Assembly of the membrane domain of ATP synthase in human mitochondria

Jiuya Hea,1, Holly C. Forda,1, Joe Carrolla, Corsten Douglasa, Evvia Gonzalesa, Shujing Dinga, Ian M. Fearnleya, and John E. Walkera,2

aMedical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, United Kingdom

Contributed by John E. Walker, January 16, 2018 (sent for review December 20, 2017; reviewed by Ulrich Brandt and Nikolaus Pfanner) The ATP synthase in human mitochondria is a membrane-bound mitochondria in human ρ0 cells lack organellar DNA and contain assembly of 29 proteins of 18 kinds. All but two membrane another incomplete ATP synthase, necessarily with no ATP6 or components are encoded in nuclear genes, synthesized on cyto- ATP8 (7, 9). These incomplete vestigial ATP synthases provide plasmic ribosomes, and imported into the matrix of the organelle, insight into the pathway of assembly of the complete enzyme. where they are assembled into the complex with ATP6 and ATP8, Here we investigated the effects of the selective removal of su- the products of overlapping genes in mitochondrial DNA. Disrup- pernumerary membrane subunits e, f, g, DAPIT, and 6.8PL on tion of individual human genes for the nuclear-encoded subunits assembly of human ATP synthase. in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a descrip- Results tion of the pathway of assembly of the membrane domain. The Human Cells Lacking Supernumerary Subunits. The human genes key intermediate complex consists of the F1-c8 complex inhibited ATP5I, ATP5J2, ATP5L, USMG5, and C14orf2 (SI Appendix, Fig. by the ATPase inhibitor protein IF1 and attached to the peripheral S2) encoding, respectively, subunits e, f, g, DAPIT, and 6.8PL of stalk, with subunits e, f, and g associated with the membrane ATP synthase were disrupted in HAP1-WT (wild-type) cells (SI domain of the peripheral stalk. This intermediate provides the Appendix, Fig. S3 and Tables S1 and S2). Each of the mutant template for insertion of ATP6 and ATP8, which are synthesized cells, HAP1-Δe, -Δf, -Δg, -ΔDAPIT, and -Δ6.8PL, did not ex- on mitochondrial ribosomes. Their association with the complex is press the eponymous subunit (Fig. 2). The removal of subunits e, BIOCHEMISTRY stabilized by addition of the 6.8 proteolipid, and the complex is f, g, or DAPIT had little effect on cellular growth (SI Appendix, coupled to ATP synthesis at this point. A structure of the dimeric Fig. S4), except that HAP1-Δe cells reached a similar level of yeast Fo membrane domain is consistent with this model of assem- confluence as HAP1-WT cells and then declined. In addition, bly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and the growth of HAP1-Δg cells was slightly retarded but reached a ATP8 into the membrane assembly, and the monomeric complexes maximum growth similar to that of controls. In contrast, the then dimerize via interactions between ATP6 subunits and be- removal of 6.8PL retarded cellular growth, but there is no extant tween 6.8 proteolipids (j subunits). The dimers are linked together explanation for this. The removal of any one of subunits e, f, or g back-to-face by DAPIT (diabetes-associated protein in insulin- had a severe effect on respiration (SI Appendix, Fig. S4), similar sensitive tissue; yeast subunit k), forming long oligomers along to the effect of removing the OSCP or subunit b or c (7, 8). In the edges of the cristae. HAP1-Δe, -Δf, or -Δg cells, the lack of effect of oligomycin in- dicated that the vestigial ATP synthase is uncoupled from pmf. human mitochondria | ATP synthase | assembly | membrane subunits In HAP1-Δ6.8PL cells, there was evidence of coupled ATP

