Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome b in yeast mitochondria

Abstract

Cytochrome (Cyt) is the only mitochondrial encoded subunit from the complex. Cbp3 and Cbp6 are chaperones necessary for translation of the mRNA and Cyt hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cyt hemylation seems to be universally conserved. BY4742 yeast requires Cbp3 and Cbp6 for efficient mRNA translation, whereas the D273-10b strain synthesizes Cyt at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cyt are close to wildtype in mutant D273-10b cells, and Cyt forms non-functional, supercomplex-like species with cytochrome oxidase, in which at least core 1, cytochrome, and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cyt synthesis is of nuclear origin rather than mitochondrial, and Smt1, a mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cyt hemylation and demonstrate that in the absence of heme , Cyt can form non-functional supercomplexes with cytochrome oxidase. Our observations support that an additional protein or proteins are involved in Cyt synthesis in some yeast strains.

Mitochondrial microproteins link metabolic cues to respiratory chain biogenesis

“…CIII assembly in yeast begins with the translation of COB mRNA from the mitochondrial genome. COB mRNA is translated to produce cytochrome b (Cytb) under the control of two sets of regulators-Cbs1, Cbs2, Cbp1, and the Cbp3/ Cbp6 complex (Garcı ´a- Guerrero et al, 2018;Gruschke et al, 2011Gruschke et al, , 2012. Cbp3/Cbp6 competes with Cbs1 at the mito-ribosome exit tunnel to stimulate Cytb translation (Gruschke et al, 2011(Gruschke et al, , 2012Salvatori et al, 2020).…”

“…CYTB, the mtDNA-encoded core enzymatic subunit of CIII, is the first protein to be synthesized during assembly. Assembly factors required for CYTB biogenesis have been extensively studied in yeast (Garcı ´a-Guerrero et al, 2018;Gruschke et al, 2011Gruschke et al, , 2012Salvatori et al, 2020). While they have been identified in mammals (Tucker et al, 2013;Wanschers et al, 2014), how these early steps of CYTB synthesis are achieved in mammalian CIII biogenesis is unknown, along with the identities of the proteins and complexes involved.…”

Pathways to balance mitochondrial translation and protein import

“…Interestingly, the Cbp3/ Cbp6 complex is present in two pools. One population of Cbp3/Cbp6 interacts with mitoribosomes in close proximity to the polypeptide exit tunnel to stimulate synthesis of cytochrome b by an unknown mechanism (Gruschke et al 2011;García-Guerrero et al 2018). A second pool of Cbp3/Cbp6 remains associated with cytochrome b after its synthesis is completed to keep the protein in a conformation that allows insertion of the first heme b molecule and binding of the assembly factor Cbp4 ( Fig.…”

The cytochromebcarboxyl-terminal region is necessary for mitochondrial Complex III assembly

“…Cbp3 and Cbp6 are chaperones that interact with the mitoribosome tunnel exit and with the newly synthesized Cyt b (Gruschke et al , 2011). In some yeast strains, Cbp3/Cbp6 are necessary for COB mRNA efficient translation (Dieckmann & Tzagoloff, 1985; García-Guerrero et al , 2018; Gruschke et al ., 2011; Gruschke et al ., 2012; Wu & Tzagoloff, 1989), although the main role of these proteins is to trigger heme b L addition to Cyt b , through a mechanism that is not completely understood (García-Guerrero et al ., 2018; Hildenbeutel et al , 2014). The current model states that interaction of Cyt b , Cbp3 and Cbp6 form intermediate 0 (Hildenbeutel et al ., 2014).…”