of Myo2 to Ypt31/32 were mutated and the mutants tested for interaction with the constitutively active mutants of the indicated Rab GTPases by yeast two-hybrid assay. (F) Schematic diagram of myosin Va indicating the identified Rab-binding domains. Asterisk indicates the location of the Y1203A mutation; double asterisks indicate the location of the Q1753R mutation.
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Figure 1: Myosin Va interacts with multiple Rab GTPases. (A) Schematic diagram depicting the extended “active” structure of myosin Va. The location of the alternatively spliced exons B, D, and F are indicated. GTD, globular-tail domain; MD, motor domain. (B) HeLa cells expressing dominant-active (DA) or dominant-negative (DN) mutants of the indicated Rab GTPases fused to GFP were lysed, and the fusion proteins were immunoprecipitated with an anti-GFP antibody. The ability of myosin Va to form a complex with these Rabs was tested by Western blot analysis using anti-myosin Va antibody. Input represents 10% of the starting material used in all conditions. (C) Regions of the myosin Va tail tested using the yeast two-hybrid technique. The constitutively active mutant of each Rab GTPase was used. CC, coiled coil; GTD, globular tail domain. (D) Identification of a critical amino acid for binding to Rab6 and Rab14. ClustalW alignment of the Rab6/Rab14-binding regions of human myosin Va and myosin Vb. Arrows indicate the conserved amino acids that were mutated to alanine, and the red arrow indicates tyrosine 1203, which, when mutated, abolishes the Rab6 and Rab14 interaction. The table indicates the strength of interaction of each mutant with constitutively active Rab6A, Rab11A, and Rab14 using the yeast two-hybrid HIS3 and LacZ reporter assays. (E) Identification of critical amino acids that mediate binding to Rab3A, Rab11A, and Rab39B. The amino acids in myosin Va that correspond to those identified by Lipatova et al. (2008) that mediate the binding of Myo2 to Ypt31/32 were mutated and the mutants tested for interaction with the constitutively active mutants of the indicated Rab GTPases by yeast two-hybrid assay. (F) Schematic diagram of myosin Va indicating the identified Rab-binding domains. Asterisk indicates the location of the Y1203A mutation; double asterisks indicate the location of the Q1753R mutation.

Mentions: In a systematic approach to investigate the ability of the entire complement of human Rab GTPases to interact with myosin Va, we performed a yeast two-hybrid “living chip” assay (see Materials and Methods) in which a library of the Y187 yeast strain transformed with the wild-type (wt), constitutively active (DA; GTP bound), and constitutively inactive (DN; GDP bound) forms of each human Rab in the pLexA bait vector was mated with the L40 yeast strain transformed with a prey vector expressing the tail region of the F isoform of myosin Va (see later discussion). Colonies containing interacting bait and prey constructs were selected for by growth on synthetic medium lacking histidine (Supplemental Figure S1) and further assayed for β-galactosidase activity (Supplemental Table S1). We observed the previously reported interactions with active Rab3A and Rab11A (Roland et al., 2009; Wollert et al., 2011), which validated our screen (Supplemental Table S1). In addition, we also found interactions between myosin Va and the activated forms of Rabs 3B, 3C, 3D, 6A, 6A′, 6B, 9B, 11B, 14, 29, 30, and 39B, as well as with wild-type Rab25, which naturally contains a leucine residue at position 71 (Supplemental Table S1). The Rab29 and Rab30 interactions were dismissed as false positives because they scored positive with the majority of preys tested (Supplemental Table S1 and unpublished data). The remaining interactions were retested individually in the yeast two-hybrid system (Figure 1C), and only Rab9B was eliminated in this more stringent round of assays (Supplemental Figure S3). Cells expressing green fluorescent protein (GFP)–fused DA or DN mutants of representative members from each of its partner Rab subfamilies were lysed and the fusion proteins immunoprecipitated with an anti-GFP antibody. Coimmunoprecipitating myosin Va proteins were then revealed with an anti-myosin Va antibody. The data revealed that endogenous myosin Va forms a complex with each of the DA mutants of Rabs 39B, 14, 11A, 8A, 6A, and 3A, whereas only weak binding was observed with their DN mutants (Figure 1B). Of importance, no binding was observed with the DA or DN mutants of Rab7A or Rab4A, which were used as negative controls (Figure 1B). These results are consistent with our yeast two-hybrid studies, which demonstrated that myosin Va preferentially associates with a subset of GTP-bound Rabs.


