BOLA1 is an aerobic protein that prevents mitochondrial morphology changes induced by glutathione depletion

Abstract

Aims: The BolA protein family is widespread among eukaryotes and bacteria. In Escherichia coli, BolA causes a spherical cell shape and is overexpressed during oxidative stress. Here we aim to elucidate the possible role of its human homolog BOLA1 in mitochondrial morphology and thiol redox potential regulation.

Results: We show that BOLA1 is a mitochondrial protein that counterbalances the effect of L-buthionine-(S,R)-sulfoximine (BSO)-induced glutathione (GSH) depletion on the mitochondrial thiol redox potential. Furthermore, overexpression of BOLA1 nullifies the effect of BSO and S-nitrosocysteine on mitochondrial morphology. Conversely, knockdown of the BOLA1 gene increases the oxidation of mitochondrial thiol groups. Supporting a role of BOLA1 in controlling the mitochondrial thiol redox potential is that BOLA1 orthologs only occur in aerobic eukaryotes. A measured interaction of BOLA1 with the mitochondrial monothiol glutaredoxin GLRX5 provides hints for potential mechanisms behind BOLA1's effect on mitochondrial redox potential. Nevertheless, we have no direct evidence for a role of GLRX5 in BOLA1's function.

Innovation: We implicate a new protein, BOLA1, in the regulation of the mitochondrial thiol redox potential.

Conclusion: BOLA1 is an aerobic, mitochondrial protein that prevents mitochondrial morphology aberrations induced by GSH depletion and reduces the associated oxidative shift of the mitochondrial thiol redox potential.

FIG. 1.

Phylogenetic distribution of the BolA family among eukaryotes and Escherichia coli and conservation of key residues. A phylogeny was determined for BolA1, 2, and 3 among species that cover the major eukaryotic taxa, including all anaerobic species (indicated in red), and the Bola family members for which experimental knowledge is available (in bold). The phylogeny clearly separates a BolA1 clade (top) from the other two BolA clades (BolA3 on the left, BolA2 on the right-bottom). The C-terminal histidine in the figure (green symbol) represents His-103 in Fra2, an Fe-S cluster ligand in the BolA2-Grx3 complex (25). This histidine is conserved throughout the BolA family in Eukaryotes (Supplementary Fig. S4). A cysteine (Cys-97 in BOLA2, red symbol) that has also been implicated in Fe-S binding in the BolA2-Grx3 complex, although not directly (25), is only conserved in BolA2 and BolA3. In the BolA1 clade, this cysteine appears to be replaced by a conserved histidine (His-67 in BOLA1). The phylogeny indicates that the Arabidopsis thaliana protein that is fused with a SufE domain (40) is a member of the BolA1 clade.

FIG. 2.

BOLA1 is a mitochondrial protein. (A) Overlap in BOLA1-GFP (left panel) and TMRM (middle panel) fluorescence, demonstrating mitochondrial localization of BOLA1 in human skin fibroblasts (right panel). Note the absence of any TMRM fluorescence bleed through in GFP-negative cells. (B) BOLA1-GFP is exclusively present in the mitochondrial fraction of doxycycline-induced HEK293 cells. (C) Mitochondria-associated BOLA1-GFP and GLRX5-GFP are proteinase K-resistant, suggesting their presence within the limits of the inner-membrane. In (A), images were contrast optimized for visualization purposes.CK-B, cytosolic creatine kinase B; TOM20, import receptor of the outer-membrane translocator; prohibitin 1, mitochondrial inner membrane associated IMS protein; SDHA, A-subunit of the succinate dehydrogenase complex of the mitochondrial matrix; TMRM, tetramethylrhodamine methyl ester.

FIG. 3.

BOLA1 is complexed to GLRX5. (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis of a single-step affinity purification performed with doxycycline-induced HEK293 cells expressing GLRX5-TAP. Membranes were probed with antibodies against BOLA1, core 2 protein of mitochondrial complex III, A-subunit of the succinate dehydrogenase complex of the mitochondrial matrix (SDHA), and CBP for detection of the TAP-tag. As a control, noninduced cells were used. (B) Analysis of BOLA1-TAP purification with cell lysates containing GLRX5-GFP or GFP. Membranes were probed with anti-GFP to check for copurification and to determine the input (upper and middle panel, respectively) and anti-CBP to test the efficiency of the pull down. Molecular mass marker (kDa) is shown on the left. CBP, calmodulin-binding protein.

