Mitochondrial versus nuclear gene expression and membrane protein assembly: the case of subunit 2 of yeast cytochromecoxidase

Abstract

The diminished cytochrome c oxidase (CcO) steady-state levels in a yeast strain expressing an allotopic cCox2W56R subunit are due to inefficient translocation through TIM23. When nuclear cCox2W56R and mitochondrial Cox2 (mtCox2) were coexpressed, they gave rise to a mixed population of CcO, with most complexes containing mtCox2.

Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochromecoxidase

“…Similarly to what was observed in in vitro import assays in legumes (Daley et al , 2002b), each single-substitution V49Q or L51G was insufficient to restore respiration. In line with the cytosol-synthesized Cox2 biogenesis model (Daley et al , 2002a; Jiménez-Suárez et al , 2012; Rubalcava-Gracia et al , 2018), the first TMS (TMS1) of Cox2 is transferred to the matrix through the TIM23 translocase, while the second TMS is laterally released into the inner membrane by TIM23 (Figure 4D). As a final step, TMS1 is inserted from the matrix in the inner membrane by Oxa1, positioning Cox2 in its correct topology (Figure 4D).…”

“…Previous work evidenced that most of the allotopically synthesized Cox2 W56R protein accumulates as an unprocessed precursor (i.e., still carrying its MTS) detected in the mitochondrial fraction (Supekova et al , 2010; Cruz-Torres et al , 2012; Rubalcava-Gracia et al , 2018). We thus performed cellular fractionations to test the cytosolic versus mitochondrial distribution of Cox2 WT , Cox2 W56K , and Cox2 W56Q .…”

“…Next, we questioned whether Cox2 V49Q/L51G follows the same biogenesis pathway as that of allotopically produced Cox2 W56R . In contrast to orthodox Cox2, synthesized in mitochondria, allotopically synthesized Cox2 W56R does not require the Cox18 translocase for the correct insertion of its second TMS into the inner membrane (Elliott et al , 2012), and it was postulated to be released laterally into this membrane directly from the TIM23 complex (Rubalcava-Gracia et al , 2018). We observed that, similarly to the Cox2 W56R strain, the Cox2 V49Q/L51G variant is able to restore respiration when expressed in a cox18 -null (Figure 2D), indicating that the Cox18 translocase is completely dispensable.…”

“…The representative species of this lineage encode Cox2 in the nuclear DNA, in the mitochondrial DNA, or in both genomes, depending on which copy was activated and/or inactivated (Adams et al , 1999; Daley et al , 2002a,b). Also, the COX2 gene has been relocalized experimentally to the yeast nucleus (Supekova et al , 2010), and subsequent studies have provided supporting genetic and biochemical evidence for successful allotopic expression (Cruz-Torres et al , 2012; Elliott et al , 2012; Rubalcava-Gracia et al , 2018). The substitution of a tryptophan for an arginine in position 56 (Cox2 W56R ), within the first transmembrane segment (TMS) of allotopically produced Cox2, decreases its hydrophobicity and allows the partial respiratory rescue of a cox2- null yeast strain, with an estimated ∼60% recovery of C c O levels as compared with the wild-type strain (Cruz-Torres et al , 2012).…”

“…The substitution of a tryptophan for an arginine in position 56 (Cox2 W56R ), within the first transmembrane segment (TMS) of allotopically produced Cox2, decreases its hydrophobicity and allows the partial respiratory rescue of a cox2- null yeast strain, with an estimated ∼60% recovery of C c O levels as compared with the wild-type strain (Cruz-Torres et al , 2012). This partial complementation can be attributed solely to diminished import, since a mitochondrial COX2 gene producing the W56R variant cannot be distinguished from wild-type (Rubalcava-Gracia et al , 2018). The decrease in hydrophobicity of the first TMS in Cox2 also occurred during the natural relocation of the COX2 gene to the nucleus in legumes (Daley et al , 2002b).…”