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)

Myosin Va interacts with multiple Rab GTPases. (A) Schematic diagram depicting the extended “active” structure of myosin Va. The location of the alternatively spliced exons B, D, and F are indicated. GTD, globular-tail domain; MD, motor domain. (B) HeLa cells expressing dominant-active (DA) or dominant-negative (DN) mutants of the indicated Rab GTPases fused to GFP were lysed, and the fusion proteins were immunoprecipitated with an anti-GFP antibody. The ability of myosin Va to form a complex with these Rabs was tested by Western blot analysis using anti-myosin Va antibody. Input represents 10% of the starting material used in all conditions. (C) Regions of the myosin Va tail tested using the yeast two-hybrid technique. The constitutively active mutant of each Rab GTPase was used. CC, coiled coil; GTD, globular tail domain. (D) Identification of a critical amino acid for binding to Rab6 and Rab14. ClustalW alignment of the Rab6/Rab14-binding regions of human myosin Va and myosin Vb. Arrows indicate the conserved amino acids that were mutated to alanine, and the red arrow indicates tyrosine 1203, which, when mutated, abolishes the Rab6 and Rab14 interaction. The table indicates the strength of interaction of each mutant with constitutively active Rab6A, Rab11A, and Rab14 using the yeast two-hybrid HIS3 and LacZ reporter assays. (E) Identification of critical amino acids that mediate binding to Rab3A, Rab11A, and Rab39B. The amino acids in myosin Va that correspond to those identified by Lipatova et al. (2008) that mediate the binding of Myo2 to Ypt31/32 were mutated and the mutants tested for interaction with the constitutively active mutants of the indicated Rab GTPases by yeast two-hybrid assay. (F) Schematic diagram of myosin Va indicating the identified Rab-binding domains. Asterisk indicates the location of the Y1203A mutation; double asterisks indicate the location of the Q1753R mutation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Myosin Va interacts with multiple Rab GTPases. (A) Schematic diagram depicting the extended “active” structure of myosin Va. The location of the alternatively spliced exons B, D, and F are indicated. GTD, globular-tail domain; MD, motor domain. (B) HeLa cells expressing dominant-active (DA) or dominant-negative (DN) mutants of the indicated Rab GTPases fused to GFP were lysed, and the fusion proteins were immunoprecipitated with an anti-GFP antibody. The ability of myosin Va to form a complex with these Rabs was tested by Western blot analysis using anti-myosin Va antibody. Input represents 10% of the starting material used in all conditions. (C) Regions of the myosin Va tail tested using the yeast two-hybrid technique. The constitutively active mutant of each Rab GTPase was used. CC, coiled coil; GTD, globular tail domain. (D) Identification of a critical amino acid for binding to Rab6 and Rab14. ClustalW alignment of the Rab6/Rab14-binding regions of human myosin Va and myosin Vb. Arrows indicate the conserved amino acids that were mutated to alanine, and the red arrow indicates tyrosine 1203, which, when mutated, abolishes the Rab6 and Rab14 interaction. The table indicates the strength of interaction of each mutant with constitutively active Rab6A, Rab11A, and Rab14 using the yeast two-hybrid HIS3 and LacZ reporter assays. (E) Identification of critical amino acids that mediate binding to Rab3A, Rab11A, and Rab39B. The amino acids in myosin Va that correspond to those identified by Lipatova et al. (2008) that mediate the binding of Myo2 to Ypt31/32 were mutated and the mutants tested for interaction with the constitutively active mutants of the indicated Rab GTPases by yeast two-hybrid assay. (F) Schematic diagram of myosin Va indicating the identified Rab-binding domains. Asterisk indicates the location of the Y1203A mutation; double asterisks indicate the location of the Q1753R mutation.
Mentions: In a systematic approach to investigate the ability of the entire complement of human Rab GTPases to interact with myosin Va, we performed a yeast two-hybrid “living chip” assay (see Materials and Methods) in which a library of the Y187 yeast strain transformed with the wild-type (wt), constitutively active (DA; GTP bound), and constitutively inactive (DN; GDP bound) forms of each human Rab in the pLexA bait vector was mated with the L40 yeast strain transformed with a prey vector expressing the tail region of the F isoform of myosin Va (see later discussion). Colonies containing interacting bait and prey constructs were selected for by growth on synthetic medium lacking histidine (Supplemental Figure S1) and further assayed for β-galactosidase activity (Supplemental Table S1). We observed the previously reported interactions with active Rab3A and Rab11A (Roland et al., 2009; Wollert et al., 2011), which validated our screen (Supplemental Table S1). In addition, we also found interactions between myosin Va and the activated forms of Rabs 3B, 3C, 3D, 6A, 6A′, 6B, 9B, 11B, 14, 29, 30, and 39B, as well as with wild-type Rab25, which naturally contains a leucine residue at position 71 (Supplemental Table S1). The Rab29 and Rab30 interactions were dismissed as false positives because they scored positive with the majority of preys tested (Supplemental Table S1 and unpublished data). The remaining interactions were retested individually in the yeast two-hybrid system (Figure 1C), and only Rab9B was eliminated in this more stringent round of assays (Supplemental Figure S3). Cells expressing green fluorescent protein (GFP)–fused DA or DN mutants of representative members from each of its partner Rab subfamilies were lysed and the fusion proteins immunoprecipitated with an anti-GFP antibody. Coimmunoprecipitating myosin Va proteins were then revealed with an anti-myosin Va antibody. The data revealed that endogenous myosin Va forms a complex with each of the DA mutants of Rabs 39B, 14, 11A, 8A, 6A, and 3A, whereas only weak binding was observed with their DN mutants (Figure 1B). Of importance, no binding was observed with the DA or DN mutants of Rab7A or Rab4A, which were used as negative controls (Figure 1B). These results are consistent with our yeast two-hybrid studies, which demonstrated that myosin Va preferentially associates with a subset of GTP-bound Rabs.

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