he ATP synthase in human mitochondria provides most Significance Tcellular ATP under aerobic conditions. Energy derived from oxidative metabolism generates a proton-motive force (pmf) Mitochondria generate the cellular fuel ATP to sustain complex across the inner membrane of the organelle, and the ATP syn- life. Production of ATP depends on the oxidation of energy-rich thase harnesses the pmf to make ATP from ADP and phosphate compounds to produce the proton motive force (pmf), a (1, 2). Human ATP synthase is an assembly of 29 subunits of chemical potential difference for protons, across the inner 18 types (including the regulatory protein IF1) with a combined membrane. The pmf drives the ATP synthase, a molecular molecular weight of 592 kDa (3). The subunits are organized into machine with a rotary action, to synthesize ATP. We have membrane-extrinsic and membrane-intrinsic domains, linked by shown that the assembly of human ATP synthase in the inner central and peripheral stalks (Fig. 1). organellar membrane involves the formation of a monomeric The protein subunits emanate from two genomes. In humans intermediate made from 25 nuclear-encoded proteins into and other vertebrates, ATP6 and ATP8 are encoded in over- which the two mitochondrially encoded subunits are inserted lapping genes in mitochondrial DNA (4) and are translated in- and then sealed by association of another nuclear-encoded side mitochondria. The other subunits, produced from nuclear protein, thereby dimerizing the complex. Association of a final genes, are translated in the cellular cytoplasm, imported into the nuclear protein oligomerizes the dimers back-to-face along the organelle, and assembled together with ATP6 and ATP8 to cristae edges. produce the complete ATP synthase. Although the assembly of the yeast ATP synthase has been studied extensively (5, 6), until Author contributions: J.E.W. designed research; J.H., H.C.F., J.C., C.D., E.G., S.D., and I.M.F. recently, relatively little was known about how human ATP performed research; J.H., H.C.F., J.C., C.D., S.D., I.M.F., and J.E.W. analyzed data; and synthase is assembled. However, gene editing now permits re- J.E.W. wrote the paper. moval of individual subunits of the human enzyme and the Reviewers: U.B., Radboud University Medical Center; and N.P., University of Freiburg. subsequent examination of the consequences for assembly. Thus, The authors declare no conflict of interest. in the absence of subunit c, an incomplete ATP synthase is Published under the PNAS license. 1 formed lacking the c8 ring, ATP6, ATP8, DAPIT, and 6.8PL (7). J.H. and H.C.F. contributed equally to this work. Likewise, removal of either the OSCP or subunit b leads to loss 2To whom correspondence should be addressed. Email: [email protected]. of the entire peripheral stalk plus ATP6, ATP8, and the five This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. supernumerary subunits, leaving F1-c8 inhibited by IF1 (8). The 1073/pnas.1722086115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1722086115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 WT eWTfWTg WT DAPIT WT 6.8PL OSCP cs f1  e f2 g DAPIT 6.8PL

Fig. 2. Absence of individual supernumerary subunits of ATP synthase from clonal HAP1 cells. Mitoplasts were prepared from HAP1-WT cells and HAP1- Δe, -Δf, -Δg, -ΔDAPIT, and -Δ6.8PL clonal cells. In the HAP1-Δf cells, f1 and F6 f2 denote isoforms of subunit f. The mitoplasts were extracted with n-  Dodecyl-β-D-maltoside. The extracts were fractionated by SDS/PAGE and  d Western blotted with antibodies against the corresponding subunits. Citrate synthase (cs) served as a loading control.  b S11–S20) were consistent with the following conclusions. Re- moval of subunit e or g was accompanied by loss of all other supernumerary subunits, plus ATP6 and ATP8. The peripheral  stalk subunits were partially depleted, but the rest of the complex  was unaffected. The vestigial complex from HAP1-Δf cells   lacked ATP6, ATP8, DAPIT, and 6.8PL but otherwise had a full Matrix complement of subunits. In contrast, when DAPIT was removed, all other subunits were fully retained. The removal of 6.8PL led ATP6 to partial losses of ATP6, ATP8, and DAPIT only. In all but one instance, formation of the vestigial complexes was accompanied c f g e by an elevation in the relative level of IF1-M1 (7). The exception DAPIT

