The P174L Mutation in Human Sco1 Severely Compromises Cox17-dependent Metallation but Does Not Impair Copper Binding

Paul A Cobine,Fabien Pierrel,Scot C Leary,Florin Sasarman,Yih-Chern Horng,Eric A Shoubridge,Dennis R Winge

doi:10.1074/jbc.m600496200

Paul A Cobine, Fabien Pierrel + Show 5 more

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https://doi.org/10.1074/jbc.m600496200

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Abstract

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.

Highlights

Immortalized fibroblasts from a SCO1 patient with the P174L substitution exhibited a severe deficiency in Cytochrome c oxidase (CcO), which is reflected in both low residual enzyme activity and low steady-state levels of CoxII protein (Fig. 1, A and C)
Residual CcO activity in P174L-overexpressing SCO1 patient fibroblasts could be further increased by supplementing the growth media with Cu-His (Fig. 1A), an effect that was not attributable to altered levels of Sco1 protein These data demonstrate that the P174L mutant Sco1 retains sufficient residual function to allow for some assembly of the CcO holoenzyme
The reduced CcO activity in SCO1 patient cells could be due in part to reduced Sco1 protein levels [10] (Fig. 1C), the inability of overexpressed P174L Sco1 mutant to fully restore CcO activity argues that some aspect of its function is impaired

Summary

Introduction

Isolated CcO deficiency, one of the most commonly recognized causes of respiratory chain defects in humans, is associated with a wide spectrum of clinical phenotypes [3, 4], and autosomal recessive mutations have been identified in six nuclear genes that encode CcO assembly factors in these patients [3,4,5,6,7,8,9] Two such factors, SCO1 and SCO2, encode metallochaperones that play a role in the copper ion metallation of the CuA site in CoxII.

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