Phosphorylation of the F(1)F(o) ATP synthase beta subunit: functional and structural consequences assessed in a model system

Abstract

Rationale: We previously discovered several phosphorylations to the beta subunit of the mitochondrial F(1)F(o) ATP synthase complex in isolated rabbit myocytes on adenosine treatment, an agent that induces cardioprotection. The role of these phosphorylations is unknown.

Objective: The present study focuses on the functional consequences of phosphorylation of the ATP synthase complex beta subunit by generating nonphosphorylatable and phosphomimetic analogs in a model system, Saccharomyces cerevisiae.

Methods and results: The 4 amino acid residues with homology in yeast (T58, S213, T262, and T318) were studied with respect to growth, complex and supercomplex formation, and enzymatic activity (ATPase rate). The most striking mutant was the T262 site, for which the phosphomimetic (T262E) abolished activity, whereas the nonphosphorylatable strain (T262A) had an ATPase rate equivalent to wild type. Although T262E, like all of the beta subunit mutants, was able to form the intact complex (F(1)F(o)), this strain lacked a free F(1) component found in wild-type and had a corresponding increase of lower-molecular-weight forms of the protein, indicating an assembly/stability defect. In addition, the ATPase activity was reduced but not abolished with the phosphomimetic mutation at T58, a site that altered the formation/maintenance of dimers of the F(1)F(o) ATP synthase complex.

Conclusions: Taken together, these data show that pseudophosphorylation of specific amino acid residues can have separate and distinctive effects on the F(1)F(o) ATP synthase complex, suggesting the possibility that several of the phosphorylations observed in the rabbit heart can have structural and functional consequences to the F(1)F(o) ATP synthase complex.

Figure 1. The amino acid residues of interest mapped onto the 3D structure of the α/β hexamer of S. Cerevisiae (PDB file 2HLD)

Amino acid residues are color-coded, with numbers given for the mature protein (with known mitochondrial targeting sequence removed). Two of the residues (T58, yellow and S213, red) are located on the matrix-facing portion of the β subunit while the other two (T262, green and T318, blue) are located within the center of the complex. Both buried (T262) and accessible (T58) residues had observed assembly and functional differences.

Figure 2. Blue Native PAGE (BN-PAGE) of isolated WT and mutant mitochondria

A) 1D SDS-PAGE (4–20%), denaturing/reducing gel. The total protein stain shows equal protein loading and β subunit content of all lanes (except atp2Δ, which lacks the β subunit). Total protein stain of BN-PAGE gels shows equal protein content in all lanes for lauryl maltoside (B) and digitonin (D) solubilized mitochondria. (C) and (E) display representative western blots of the ATP synthase β subunit (n=3) for lauryl maltoside and digitonin solubilized mitochondria, respectively. Bands are labeled according to the complex that is present; F₁F_o dimers, F₁F_o monomers, the F₁ portion of the complex and α/β lower molecular weight bands (For intensity data values see Online Table II).

Figure 3. Two-dimensional BN/SDS-PAGE of isolated WT and mutant mitochondria

A) Lauryl maltoside solubilized mitochondria from WT, T262A and T262E yeast strains and B) digitonin solubilized mitochondria from WT, T58A, T58E, T262A and T262E were subjected to BN-PAGE in the first dimension and SDS-PAGE in a second dimension (4–12% gels). This technique allows for the separation of complexes observed on 1D BN-PAGE into individual subunits. Circled protein gel spots were identified by MS/MS as indicated. These 2D BN/SDS-PAGE gels confirm the presence of the F_o and/or F₁ subunits at the correct location in the BN-PAGE. (For MS identification data see Online Table III).

Figure 4. In-solution ATPase assays

A) Mitochondria were solubilized in lauryl maltoside and the complexes were separated on a sucrose gradient to isolate the ATP synthase complex. A representative gel of the combined ATP synthase fractions from each of the strains is shown. Arrows indicate the contaminating bands also present in the atp2Δ lanes. ATPase assays were performed on this deletion strain as a negative control. B) In-solution ATPase assays were performed on fractions from each of the yeast mutant strains and were compared to WT and the negative control (atp2Δ). ND = no detectable signal, *represents significant difference from WT, and ^§represents significant difference between the non-phosphorylatable and the phospho-mimetic strains (both based on Mann-Whitney test p<0.05). n=6 all observed values fell within the linear range of the assay.

Figure 5. In-gel ATPase assays

In-gel ATPase assays were performed on BN-PAGE of digitonin solubilized mitochondria from all strains. Left panel is a representative Coomassie stained gel to indicate protein load. The right panel illustrates the ATPase activity of each of the bands as white lead phosphate precipitate on the gel. These assays mirror the results observed in Figure 4 for in-solution ATPase assays (n=3).

Figure 6. Schema of ATP synthase structures and the effects of the phospho-mimetic mutations

The various structures observed by BN-PAGE are listed and the subunit interactions that occur within them. Mutants that caused defects at any stage are listed.