The Role of Subunit VIII in the Structural Stability of the bc₁ Complex from Saccharomyces cerevisiae Studied Using Hybrid Complexes

Abstract

The QCR8 genes encoding subunit VIII of the bc, complex from Kluyveromyces lactis and Schizosaccharomyres pombe partially complement the respiratory-deficient phenotype of a S. cerevisiae QCR8-null mutant. This implies that the heterologous Qcr8 subunits can be imported by S. cerevisiae mitochondria and that they assemble to form a hybrid bc, complex that is sufficiently active to support growth. In contrast, the QCR8 gene from bovine heart, encoding the 9.5-kDa subunit, is not able to restore respiratory function to the S. cerevisiae null mutant. This lack of functional complementation is directly attributable to the inability of S. cerevisiae mitochondria to import this protein as shown by in vitro assays. However, a hybrid gene encoding the N-terminal 26 residues of S. cerevisiae subunit VIII and the rest of the 9.5-kDa bovine heart homologue, was able to functionally complement the QCR8-null mutant, albeit to a very low extent. Successful import into S. cerevisiae mitochondria was confirmed by in vitro import experiments. Surprisingly, although assembly of these hybrid complexes is reduced to an extent that is proportional to the evolutionary distance of the homologue to S. cerevisiae, the specific activities of the assembled complexes are the same as for the wild-type hc, complex. After solubilisation of the mitochondria1 membranes with the mild detergent dodecyl maltoside, the wild-type enzyme can be inactivated by incubation at increased temperature, independent of protease activity. The rate of inactivation can be significantly increased by the addition of o-phenanthroline [Boumans, H., Grivell, L. A. & Berden, J. A. (1997) J. Biol. Chem. 272, 16753-16760]. The hybrid complexes are much more sensitive to both types of treatment. We conclude that substitution of subunit VIII by a homologous counterpart results in a loosening of the structure of the be, complex on the intermembrane space side, resulting in a less stable insertion of the Rieske Fe-S protein in vivo and therefore a lower stability of the assembled enzyme under certain in vitro conditions, but without an effect on catalytic activity.

Intermediate Length Rieske Iron-Sulfur Protein Is Present and Functionally Active in the Cytochrome bc 1Complex of Saccharomyces cerevisiae

“…5B). This suggests that o-phenanthroline destabilizes the binding of ironsulfur protein to the rest of the bc 1 complex, probably as a result of disruption of the iron-sulfur cluster as described by Boumans and co-workers (26,27). DISCUSSION We have investigated the order of processing and assembly steps during import of the Rieske iron-sulfur protein into S. cerevisiae mitochondria.…”

“…Boumans and co-workers (26,27) showed that o-phenanthroline binds to solubilized bc 1 complex and disrupts the ironsulfur cluster. This can explain our finding that coimmunoprecipitation of iron-sulfur protein with the bc 1 complex in the presence of o-phenanthroline is much less efficient, if after o-phenanthroline has disrupted the iron-sulfur cluster the protein is less stably associated with the complex.…”

Functional complementation analysis of yeastbc1mutants

“…The S. cerevisiae strain DALU273 was made as described by Boumans et al . [11]. Disruption of the QCR8 gene was performed as described previously [10], giving DALU80 ( MATa, ura3, lys2, qcr8::LEU2 ) .…”

“…Disruption of the QCR8 gene was performed as described previously [10], giving DALU80 ( MATa, ura3, lys2, qcr8::LEU2 ) . S. cerevisiae strains DLL70 ( MATα , his3 , ura3 , qcr7::LEU2 ) [20], DLL80 ( MATα, his3, ura3, qcr8::LEU2 ) [10] and DALU80 [11] were used for the transformation of plasmids. Transformation of yeast was performed by the method of Klebe et al .…”

“…Both subunits are essential for correct assembly of the complex [9,10]. Complementation studies, designed to analyze the functionality in yeast of orthologous subunits from other organisms, suggested that, at increasing evolutionary distance, the import into S. cerevisiae mitochondria plays a crucial role and should be further analysed [11,12].…”