FIG. 4.

Knockdown of BOLA1 increases thiol redox potential in the mitochondrial matrix of HeLa cells. (A) Knockdown of BOLA1 by siRNAs in HeLa cells. Cells were transfected with three different BOLA1 siRNA duplexes (left panel) and a control siRNA targeting cyclophilin B (right panel). The left upper immunoblot probed with anti-BOLA1 demonstrates the effectiveness of the siRNAs. Beta-tubulin was used as protein-loading control. Molecular mass markers (kDa) are indicated on the left. Note that siRNA against cyclophilin B did not alter BOLA1 expression and BOLA1 siRNAs did not affect cyclophilin B expression. (B) BOLA1 knockdown is without effect on GLRX5 expression. (C) Cells, transfected with the indicated siRNA, were transduced with a baculovirus for expression of mito-roGFP. The percent oxidation was calculated after establishment of the maximum and minimum oxidation levels with 1 mM H₂O₂ and 10 mM DTT, respectively, as described before (35). Numerals indicate the number of cells analyzed. a and b, significantly different from the value of the corresponding bar (p<0.05). DTT, dithiothreitol.

FIG. 5.

Effect of BOLA1 overexpression on the redox potential of mitochondrial thiol groups and mitochondrial morphology under various (oxidizing) conditions. (A) BOLA1 overexpression attenuates the BSO-induced increase in oxidizing potential of the mitochondrial matrix. Fibroblasts, cotransduced for expression of mito-roGFP1 and either BOLA1-RFP or COX8-RFP, were treated without or with 1 μM or 12.5 μM BSO for 72 h. The percent oxidation in COX8- and BOLA1-RFP-positive cells was calculated as described in the legend to Figure 4. (B) BOLA1 overexpression does not interfere with the BSO-induced increase in oxidizing potential of the cytosol. (C) BOLA1 overexpression has no effect on mitochondrial morphology and number. Fibroblasts, transduced for expression of BOLA1-GFP were loaded with TMRM and subjected to digital-imaging microscopy of GFP and TMRM fluorescence. Quantitative image analysis revealed that compared to BOLA1-GFP-negative cells, BOLA1-GFP-positive cells contained mitochondria that were similar in size (mitochondrial area), length (AR), length and degree of branching (form factor, F), and of mitochondria per cell (Nc), showing that BOLA1 overexpression does not affect mitochondrial morphology and number. (D) BOLA1 prevents the BSO-induced change in mitochondrial morphology. Fibroblasts, transduced for expression of BOLA1-GFP were treated with 12.5 μM BSO for 72 h. Compared to vehicle-treated cells, BSO-treated BOLA1-GFP-negative cells contained mitochondria that were smaller in size, less elongated, and less branched. In sharp contrast, BOLA1-GFP-positive cells, present on the same coverslip, did not display these BSO-induced differences in mitochondrial morphology. The number of mitochondria per cell did not significantly differ between the different conditions. The value obtained with vehicle-treated cells was set at 100%. (E) BOLA1 prevents the SNOC-induced change in mitochondrial morphology. Fibroblasts, transduced for expression of either COX8-GFP or BOLA1-GFP were treated with 100 μM SNOC for 72 h. Subsequent analysis showed that SNOC induced a significant decrease in mitochondrial area, length, and degree of branching in COX8-GFP-positive cells, but not in BOLA1-GFP-positive cells. The value obtained with vehicle-treated COX8-GFP-positive cells was set at 100%. (F) DTT prevents the BSO-induced change in mitochondrial morphology. Fibroblasts were treated with BSO (12.5 μM), alone and in combination with DTT (100 μM), for 72h, and analyzed for mitochondrial morphology. The value obtained with vehicle-treated cells was set at 100%. Numerals indicate the number of cells analyzed. a, b, and c, significantly different (p<0.05) from the corresponding bar. BSO, L-buthionine-(S,R)-sulfoximine; SNOC, S-nitrosocysteine; AR, aspect ratio; COX8, cytochrome c oxidase subunit VIII.