Figure 2. Hierarchical assembly of TBCC with TBC-DEG and soluble αβ-tubulin dimer binding in the GDP·Pi state.

(A) Size exclusion chromatography (SEC) intensity traces of TBC-DEG (black), TBC-DEG:αβ-tubulin (cyan), αβ-tubulin (red), and TBCC (purple). (B) SEC intensity traces of TBC-DEG+TBCC+αβ-tubulin-GDP·ALF_x (green), TBC-DEG+TBCC+αβ-tubulin-GTP (gray), TBC-DEG+TBCC-GTP-ALF_x (black), TBCC+αβ-tubulin (blue), and αβ-tubulin+TBCC (blue). Additional states are described in Figure 2—figure supplement 1C,D. (C) Composition of SEC fractions shown in A and B using SDS-PAGE. Panel I, TBC-DEG; panel II, TBC-DEG:αβ-tubulin; panel III, TBC-DEG+TBCC-GDP·ALF_x; panel IV, TBCC+αβ-tubulin; panel V, TBC-DEG+TBCC+αβ-tubulin-GTP; and panel VI, TBC-DEG+TBCC+αβ-tubulin-GDP·ALF_x. TBC-DEG forms an active heterotrimeric complex, and TBCC forms a complex that co-migrates with TBC-DEG upon αβ-tubulin binding in the presence of GDP·ALF_x (panel IV). The protein standard is shown on the left and proteins are marked on the right. TBC-DEG complexes interact weakly with the resin media leading to wide elution SEC profiles in most conditions. (D) Molecular masses of TBC-DEG, αβ-tubulin, TBCC, and their complexes measured using size exclusion chromatography with multi-angle light scattering (SEC-MALS). Solid lines represent SEC intensity traces on an intensity scale shown on the right y-axis, and dotted lines represent masses calculated on the mass scale shown on the left y-axis; TBC-DEG (black), αβ-tubulin (red), TBCC (purple), TBC-DEG:αβ-tubulin (cyan), and TBC-DEG:αβ-tubulin:TBCC-GDP·ALF_x (green). Masses and elution volumes are detailed in Table 2. (E) Scheme for the hierarchical assembly of TBC-DEG with TBCC and αβ-tubulin and the role of nucleotide. TBCD, TBCE, and Arl2 form TBC-DEG complexes (TBC-DEG) and bind a single αβ-tubulin dimer (αβ-tub) to form TBC-DEG:αβ-tubulin (TBC-DEG:αβ-tub), which recruits TBCC in the GTP-like state to form TBC-DEG:αβ-tubulin:TBCC (TBC-DEG:αβ-tub:TBCC).

DOI: http://dx.doi.org/10.7554/eLife.08811.004

Figure 2—figure supplement 1. Tubulin cofactor-Arl2 co-expression and biochemical studies on TBC-DEG constructs.

(A) Domain structures of tubulin cofactors and Arl2. Top, TBCD (gray) composed of HEAT repeats. Second, TBCE, composed of Cap-Gly (blue), LRR (cyan), and ubiqutin-like (light blue) domains. Third, the Arl2-GTPase composed of ARF-like G protein fold (red) and outer unique termini (orange). Fourth, TBCC composed of an N-terminal spectrin homology domain (light green), and a β-sheet domain (dark green). (B) Summary of co-expression experiments, TBC, and Arl2 proteins. The masses of each of the proteins are shown on the left and correspond to the order shown in A. The effects of deletion (Δ) or addition of GFP (GFP) or 6Xhis-tags (his) at the N-termini and C-termini of each of the TBC and Arl2 proteins on TBC-DEG complexes are described, where check marks describe no effect on TBC-DEG expression, while a cross mark describes loss of TBC-DEG expression. (C) Size exclusion chromatography (SEC) intensity traces of TBC-DEG:αβ-tubulin (cyan), TBC-DEG:αβ-tubulin 1:2 molar ratio (blue), αβ-tubulin (red), and TBCC (purple). (D) Composition of SEC fractions shown in C using SDS-PAGE. Panel I, αβ-tubulin; panel II, TBCC; panel III, TBC-DEG+αβ-tubulin 2:1 molar ratio; and panel IV, TBC-DEG+αβ-tubulin+TBCC+GTPγS. The protein standard is shown on the left and proteins are marked on the right. (E) Size exclusion chromatography (SEC) intensity traces of TBC-DE(N-GFP)G (black). (F) Composition of SEC fractions shown in E using SDS-PAGE for TBC-DE(N-GFP)G. The protein standard is shown on the left and protein positions are marked on the right.

DOI: http://dx.doi.org/10.7554/eLife.08811.005