Dynamitin mutagenesis reveals protein-protein interactions important for dynactin structure

Abstract

Dynactin is a highly conserved, multiprotein complex that works in conjunction with microtubule-based motors to power a variety of intracellular motile events. Dynamitin (p50) is a core element of dynactin structure. In the present study, we use targeted mutagenesis to evaluate how dynamitin's different structural domains contribute to its ability to self-associate, interact with dynactin and assemble into a complex with its close binding partner, p24. We show that these interactions involve three distinct structural elements: (i) a previously unidentified dimerization motif in the N-terminal 100 amino acids, (ii) an alpha-helical motif spanning aa 106-162 and (iii) the C-terminal half of the molecule (aa 213-406), which is predicted to fold into an antiparallel alpha-helix bundle. The N-terminal half of dynamitin by itself is sufficient to disrupt dynactin, although very high concentrations are required. The ability of mutations in dynamitin's interaction domains to disrupt dynactin in vitro was found to correlate with their inhibitory effects when expressed in cells. We determined that the dynactin subunit, p24, governs dynamitin oligomerization by binding dynamitin along its length. This suppresses aberrant multimerization and drives formation of a protein complex that is identical to the native dynactin shoulder.

Figure 1

Schematic of the predicted structural features of human dynamitin and the mutants used in the analytical ultracentrifugation studies. The cartoons represent full length dynamitin and engineered fragments and indicate the positions of point mutants. The strongly predicted coiled-coil motifs C1 (AA 105–135), C2 (AA 219–251) and C3 (AA 281–308) are gray; C3, which also has a high propensity for multicoil formation, is indicated in black. The stippled box (AA 186–213) indicates a region that is predicted to be natively disordered. The three a-helices (H1, AA 221–243; H2, AA 261–299; and H3, AA 308–406) that are predicted to assume an anti-parallel, spectrin-like fold are indicated at the top. The binding sites for MacMARCKS, calmodulin and ZW10 map are indicated as M (AA 1–58), C (AA 59–83), and Z (AA 121–143), respectively (13, 20).

Figure 2

Identification of a protease-stable domain of dynamitin. Dynamitin was subjected to in-gel proteolysis (12) using endoproteinase-Glu-C (Endo-Glu-C, also known as “V8” protease; left) or proteolysis in solution using chymotrypsin (right). Left panel: Chick brain dynactin (left lane) was subjected to SDS-PAGE to isolate individual subunits, then gel bands corresponding to dynamitin (DM) were excised and re-electrophoresed in the presence of Endo-Glu-C (5 or 10 ng as indicated). Right panel: Purified recombinant dynamitin (1 nmol) was mixed with 10 pmol chymotrypsin and incubated for the times indicated, then the samples were subjected to SDS-PAGE. Both gels are stained with Coomassie blue. The asterisks mark the positions of the predominant digestion products (≈ 30 kDa).

Figure 3

Dynactin disruption mediated by wild-type, full length dynamitin and representative dynamitin mutants. A: Fifteen μg of purified bovine brain dynactin was left untreated (top panel) or mixed with a 25X molar excess of recombinant, full length, human dynamitin (DM), either wild type (WT) or carrying one or more point mutations, as indicated (C1/C2/C3 carried the V122P mutation). Following incubation, the samples were subjected to velocity sedimentation into 5 – 20% sucrose gradients and the gradient fractions were evaluated by immunoblotting. The behavior of the dynactin subunit p150^Glued is shown here. In all cases, Arp1 sedimented at 19–20S (as seen in B; data not shown for the other mutants). B: Purified dynactin was treated with a 25X molar excess of full length dynamitin (top) or 100X molar excess of the C-terminal half (bottom; AA 213–406), then subjected to velocity sedimentation as in A. p150^Glued and Arp1 were detected by immunoblotting. The C-terminal half was detected using Coomassie blue staining. C: Purified dynactin was treated with a 100X molar excess of the N-terminal half (AA 1–212). The behaviors of p150^Glued, endogenous dynamitin (DM) and the AA 1–212 fragment were followed by immunoblotting. Arp1 sedimented at 19–20S in this experiment (as in B; data not shown).

Figure 4

Effects of wild type and mutant dynamitin in vivo. Cells were transfected with plasmids encoding GFP-tagged wild type or the L118P mutant forms of dynamitin, then fixed and stained for the Golgi complex marker, giantin. Note the compact Golgi complex in the control cell in each panel. All dynamitin mutants that caused Golgi complex dispersion yielded similar results.

Figure 5

Reconstitution of recombinant dynactin shoulder (dynamitin:p24 complex). His-tagged p24 was denatured and renatured in the presence of excess untagged dynamitin as described in the text. The resulting soluble protein was analyzed by column chromatography (A and B) or (C) velocity sedimentation into a sucrose gradient. (A) A HiTrap column was used to separate dynamitin/p24 complexes (fractions 15–29) from free dynamitin (fraction 6). (B) Dynamitin/p24 complexes isolated as in B were purified further by MonoQ chromatography. For both panels, the lane labeled L is the column load and the numbers indicate different column fractions. (C) The sedimentation profile of the dynamitin/p24 mixture prior to any chromatography is shown at the top; dynamitin alone is shown below for comparison. The bottom of the sucrose gradient is at the left. BSA (4.6S) was added to both samples prior to sedimentation to provide an internal standard; this did not alter the sedimentation behavior of dynamitin or the dynamitin/p24 complex. All gels were stained with Coomassie blue.

Figure 6

Model of dynamitin structural domains (see box; shading is as in Figure 1), plus proposed modes of self-association of wild type and mutant species. The N-terminal dimerization domain is depicted as a shaded oval, the N-terminal α-helix as a thin zig-zag, the disordered region as a dashed line, and the C-terminal domain as a thick zig-zag. The different sized arrows indicate steps that are more or less highly favored.