Aggregates of yeast mitochondrial cytochromeb observed after electrophoresis

Mitochondrial translation products obtained from yeast cells labeledin vivo in the presence of cycloheximide were separated by dodecylsulfate polyacrylamide gel electrophoresis. The labeled band, with a molecular weight of 30,000 corresponding to cytochromeb, was excised and subsequently transferred to a second gel. After electrophoretic separation, two labeled polypeptides with apparent molecular weights of 67,000 and 27,000 became visible in addition to the cytochromeb band of 30,000 molecular weight. Heating of the cytochromeb band prior to transfer resulted in an increase in the amount of the labeled polypeptides migrating with a molecular weight of 67,000.Longer exposure during autoradiography of the gels of mitochondrial translation products resulted in the appearance of a double band with an apparent molecular weight of 67,000. Limited proteolysis of this 67,000 dalton protein withStaphylococcus aureus V8 protease revealed a peptide map similar to that obtained after proteolysis of cytochromeb. These results suggest that the polypeptide with an apparent molecular weight of 67,000 represents an aggregate of cytochromeb that is either present as such in the membrane or is formedin vitro during the experimental manipulations to prepare mitochondria for gel electrophoresis.Abbreviations used: SDS, sodium dodecylsulfate.This work is in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the City University of New York.