Characterization of Assembly Intermediates Containing Subunit 1 of Yeast Cytochrome Oxidase

McStay, Gavin P.; Su, Chen-Hsien; Thomas, Susan M.; Xu, Jonathan; Tzagoloff, Alexander

doi:10.1074/jbc.m113.498592

Cited by 23 publications

(39 citation statements)

References 34 publications

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“…2B). This region was previously shown to contain the D3 and D4 assembly intermediates of Cox1p (13,21). Two-dimensional electrophoresis of the Cox16-CH enriched fraction confirmed a large increase of labeled Cox1p in the region corresponding to the D3 and D4 assembly intermediates of COX ( Fig.…”

Section: Cox16p Is Associated With the Cox1p Modulesupporting

confidence: 54%

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Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase

Tzagoloff

2017

Journal of Biological Chemistry

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Mitochondrial cytochrome oxidase (COX) catalyzes the last step in the respiratory pathway. In the yeast , this inner membrane complex is composed of 11 protein subunits. Expression of COX is assisted by some two dozen ancillary proteins that intercede at different stages of the assembly pathway. One such protein, Cox16p, encoded by, was shown to be essential for the activity and assembly of COX. The function of Cox16p, however, has not been determined. We present evidence that Cox16p is present in Cox1p assembly intermediates and in COX. This is based on the finding that Cox16p, tagged with a dual polyhistidine and protein C tag, co-immunopurified with Cox1p assembly intermediates. The pulldown assays also indicated the presence of Cox16p in mature COX and in supercomplexes consisting of COX and the complex. From the Western signal strengths, Cox16p appears to be substoichiometric with Cox1p and Cox4p, which could indicate that Cox16p is only present in a fraction of COX. In conclusion, our results indicate that Cox16p is a constituent of several Cox1p assembly intermediates and of COX.

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Section: Cox16p Is Associated With the Cox1p Modulesupporting

confidence: 54%

“…2C). These results indicate that Cox16p is a component of the Cox1p module (13,21). The two-dimensional gels also revealed a large chloramphenicol-induced increase of radiolabeled cytochrome b in the supercomplexes (Fig.…”

Section: Cox16p Is Associated With the Cox1p Modulementioning

confidence: 66%

Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase

Tzagoloff

2017

Journal of Biological Chemistry

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“…Biogenesis of an active mitochondrial oxidase complex [6][7][8] requires (a) cross-talk between the expression systems for the nuclear and mitochodrially encoded subunits, (b) import into the mitochondrion, (c) recruitment or synthesis of the redox-active cofactors, and (d) their insertion into the relevant subunits. At the same time, any deleterious side reactions due to exposed or free cofactors, such as the generation of reactive oxygen species, must be avoided.…”

Section: Introductionmentioning

confidence: 99%

Deciphering protein–protein interactions during the biogenesis of cytochrome c oxidase from Paracoccus denitrificans

Gurumoorthy

Ludwig

2014

The FEBS Journal

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Biogenesis of the mitochondrial cytochrome c oxidase (COX) is a complex process due to its numerous subunits encoded by two genomes, as well as the localization of redox centers deep within the membrane. Here, we have assessed the biogenesis of the homologous aa 3 -type oxidase of the soil bacterium Paracoccus denitrificans. First, protein partners were analyzed using various membrane solubilization strategies to show interactions between COX and CtaG, a chaperone implicated in Cu B site metallation. Using an unbiased MS approach after immunological pull-down from untreated or cross-linked membranes, we then extend our view towards a hypothetical 'biogenesis complex' by identifying two further metal-inserting chaperones, Surf1c and Sco, together with enzymes catalyzing heme a synthesis. Our study also tentatively supports previous speculation regarding the existence of a predominantly co-translational mechanism for cofactor insertion during COX biogenesis.

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“…Pulse-chase analysis of COX intermediates in isolated mitochondria have revealed that the core Cox1p and Cox3p subunits assemble independently with their own proprietary set of nuclear-encoded subunits before the resultant modules assemble with each other. Most, if not all, structural subunits have been found to be associated with Cox1p and Cox3p (11,12). In addition to the structural subunits, the Cox1p module also contains translational regulators Mss51p, Cox14p, and Coa3p as well as several other factors involved in the maturation of the protein (13)(14)(15)(16)(17)(18)(19).…”

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confidence: 99%

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules

Franco

McStay

et al. 2018

Journal of Biological Chemistry

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Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast , COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450-550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

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Characterization of Assembly Intermediates Containing Subunit 1 of Yeast Cytochrome Oxidase

Cited by 23 publications

References 34 publications

Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase

Cox16 protein is physically associated with Cox1p assembly intermediates and with cytochrome oxidase

Deciphering protein–protein interactions during the biogenesis of cytochrome c oxidase from Paracoccus denitrificans

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules

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