Proteomics approach to study the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins

Abstract

Myosin VI is a molecular motor implicated in many processes, and it likely associates with a variety of cargoes that specify its functions. Although it is critical to Drosophila development, little is known about its cellular roles. To reveal its involvement in specific pathways, we sought to identify the binding partners of Drosophila myosin VI. We used affinity chromatography and mass spectrometry to discover interacting proteins, which we tested for direct binding. Using this approach, we found that the microtubule-associated protein Cornetto bound myosin VI, and we demonstrated a role for both in secretion of the lipidated morphogen Hedgehog. We also identified a number of other binding proteins, and further characterization of their interactions with myosin VI will advance our understanding of the roles of these complexes in cellular and developmental processes. Thus, our method has provided us the means to gain valuable insight into the multifaceted roles of a motor protein in vivo.

Fig. 1.

Affinity column construction and analysis. (A) Schematic of protein constructs used to make affinity columns with residue numbers indicated. (B) Coomassie-stained SDS-PAGE of purified proteins used for column construction after affinity and ion exchange chromatography. (C) Extract preparation, column application, and elution sample handling workflow. Elutions included sequential steps of 250 mM, 500 mM, and 1 M KCl. (D) SDS-PAGE of TCA-precipitated fractions. (Left) Coomassie-stained 100 mM KCl washes. (Right) Silver-stained 1 M KCl elutions.

Fig. 2.

M6 binds novel cargoes and is likely present in larger complexes. (A) Anti-myc Western blots of tagged candidate proteins retained by immobilized M6 or M5 after washing with 300 mM salt. Candidate names are indicated above each set of comparisons. Molecular weight markers and their sizes in kilodaltons are indicated on the left of each set. Lava lamp is listed as Lva, and only a construct containing the N-terminus was translated. CG11092 and CG2774 were chosen as specificity controls and were present in both M6 and M5 elution samples. Experiments were repeated several times each, and consistent results were obtained. (B) M6 interaction map constructed in Cytoscape (52). Included are novel (bold lines) and known (thin lines) physical interactions, culled from the DroID database (31) and primary sources (17, 25); all were present in and specific to the M6 elution. Those proteins with the highest UPR are colored the darkest shade of green, fading to white for the lowest-ranked. Not all interactions are shown for some very highly connected proteins (Emb, βTub60D, Nup107), and CG7671 is represented as Nup43. Kermit has been linked genetically to M6 (28), but their direct binding has not been previously demonstrated.

Fig. 3.

Biochemical analysis of Cornetto binding to M6. (A) Schematic of full-length Cornetto and constructs used to make five fragments spanning the protein, with residue numbers indicated. (B) Anti-myc Western blots showing the binding of myc-tagged Cornetto, or Cornetto fragments, to M6. (Left) Full-length Cornetto binds to immobilized M6 (lane 1), and is effectively competed off the resin upon addition of an excess of soluble M6 (lane 2). (Right) A binding assay with each of the five fragments of Cornetto to M6. Only fragment 5 retains full binding to M6, and it does not bind to M5 (last lane). (C) Coomassie-stained SDS-PAGE showing an in vitro binding assay with recombinant purified Cornetto 5 and M6. M6 immobilized on resin (approximately 5 μM as a bead suspension) was incubated with a range of Cornetto 5 concentrations (from 0–75 μM), and the protein remaining bound to the resin after washing was loaded on the gel. (D) Bands from the binding assay were quantified in ImageJ, and values were plotted in Matlab and fit to a curve with the equation indicated. Based on the fit, the estimated dissociation constant (K_D) is between 5 and 10 μM.

Fig. 4.

M6 and Cornetto are involved in the secretion of processed Hedgehog. (A) Immunoprecipitations from S2R+ cell lysates with the antibodies indicated. Anti-M6, anti-Cornetto, and anti-M5 antibodies all coimmunoprecipitate M6, whereas a nonspecific antibody (anti-FLAG) does not. The input lane represents 1% of each total sample. Similar results were obtained from immunoprecipitations of whole fly embryo lysates. (B) Representative anti-Hedgehog Western blot of conditioned media from cells transfected with full-length Hedgehog and each of the dsRNAs as indicated: YFP (nonspecific control), Dispatched (Disp), M6, or Cornetto (Corn). Bands were quantitated and normalized to the signal in the YFP sample. Three independent experiments were averaged, and results are graphed on the Right (± SEM). ^∗P < 0.01, very significant compared with nonspecific control, as determined by t test. (C) Representative anti-Hedgehog Western blots of conditioned media from cells transfected with a truncated version of Hedgehog (HhN) and each of the dsRNAs as before. Bands were quantitated and normalized to the signal in the YFP sample as before, and the averages are shown on the Right (± SEM, n≥2). (D) Cuticles prepared from crosses between engrailed-GAL4 and control (y¹w^67c23), M6 dominant negative (UAS-jaguar tail-CBD), or Cornetto RNAi (UAS-cornetto RNAi) lines. Examples are representative of the normal pattern (control) and the 5–10% fraction affected (M6 DN and corn RNAi).