8 6.8PL ATP8 was the removal of DAPIT, where the levels of all forms of IF1 were unchanged. In mitoplasts from HAP1-Δe, -Δf, -Δg, and -Δ6.8PL cells, the relative levels of DAPIT were decreased to a IMS lesser extent than in vestigial complexes, indicating the presence of the free protein unassociated with the vestigial ATPase Fig. 1. Organization of subunits in monomeric ATP synthase in mammalian complex (SI Appendix, Fig. S5). Conversely, the similarities of mitochondria. Black horizontal lines represent the inner membrane. IMS, changes in the content of other subunits in mitoplasts and ves- intermembrane space. The peripheral stalk (subunits OSCP, F6, b, and d) is on tigial ATP synthase complexes suggests that there is no signifi- α the right. Subunit b has two N-terminal transmembrane -helices interacting cant pool of unassembled forms of these subunits. directly or indirectly with subunits e, f, g, DAPIT, and 6.8PL, which have no known roles in synthesis or hydrolysis of ATP. Each has a single predicted Oligomeric State of Vestigial ATP Synthases. The oligomeric states transmembrane α-helix (SI Appendix, Fig. S1). The membrane domain of subunit b is associated with mitochondrially encoded subunits ATP6 and of the WT and vestigial ATP synthases became less stable with ATP8. The N-terminal region of ATP8 has a single transmembrane α-helix, increasing concentrations of digitonin (Fig. 5). There was no and its C-terminal region extends into the peripheral stalk. ATP8 and subunit evidence of persistence in any of the mutant cells of the oligo-

b keep ATP6 in contact with the c8 ring. The pmf drives the rotation of this meric complexes observed in the WT enzyme, with the exception ring and the attached central stalk (subunits γ, δ, and e) by the translocation of HAP1-ΔDAPIT cells (Fig. 5 and SI Appendix, Fig. S7) and of protons through the interface between the c8 ring and ATP6. The rotation possibly -Δf cells (Fig. 5A, Left) of the dimeric complex. The of the central stalk carries energy into the three catalytic sites in the F1 dimeric state in HAP1-ΔDAPIT cells was reduced relative to domain (subunit composition α3β3γδe). An α subunit has been removed to γ that in WT cells. In contrast, in all cases, a vestigial monomeric expose the central subunit. complex was present, although in HAP1-Δe, -Δf, and -Δg cells, but not in HAP1-Δ6.8PL and HAP1-ΔDAPIT cells, its apparent synthase, albeit at reduced levels, as well as a residual functional respiratory chain. The HAP1-ΔDAPIT cells retained almost normal levels of coupled ATP synthase and respiration. The WT e f g WT DAPIT 6.8PL effects of removal of 6.8PL and DAPIT differ from earlier proposals (10, 11).     Vestigial ATP Synthases. HAP1 cells lacking any one of subunits e, f, g, DAPIT, and 6.8PL all assemble vestigial ATP synthase   IgG IgG complexes (Fig. 3). Each has a full complement of the α, β, γ, δ, b OSCP b and e subunits forming the F1 domain, as well as of the periph- d OSCP ATP6 d eral stalk subunits OSCP, b, d, and F6. The HAP1-Δe, -Δf, and ATP6 Δ Δ Δ  - g cells, but not the HAP1- DAPIT and - 6.8PL cells, also  lack ATP6 and ATP8. The effects of deleting one supernumerary f-1 f-1 IF1 IF1 subunit on the absence or presence of another are more difficult f-2, g, F6 f-2, g, F6 to discern, but removal of either subunit e or g is accompanied by e, DAPIT e, DAPIT 6.8PL, ATP8 6.8PL, ATP8 the loss of all other supernumerary subunits, and the loss of c,  c,  6.8PL accompanies the removal of subunit f. Fig. 3. Subunit compositions of vestigial ATP synthase complexes produced The subunit compositions of purified vestigial complexes de- by removal of individual supernumerary membrane subunits. The complexes fined by quantitative MS were in agreement with the gel profiles were immunopurified from mitoplasts derived from HAP1-WT, -Δe, -Δf, -Δg, (Fig. 4, SI Appendix, Fig. S5, and Datasets S1–S10), and the -ΔDAPIT, and -Δ6.8PL cells, fractionated by SDS/PAGE, and stained with analyses of mitoplasts (SI Appendix, Figs. S5 and S6 and Datasets Coomassie blue. The positions of subunits are indicated.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1722086115 He et al. Downloaded by guest on October 1, 2021 6810 A -M1 -M2 A 1 1 c b d F 6 OSCPe g f-1 ATP6ATP86.8PLDAPITIF IF 2      f-2 6810 Δf Δf WT Δe WT Δe WT Δe Δf Δg Δg Δg ΔDAPIT Δ6.8PL ΔDAPIT Δ6.8PL WT WT WT Δ6.8PL 0 ΔDAPIT o o -2 d d protein ratio 2 -4 m m

