Mitochondrial Complex I Plays an Essential Role in Human Respirasome Assembly

Abstract

SUMMARY The assembly and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects lead to severe human disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the functional significance of the respirasomes for RC function.

A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle

“…We also noted that Cox4 signal in monomeric complex IV was increased, whereas the signal in dimeric complex IV was decreased in Cox7rpKO mitochondria. In terms of complex III assembly, the signal of Rieske iron-sulfur protein (Risp), a late-stage subunit of supercomplex 25 , was decreased in I/III 2 /IV n in Cox7rpKO muscle compared with WT muscle (Fig. 4c,d).…”

“…Digitonin (digitonin/protein ratio of 0.5-2 g g À 1 ) was added to solubilize the mitochondria. As RISP is known as a late-stage assembly subunit in mitochondrial respiratory supercomplex 25 , a milder solubilization condition of 0.5 g digitonin per gram protein was employed to detect RISP. After 30-min incubation at 4°C, the solubilized proteins were obtained as supernatant fraction by centrifugation at 22,000g.…”

Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes

“…It is difficult to envisage mechanistically how supercomplexes could exert primary effects on the formation of reactive oxygen species, and only limited circumstantial evidence has been presented to support this proposal (33). Effects on complex stability and assembly (34) have been proposed but support for the stability hypothesis is based on genetic ablation of "partner" enzymes (35)(36)(37), causing substantial disruption that impedes respiratory-chain turnover. This decreased turnover causes upstream species such as NADH to accumulate and oxidative stress to increase, thus providing a plausible supercomplex-free explanation of the results (38).…”

Mitochondrial respiratory chain dysfunction: Implications in neurodegeneration

“…The synthesis of mitochondrialencoded subunits resumed after the drug removal, and the time-course incorporation of nuclear and mitochondrial RC subunits into supercomplex assembly intermediates was investigated. Based on the alignment and analysis of these intermediates we proposed a multistep assembly model of mitochondrial supercomplexes (Moreno-Lastres et al, 2012) [ Figure 1]. The conceptual novelty of this model is that the respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of free subunits and subcomplexes from complexes III and IV.…”

“…Accordingly, alterations in the assembly and enzyme activities of the RC complexes I and IV were demonstrated in rat choroid plexus epithelial cells treated with Aβ, and also in human choroid plexus cells from patients suffering AD (Vargas et al, 2010). The import blockage of complex IV constituents towards mitochondria could lead to an abnormal assembly of complex IV and the respirasome, which in turn would be responsible for the partial loss of complex I activity (Schagger et al, 2004;Li et al, 2007;Moreno-Lastres et al, 2012). APP can not only alter mitochondrial function by blocking the mitochondrial protein import system, but can also exert its deleterious effects inside the organelle.…”