Unc45b forms a cytosolic complex with Hsp90 and targets the unfolded myosin motor domain

Abstract

Myosin folding and assembly in striated muscle is mediated by the general chaperones Hsc70 and Hsp90 and a myosin specific co-chaperone, UNC45. Two UNC45 genes are found in vertebrates, including a striated muscle specific form, Unc45b. We have investigated the role of Unc45b in myosin folding. Epitope tagged murine Unc45b (Unc45b(Flag)) was expressed in muscle and non-muscle cells and bacteria, isolated and characterized. The protein is a soluble monomer in solution with a compact folded rod-shaped structure of approximately 19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45b(Flag) fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45b(Flag) re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45b(Flag)/Hsp90 complex binds the partially folded myosin motor domain when incubated with myosin subfragments synthesized in a reticulocyte lysate. This binding is independent of the myosin rod or light chains. Unc45b(Flag) does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45b(Flag) enhances myosin motor domain folding during de novo motor domain synthesis indicating that it has a direct role in myosin maturation. Thus, mammalian Unc45b is a cytosolic protein that forms a stable complex with Hsp90, selectively binds the unfolded conformation of the myosin motor domain, and promotes motor domain folding.

Figure 1. Flag-tagged Unc45b was expressed in C2C12 muscle cells using recombinant adenovirus vectors then extracted, fractionated and affinity purified.

A. Adenovirus driven over-expression of Unc45b^Flag in C2C12 myotubes results in the accumulation of an abundant 95 kDa band (asterisk) corresponding to Unc45b^Flag in whole cell lysates (WCL). The protein is completely extracted into the triton soluble cytosolic fraction (TSE), and fractionates by ammonium sulfate into the 30–60% saturation precipitate (60% ppt). Little is found in the 0–30% precipitate (30% ppt). Affinity purification on anti-Flag beads (mAb M2) followed by dissociation with SDS-gel loading buffer (bound) or elution with 3× Flag peptide (eluted) reveals that Unc45b^Flag associates with a 90 kDa protein. The lane labeled DT is the drop-through fraction from the anti-Flag beads containing the protein that did not bind. B. Western blots identify the 90 kDa protein as Hsp90. Samples are: Lane 1, purified Unc45b^Flag; Lane2, pure Hsp90; and Lane 3, Unc45b fraction eluted from anti-Flag beads. The blots were developed with anti-Unc45b and anti-Hsp90β antibodies. The lower protein loads used for the Western blot experiment resolve the Hsp90 into a doublet, demonstrating that both Hsp90 isoforms (α & β) are bound by Unc45b^Flag. This was confirmed by Western blots with anti-Hsp90α (data not shown). The Unc45b^Flag sample in Lane 2 was expressed in bacteria and purified to homogeneity (see Fig. S1).

Figure 2. Purified Unc45b^Flag binds human and rabbit Hsp90.

Unc45b^Flag was bound to anti-Flag beads. Aliquots of anti-Flag beads alone or with Unc45b^Flag were incubated with purified human Hsp90 or rabbit reticulocyte lysate, and proteins pulled down (p) with the beads and the supernatants (s) were analyzed by SDS-PAGE. Unc45b^Flag readily binds purified human Hsp90 in this assay. Unc45b^Flag selectively bind Hsp90 when incubated with rabbit reticulocyte lysate (Retic Lysate). The complex recovered from the lysate did not include other Hsp90 binding partners suggesting that the interaction is characterized by a high degree of selectivity.

Figure 3. Analysis of Unc45b in muscle cell lysates by gel filration and immunoprecipitation.

A cytosolic lysate was prepared from C2C12 myotubes and concentrated by ammonium sulfate precipitation. A. Analysis of the lysate by gel filtration followed by Western blotting shows that the elution of Unc45b and Hsp90 overlap. The muscle Unc45b elutes at an apparent molecular weight that is greater than the 180 kDa Hsp90 dimer. B. The column was calibrated with standards including: Hsp90, Unc45b^Flag and a complex of Unc45b^Flag/Hsp90. Pure Unc45b^Flag elutes at a position consistent with a monomeric mass of about 100 kDa. Pure Hsp90 elutes as a broad double peak consistent with its higher mass and conformational heterogeneity. A synthetic complex of Unc45b^Flag and Hsp90 elutes as a single peak at the position of the Hsp90, suggesting that the complex is more compact and with less conformational heterogeneity than pure Hsp90. C. Immunoprecipitation of the endogenous Unc45b from the lysate with anti-Unc45b IgG and Protein-A beads shows that Hsp90 is bound to Unc45b in the lysate. This is confirmed by the Western blots with anti-Unc45b and anti-Hsp90. In addition to Hsp90, a 120 kDa band is detected in the antibody pull-down. So, although purified Unc45-Flag is a monomer in solution, the Unc45b in muscle lysates isolates as a cytosolic complex with Hsp90 and potentially one other protein.

Figure 4. Binding of Unc45b^Flag to myosin fragments.