log s1 s1 -6

-8

B -M1 -M2 -M3 1 1 1 cs cs 2      c b d F 6 OSCPe g f-1 ATP6ATP86.8PLDAPITIF IF IF 6810 0 B 6810 -2 WT Δe WT Δe Δf WT Δf Δe Δf Δg Δg Δg WT Δ6.8PL Δ6.8PL ΔDAPIT ΔDAPIT WT Δ6.8PL WT ΔDAPIT protein ratio

2 -4 o

log o -6 d d m m -8

C -M1 -M2 1 1 s2 6 s2 2      c b d F OSCPe g f-1 f-2 ATP6ATP86.8PLDAPITIF IF

0 BIOCHEMISTRY -2 cs cs

-4 6810 protein ratio

2 C -6

log 6810 -8 WT Δ6.8PL ΔDAPIT Δ6.8PL ΔDAPIT WT ΔDAPIT WT Δ6.8PL Δf Δe Δe Δe WT Δf WT WT Δf Δg Δg -10 Δg o o d d D -M1 -M2 1 1 2      c b d F 6 OSCPe g f-1 f-2 ATP6ATP86.8PLDAPITIF IF m m s1 s1 0

-2 protein ratio 2 -4 cs cs log -6 D 6810 -8 6810

E -M1 -M2 1 1 WT Δ6.8PL ΔDAPIT Δ6.8PL ΔDAPIT WT Δe WT Δe Δf WT Δf WT Δe Δf ΔDAPIT Δ6.8PL WT Δg Δg 2      c b d F 6 OSCPe g f-1 f-2 ATP6ATP86.8PLDAPITIF IF Δg o o 0 d d m -2 m

protein ratio -4 2 log -6

-8 cs cs

Fig. 4. Effects of removal of individual supernumerary membrane subunits on Fig. 5. Oligomeric states of ATP synthase and vestigial forms in HAP1 cells. human ATP synthase. Shown are relative abundances of subunits and forms M1, ATP synthase and vestigial complexes were extracted at various digitonin/

M2, and M3 of IF1 in immunopurified ATP synthase and vestigial complexes, protein (g/g) ratios indicated above the lanes, from mitoplasts of HAP1-WT, from clonal cells. (A) HAP1-Δe. (B)HAP1-Δf. (C) HAP1-Δg. (D) HAP1-ΔDAPIT. (E) -Δe, -Δf, -Δg, -ΔDAPIT, and -Δ6.8PL cells. Extracts were fractionated by BN- HAP1-Δ6.8PL. The histograms, derived from SI Appendix,Fig.S5and Datasets PAGE, and complexes were revealed by Western blotting with antibodies S1–S10, are the median values of both relative abundance ratios determined for against various subunits of ATP synthase: β subunit (A), b subunit (B), γ proteins found in complementary SILAC experiments. Error bars show the range subunit (C), and d subunit (D). Citrate synthase (cs) served as a loading

of the two values. IF1-M1, -M2, and -M3 are different mature forms of IF1,andf- control. The positions of the complexes are indicated on the left; d, dimers; 1 and f-2 are isoforms of subunit f (Swiss-Prot P56134). Similar experiments m, monomers; o, oligomers; s1, F1-c8 subcomplexes; s2, subcomplexes con- conducted on mitoplasts are presented in SI Appendix,Fig.S6. taining subunits b, e, and g.

