Assembly of the membrane domain of ATP synthase in human mitochondria

Abstract

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F-c complex inhibited by the ATPase inhibitor protein IF and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

Respiratory supercomplexes act as a platform for complex III ‐mediated maturation of human mitochondrial complexes I and IV

“…The graphs were generated as in Fig 3, but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al, 2018) were averaged to simplify the analysis. The graphs were generated as in Fig 3, but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al, 2018) were averaged to simplify the analysis.…”

“…A Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Fig 3, but in this case, the peptide intensity values for the individual subunits belonging to each module (He et al, 2018) were averaged to simplify the analysis. The represented values are the mean AE SEM of the two reciprocal labeling experiments.…”

“…ª 2020 The Authors The EMBO Journal 39: e102817 | 2020 detected subunits of cV belonging to its two distinct assembly modules (He et al, 2018). The F1 particle included a (ATP5A1), b (ATP5B), c (ATP5C1), and e (ATP5E) subunits, whereas the peripheral stalk included subunits b (ATP5F1), d (ATP5H), F6 (ATP5J), OSCP (ATP5O), e (ATP5I), f (ATP5J2), g (ATP5L), MT-ATP6, MT-ATP8, 6.8PL (MP68), and DAPIT.…”

MLQ is responsible for stabilisation of subunit a in the holoenzyme of mammalian ATP synthase

“…While this model still stands true in principle, recent studies on the mammalian enzyme challenged some of the assumptions. By systematically knocking out subunits e, f, g, DAPIT, MLQ (He, Ford et al 2018) and c (He, Ford et al 2017) Walker and coworkers demonstrated some level of variability regarding incorporation of individual modules. Thus, vestigial (and catalytically non-functional) complex can assemble without the c-ring, although the formation of F1-c module probably represents standard assembly intermediate.…”

Selective over-synthesis and rapid turnover of mitochondrial protein components of respiratory complexes

“…Pulse labeling with stable isotopes of essential amino acids can distinguish newlysynthesized proteins from pre-existing copies. We grew HeLa cells in "light" medium containing 12 C-lysine and arginine and switched to medium with lysine and arginine containing six 13 C residues in each to initiate pulse labeling, continued growth for defined time periods and isolated mitochondria and/or respiratory chain complexes. Labeling with stable isotopes enables mass spectrometric detection of the heavy:light or H:L ratio of a large fraction of the mitochondrial proteome as an index of the synthesis rate for a far greater number of proteins than in a radioactive labeling experiment.…”