HIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV

Daniella H Hock,Boris Reljic,Ching-Seng Ang,Linden Muellner-Wong,Hayley S Mountford,Alison G Compton,Michael T Ryan,David R Thorburn,David A Stroud

doi:10.1074/mcp.ra120.002076

Abstract

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

Highlights

Factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates
HIGD2A is thought to be the mammalian ortholog of Rcf1 and knockdown experiments in mouse myoblasts were shown to alter the distribution of complex IV-containing supercomplexes, suggesting the function of HIGD2A in supercomplex assembly is conserved from yeast to man [26]
When we followed the fate of newly translated COX3 in a chase experiment (Fig. 6D) we found no difference in the rate of COX3 turnover between HIGD2AKO and control cells

Summary

Introduction

Factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. Complexes I, III, and IV have been shown to exist in several higher order structures known as respiratory chain supercomplexes or respirasomes [1,2,3,4]. These structures have been suggested to reduce production of reactive oxygen species (ROS), improve complex stability and/or distribution throughout the protein dense environment of the IMM, and allow efficient channeling of substrates [3,4,5,6,7,8]. HIGD2A is Critical in Assembly of Human Complex IV [14, 15] implicating the importance of COX3 in complex IV and mitochondrial function

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