He et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 1, 2021 A BCOSCP OSCP DOSCP    

F6 F6 F6     d   d   d   b  b  b

IF1 IF1 IF1 IF1                

c8 c8 c8 g e f g e

H OSCP G OSCP F OSCP E OSCP    

F6 F6 F6 F6   d   d   d   d  b  b  b  b

IF1 IF1                

ATP6 ATP6

c8 f g e c f g e f g e c f g e 8 6.8PL 8 ATP8 ATP8 6.8PL DAPIT

Fig. 6. Pathway of assembly of the membrane domain of human ATP synthase. (A–D, F, and G) Intermediate vestigial complexes and subunit compositions characterized from mitochondria of clonal cells: HAP1-Δbor-ΔOSCP (A), HAP1-Δeor-Δg(B), HAP1-Δf(C), HAP1-Δc(D), HAP1-Δ6.8PL (F), and HAP1-ΔDAPIT (G). (E) Vestigial complex from the mitochondria of ρ0 cells. The horizontal black lines denote the boundaries of the inner mitochondrial membrane. (H)The complete monomeric ATP synthase.

molecular weight was slightly smaller than that of the mono- during assembly. This complex, comprising a total of 25 proteins, meric WT-ATP synthase, reflecting their subunit compositions. provides the template for the insertion of ATP6 and ATP8 sin- In HAP1-Δe, -Δf, -Δg, and -Δ6.8PL cells, a complex smaller gly, in either order, or together. Both are translated on mito- than the monomeric complexes was probably F1-c8 (s1 in Fig. chondrial ribosomes, probably close to the matrix surface of the 5). In HAP1-WT, -Δf, -ΔDAPIT, and -Δ6.8PL cells, but not -Δe inner membrane. The resulting complex F is partially coupled to or -Δg cells, there were high levels of a series of smaller sub- ATP synthesis (SI Appendix, Fig. S4E) and is somewhat unstable, complexes (s2 in Fig. 5) containing subunit b, but not F1 sub- as ATP6 and ATP8 are substoichiometric. The addition of 6.8PL units or subunit d. stabilizes ATP6 and ATP8 in complex G, producing an active enzyme coupled to ATP synthesis with no accompanying in- Turnover of Subunits. When HAP1, HEK, or fibroblast cells were crease in IF1 (Fig. 4 and SI Appendix, Fig. S4D). grown in the presence of glucose, where the cells are pre- In mitochondrial membranes, the ATP synthase is dimerized dominantly glycolytic, DAPIT was significantly less stable than via interactions in the membrane domain, and the dimers form any of the other subunits of the ATP synthase, including 6.8PL extensive front-to-back rows along the edges of the cristae (12– (SI Appendix, Fig. S8 and Datasets S21–S24). In contrast, when 14). Dimerization of the yeast enzyme depends on the presence the cells were grown in the presence of galactose, which enforces of subunits e and g (15), and these associate sequentially with a dependence on oxidative phosphorylation, the difference be- monomeric complex leading to an assembly ready to dimerize tween DAPIT and other subunits was abolished almost entirely. (16). In a structure of the dimeric Fo membrane domain of the yeast enzyme (17), neither of these subunits makes contacts Discussion across the monomer-monomer interface, which mainly involves The vestigial ATP synthase complexes characterized here and contacts between the two subunits j and the two ATP6 subunits. previously (7, 8) have been arranged in an order that describes a However, the requirement for the presence of e and g for di- branched pathway of assembly of the membrane domain of the merization to occur is explained by the assembly pathway for the enzyme (Fig. 6). In one arm, the F1-c8 complex, A, is converted human enzyme, as both subunits must be present in the growing to B by addition of the peripheral stalk. Insertion of subunits e complex before ATP6 (and ATP8) can be inserted and sub- and g into B, together or singly in either order, produces complex sequently stabilized by the association of 6.8PL (Fig. 6). C, and subsequent addition of subunit f yields complex E. In Two important conclusions can now be drawn. First, 6.8PL the second arm, complex D, consisting of the F1 domain plus the and yeast subunit j are almost certainly functional orthologs, a peripheral stalk and subunits e, f, and g, is converted to E by the view supported by the relationships between their sequences and addition of the c8 ring. In complexes A–E, ATP synthesis is positions of transmembrane α-helices (SI Appendix, Fig. S9A). uncoupled from pmf, and the hydrolytic activity of their F1 do- Second, dimerization most likely follows the insertion of mains appears to be inhibited by IF1. The two arms converge at ATP6 and ATP8 and the association of 6.8 PL. Our native gel E, a complex of 13 nuclear-encoded subunits plus IF1, each analyses (Fig. 5) and those published elsewhere (7, 9) are in imported individually into the mitochondrial matrix before or accordance with this suggestion. The final step in the assembly

