Rcf proteins and their differential specificity for respiratory chain complexes: A unique role for Rcf2 on oxygen sensitive supercomplexes?

Highlights

• Rcf2 shows an N-out C-out topology.

• Rcf2 and Rcf3 bind early subunits of the complex IV.

• Rcf1 and Rcf3 are excluded from the hypoxic III₂IV complex.

• Rcf2^N-Rcf1 can compensate for a combined rcf1∆/rcf2∆/rcf3∆ phenotype.

• Rcf2 and the endogenous Rcf2^N and Rcf2^C fragments associate with the hypoxic III₂IV complex

Abstract

The respiratory chain, embedded in the inner mitochondrial membrane, is organized as a network of individual complexes, as well as large supercomplex structures. In the yeast S. cerevisiae, these supercomplexes consist of a dimeric cytochrome bc₁-complex adjoined by one or two copies of cytochrome c oxidase. The formation of these complexes is a dynamic process and is regulated by various factors in order to adapt to environmental and metabolic changes. These adaptions occur at the level of enzyme regulation, complex assembly, as well as altered nuclear and mitochondrial transcription and translation. Members of the Rcf protein family (Rcf1, Rcf2 and Rcf3) are required for respiratory complex biogenesis and act mainly by regulating the assembly and enzyme activity of complex IV within supercomplexes. Rcf1 functions in the assembly process via the COX3 module, whereas Rcf2 and Rcf3 are responsible for enzymatic regulation. In this study, we have extended this knowledge to show that Rcf2 and Rcf3 can also associate with newly synthesized mitochondrial encoded proteins, such as Cox3, and therefore contribute to complex IV assembly. Since the Rcf proteins have overlapping regions of sequence similarities, we engineered novel fusion proteins of Rcf1 and Rcf3, with parts of Rcf2, to probe which of the Rcf protein domains can be attributed to their functions. The fusion proteins could compensate for the individual phenotypes of the complexIV assembly defect and the lack of complex IV regulation. Finally, the role of Rcf proteins for defined species of respiratory chain complexes in a hypoxic model was investigated, uncovering a unique association of Rcf2 with the hypoxic III₂IV supercomplex. We therefore suggest an involvement of Rcf2 for adaption of the respiratory chain to altering oxygen levels.