Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 30, Pages 22163-22173Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.484329
Keywords
Gel Electrophoresis; Membrane Proteins; Mitochondrial Transport; Protein Aggregation; Transporters; Detergent Micelle
Categories
Funding
- Medical Research Council
- European Membrane Protein Consortium [LSHG-CT-2004-504601]
- European Drug Initiative on Channels and Transporters [HEALTH-F4-2007-201924]
- MRC [MC_U105663139] Funding Source: UKRI
- Medical Research Council [MC_U105663139] Funding Source: researchfish
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Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two approximate to 32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from approximate to 60 to approximate to 130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is approximate to 120 kDa, but appears smaller on gels (approximate to 60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.
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