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B BIOCHEMISTRY

Fig. 7. Structure of the dimeric Fo domain of the ATP synthase from S. cerevisiae (17). The c10 ring is in gray, and other subunits are colored and identified on the structures. In A and B, the two associated monomers are viewed from the matrix and from the intermembrane space, respectively. The interface between monomers is formed mainly by the two ATP6 (or a) subunits and by the two j subunits. Here it is proposed that subunit j and the human 6.8PL (labeled 6.8) are orthologs. It is also proposed that subunit k is the ortholog of human DAPIT, and that these subunits aid the formation of oligomeric structures consisting of rows of dimers along the edges of the mitochondrial cristae.

pathway, the addition of DAPIT, produces the fully assembled associated with fission and fusion of the dynamic network of mi- enzyme, complex H (Fig. 6). DAPIT is the most peripheral of the tochondria in cells. membrane subunits, and unless it is stabilized by the addition of In S. cerevisiae, assembly factors ATP11 and ATP12 participate exogenous phospholipids, mild detergents other than digitonin in formation of the F1 domain (20). Another assembly factor, remove it from the purified complex (18, 19). The most likely formation of complex V assembly factor 1 (FMC1), participates at role of DAPIT is in the formation of links between dimers of elevated temperatures, and its loss can be compensated for by ATP synthase to produce the rows of dimeric complexes along overexpression of ATP12 (21). The human orthologs are the “edges” of the cristae (12–14). ATPAF1, ATPAF2, and C7orf55 (22, 23), but since their role is in In the model of the yeast dimeric Fo domain, the incomplete formation of the F1 domain, it is unsurprising that they were not structure of subunit k is associated with subunit ATP6 and is significantly associated with complexes A–G(Fig.6,SI Appendix, distal from the monomer-monomer interface (Fig. 7). In this Fig. S5 and Datasets S1–S20). Another assembly factor, inner position, it is well placed to make contacts between dimers in membrane assembly complex (INAC), made of components of the oligomeric rows of dimers in mitochondrial inner mem- 22 and 17 kDa, is required for assembly of the yeast peripheral branes. It is likely that DAPIT and yeast subunit k are orthologs stalk (6, 24). However, no human orthologs have been recognized (SI Appendix,Fig.S9B); therefore, the Saccharomyces cerevisiae from sequences, and how the human peripheral stalk is assembled and human ATP synthases have the same complement of sub- before being added to the F1 domain or to complex A (Fig. 6) units (SI Appendix,TableS3). The relative stability of DAPIT remains unclear. A subcomplex of subunits b, e, and g has been under conditions of oxidative metabolism may reflect its role in proposed as an assembly intermediate (25), and it may be among dimer-dimer interactions during the remodeling of mitochondria the s2 subcomplexes (Fig. 5B).