To analyze myosin binding activity of Unc45b we synthesized full-length myosin or its subfragments in a coupled reticulocyte lysate synthesis assay and measured pull-down of the radioactive proteins by Unc45b^Flag bound to anti-Flag beads. The target proteins included: full length striated muscle myosin (MHC), heavy meromyosin (HMM), a MD::GFP chimera, myosin subfragment 1 (S1) and the subfragment 2 (S2) region of the myosin rod (see also Fig. S3). The newly synthesized radioactive proteins were incubated at 0°C with proteins bound to the anti-Flag beads and analyzed by SDS-PAGE and autoradiography. A. Autoradiography translation mix (T), and the proteins bound to anti-Flag beads alone (1), beads with Unc45b^Flag (2) and Unc45b/Hsp90 complex (3). Unc45b^Flag and Unc45b/Hsp90 complex bind all myosin subfragments that include the motor domain but not to the S2 region of the rod or the myosin light chains (not shown). B. The stained gel corresponding to the autoradiograph shows that the purified Unc45b^Flag binds Hsp90 when added to the lysate. C. The stoichiometry of the Unc45b^Flag binding interaction was investigated by titration of the binding of the MD::GFP chimera with increasing concentrations of Unc45b^Flag. To detect the low concentration of Unc45b^Flag recovered in this assay the gel was silver stained before autoradiography. The upper panel shows the autoradiograph detecting the MD::GFP in the pull-down. The lower panel shows the proteins bound to the anti-Flag beads. The amount of Hsp90 detected increases linearly with added Unc45b; whereas, the MD::GFP binding saturates at the higher Unc45b^Flag concentration tested. This is consistent with binding of the motor domain by the Unc45b^Flag/Hsp90 complex. These data are plotted in Fig. S4.

Figure 5. Unc45b does not bind the native myosin motor domain.

Native HMM prepared by chymotryptic digestion of myosin was incubated with anti-Flag beads alone and in combination with Unc45b^Flag and Hsp90 at 25°C. Native HMM does not bind Unc45b or Unc45b/Hsp90 complex. This is consistent with a chaperone activity for Unc45b^Flag.

Figure 6. A smooth muscle myosin MD::GFP chimera was synthesized in reticulocyte lysate in the absence and presence of Unc45b^Flag.

Aliquots of translation reaction were analyzed by a native gel eletrophoresis and SDS-PAGE followed by autoradiography. A. The native gel resolves the folded motor domain as a discrete band (Native Conformation). The unfolded motor domain migrates more slowly as a diffuse smear is this gel system. The inclusion of Unc45b^Flag (bacterial expression) or Unc45b^Flag/Hsp90 complex (myotube expression) to the synthesis assay dramatically enhances the formation of native conformation. The SDS gel (lower panel) shows that the levels of MD::GFP synthesis and stability are unaffected by the addition of Unc45b. B. SDS PAGE of the purified proteins added to the reticulocyte lysate: Unc45b^Flag, and the Unc45b^Flag/Hsp90 complex.

Figure 7. Characterization of the domain structure of Unc45b by limited proteolysis.

A. Time course of the digestion of Unc45b^Flag with trypsin reveal a ∼37 kDa fragment that appears early in the digest and is relatively stable for up to 30 min. Other initial fragments include a 60 kDa fragment that disappears with the appearance of a 34 kDa and 26 kDa fragment. Western blotting with anti-Flag mAb shows that the 37 kDa fragments retains the C-terminal Flag epitope and corresponds most of the UCS domain. The anti-Unc45b antibody identifies the four early fragments and monitors their disappearance. B. Pull-down assay with anti-Flag beads to determine if the 37 kDa fragment bearing the Flag tag is still capable of binding the myosin motor domain synthesized in the reticulocyte lysate. The fragments produced by 2 min trypsin digestion were incubated with skeletal muscle MD::GFP chimera. The upper panel is the stained gel and the lower panel the autoradiograph. The 60, 37, 34 and 26 kDa Unc45b^Flag fragments (marked by arrows) are all pulled-down along with the 37 kDa fragment by the anti-Flag beads. Not only do they all remain associated with the 37 kDa fragment, the protease clipped protein retains myosin motor domain and Hsp90 binding activity comparable to intact Unc45b^Flag. Lane T is the translation mix alone.

Figure 8. Gallery of Unc45b and Hsp90 molecules contrasted by rotary shadowing with platinum and imaged by electron microscopy.

Each image is the class average of 25–50 particles that were selected by reference-free classification of several hundred individual particles and then aligned. These characteristic views of the 105 kDa Unc45b monomer reveal a molecule that is ∼19 nm long and 5 nm wide. Many images show at least two domains, but this interpretation is limited by the resolution of the rotary shadowing contrasting method. Class averages of Hsp90 dimers (∼180 kDa) are shown for comparison. Hsp90 is a 30 nm long dimer with a C-terminal dimerization domain and N-terminal ATPase domains linked by flexible regions.