Figure 3. COQ8A Interacts with Complex Q but Lacks Protein Kinase Activity in trans.

(A) Immunoblot (IB) analysis of interactions between COQ8A-FLAG and COQ5-HA, COQ3-HA, or COQ9-HA transfected into COS cells and immunoprecipitated (IP’d) using anti-FLAG beads.

(B) Heatmap showing the top 25 endogenous proteins most enriched by COQ8A-FLAG IP’d from HEK293 cells compared to various control IPs (mean, n = 4).

(C) Relative abundances of endogenous proteins co-purifying with COQ8A-FLAG compared to MLS-GFP-FLAG (mean, n = 4) IP’d from HEK293 cells as assessed by LC-MS/MS.

(D) Cartoon of COQ8 active site residues (based on PDB 4PED).

(E) Relative abundances of endogenous COQ proteins co-purifying with COQ8A-FLAG (log₂(mutant/WT)) IP’d from HEK293 cells as assessed by LC-MS/MS.

(F) SDS-PAGE analysis of in vitro Mg[γ-³²P]ATP autophosphorylation reactions with Coq8^NΔ41 variants or PKA. BSA, bovine serum albumin (reaction buffer component).

(G) Divalent cation dependence of Coq8^NΔ41 autophosphorylation.

(H) Time course of Coq8^NΔ41 autophosphorylation.

(I) Coq8^NΔ41 autophosphorylation sites identified by LC-MS/MS mapped onto a homology model of Coq8p (based on COQ8A structure, PDB 4PED).

(J) SDS-PAGE analysis of in vitro Mg[γ-³²P]ATP autophosphorylation reactions with combinations of Coq8^NΔ41 variants. MBP, maltose binding protein tag.

(K) SDS-PAGE analysis of in vitro Mg[γ-³²P]ATP kinase reactions with PKA or Coq8^NΔ41 and potential substrate proteins.

See also Figure S3.