He et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 1, 2021 It is noteworthy that the extrinsic membrane domain of the TMEM70 is a membrane protein that has been suggested to bovine peripheral stalk assembles from its four constituent pro- participate in the assembly of the membrane domain of human teins in vitro, and that the assembled complex will then associate ATP synthase (32, 33), but neither this protein nor any other with the bovine F1-ATPase spontaneously to produce an F1-pe- plausible assembly protein was increased in their association with ripheral stalk complex (lacking the membrane domain); both vestigial complexes A–G (Fig. 6, SI Appendix,Fig.S5and Datasets events proceed without the aid of exogenous protein factors (26). S1–S20). However, it seems likely that assembly proteins do par- The compositions of complexes B and D (Fig. 6) show that the peripheral stalk can be added to either the separate F domain ticipate in this pathway, and other methods, such as complexomics 1 (34, 35), may be more successful in identifying candidates. or the F1 domain in the F1-c8 complex, and a similar conclusion has been reached with the yeast ATP synthase (6). The yeast c10 Materials and Methods ring probably assembles separate from the F1 domain or the F1- Human genes ATP5I, ATP5J2, and ATP5L were disrupted in HAP1 cells by peripheral stalk complex, and subsequently the F1-c10 complex or the fully assembled enzyme forms (5, 6). A similar process may CRISPR-Cas9 with pairs of guide RNAs (SI Appendix, Table S1) as described Δ Δ Δ operate in human mitochondria, but the mechanisms of assembly previously (7), leading to clonal cells HAP1- e, - f, and - g, respectively. Clonal cells HAP1-Δ6.8PL (C14orf2) and HAP1-ΔDAPIT (USMG5) were pur- of the c8 ring and the S. cerevisiae c10 ring necessarily differ, as the human c subunit is encoded by three nuclear genes and is chased from Horizon Discovery. Cell proliferation was monitored with an IncuCyte HD instrument (Essen BioScience), and the oxygen consumption of imported into the organelle (27, 28), whereas the yeast c-protein e is a mitochondrial gene product made in the mitochondrial cells was measured in a Seahorse XF 24 analyzer (Agilent Technologies). The matrix (29). In addition, yeast ATP6 is made as a precursor with oligomeric state of ATP synthase and vestigial complexes was examined by an N-terminal extension, which is removed by the inner mem- BN-PAGE and Western blot analysis. ATP synthase and vestigial complexes brane protease ATP23 during assembly (30, 31); human were immunopurified from mitoplasts, analyzed by SDS/PAGE, and stained ATP6 has no precursor. Also, yeast ATP6 and ATP8 are asso- with Coomassie blue, and bands were analyzed by MS. Proteins were sub- jected to stable isotope labeling by amino acids in cell culture (SILAC) and ciated first with either the peripheral stalk or the F1-peripheral stalk subcomplex, together with assembly factors ATP23, ATP10, quantitated by MS. Details are provided in SI Appendix. and INAC, and then with the c ring to form the complete 10 ACKNOWLEDGMENTS. We thank M. G. Montgomery for producing Figs. 1, complex (5, 6). This pathway differs from the one proposed here 6, and 7 and SI Appendix, Fig. S9. This work was supported by the Medical for the human enzyme, as ATP6 and ATP8 are incorporated Research Council (MRC) through Programme Grant MR/M009858/1 (to after the F1-peripheral stalk-c8 complex has formed, and then J.E.W.). H.C.F. and C.D. have received MRC studentships, and E.G. has re- this complex is stabilized by 6.8PL (Fig. 6, complexes F and G). ceived a grant from St John’s College, Cambridge.

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