Eukaryote membrane genetics: the Fo sector of mitochondrial ATP synthase

TIG17 Ollis, D. L., Kline, C, and Steitz, T. A. (1985)Nature 313, 818819 18 Wang, S. W. et al. EMBO /I. (in press) 19 Zmudzka, B. Z. etal. (1986) Proc. NatlAcod. Sd. USA 83, 51065110 20 Steitz, T. A. Biological Organization: Macromolecular Interactions at High Resolution, Academic Press (in press) 21 Petruska, J., Sowers, L. C. and Goodman, M. F. (1986) Proc. Natl Acad. Sd. USA 83, 1559-1562 22 Davison, A. J. and Scott, J. E. (1986)]. Gon. Viral. 67, 1759-!816 23 Baer, R. et al. (1984)Nature 310, 207-211 24 Earl, P. L., Jones, E. V. and Moss, B. (1986) Proc. Natl Acad. Sd. USA 83, 3659-3663 25 Gingeras, T. R. et ai. (1982)]. Biol. Chem. 257, 13475-13491 26 Yoshikawa, H. and lto, J. (1982) Gone 17, 323-335 27 Kouzarides, T. et al. (1987)]. Viral. 61, 125-133 28 Tsurmi, T., Maeno, K. and Nishiyama, Y. (1987) Gene 52, 129-137 29 Binns, M. M. et al. (1987)Nucleic Acids Res. 15, 6563--6573

February 1988, Vol. 4, no. 2

30 Jang, G., Leavitt, M. C. and Ito, J. (1987)Nucle~ Acids Res. 15, 9O88 31 Kuzmin, E. V. and Levchenko, I. V. (1987)Nucleic Acids Res. 15, 6758 32 Hall, J. D., Gibbs, J. S., Coen, D. M. and Mount, D. W. (1986) DNA 5, 281-288 33 Quinn, J. P. and McGeoch, D.J. (1985) Nucleic Acids Res. 13, 8143-8163 34 Argos, P., Tucker, A. D. and Phih'pson, L. (1986) Virolo~ 149, 208-216 35 Larder, B. A., Kemp, S. D. and Darby, G. (1987) EMBO ]. 6, 169-175 36 Knopf, C. W. (1986)Nucleic Acids Res. 14, 8225--8226

]. D. Hall is at the Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA.

Energy-transducing enzyme complexes feature prominently in genetic approaches to understanding the structure and function of biological membranes ~. A rich background to such studies in eukaryotic cells is the membrane system of mitochondria', whose ATP synthase enzyme plays a major role in oxidative phosphorylation s. The membrane Fo The set of integral membrane proteins of the proton-translocating Fo sector of sector of this enzyme is featured in mitochondrial ATP synthaseprovides a focal point for euharyote membrane genetics this review as a focal point for from molecular, cell biological and evolutionaPyperepective$. Detailed analysis of eukaryote membrane genetics, in yeast mutants has el:scidated the assembly and functions of three mitochondrially terms of the structure, function, encoded F~ sector subunits, whereas work on filamentous fungi and mammals has biogenesis and assembly of its conrevealed that one of these subunits is encoded in the nucleargenome and delivered to stituent protein subunits. the mitochondrial inner membrane following import into the organeUe. These two Proton-translocating ATP synaspects of membrane biogenesis have now been integrated through the deliberate thase complexes are found not only in relocation of a yeast mitockondrial &ene to the nucleus; a functional mitocitondrial membranes of mitochondria, but also ATP syntkase complex has thus been assembled using an Fo sector subunit in chloroplasts and bacteria s. Known introduced into the membranefrom the non-natural side. as FoF1 ATPases, these multisubunit enzymes are characterized by a membrane- between Fo and F1 is clear in both compositional and embedded domain (Fo sector) containing a proton channel functional terms s,9. Fo can be isolated as a discrete which is functionally coupled to hydrophilic catalytic aggregate of subunits a, b and c (the stoichiometry is domains (FI sector) on which the adenine nucleotide ab2cs_l~) whose proton-translocating activity can be interconversions occur (ADP + Pi ~ ATP + H,O). Mito- readily measured. Subunit c has two transmembrane chondrial ATP synthase (mtATPase) complexes have stems separated by a hydrophilic charged loop and been analysed in several organismss. Highlighted in this contains a binding site for the inhibitor dicyclohexylcarboreview are two well-studied examples: that of bovine diimide (DCCD)~°. Subunit c is intimately involved in heart 3"4, a long-time favourite for biochemical study of proton conductance, in concert with subunit a, which has mtATPase, and that of the yeast Saccharomyces cere- at least five transmembrane spans 1~. Contacts between visiae 5-7, a eukaryotic microorganism especially suited Fo and F~ that have been recognized include those made for investigations based on molecular genetic analysis and through the hydrophilic loop~°ag-of subunit c and through manipulation. Both these mtATPase complexes contain subunit b, which has a single N-terminal transmembrane at least 11 different subunits (Fig. 1) of which five (oh, ~, stem and an extended hydrophilic domain that interacts 7, 6, c) in each case comprise the F~ sector. In the Fo with F1 sector subunitss.eal. Detailed chemical mechansector are found three hydrophobic integral membrane isms have yet to be established for the translocation of polypeptides (yeast subunits 6, 8 and 9; bovine subunits protons through Fo and for how the specific conforma6, A6L and 9) which, with the exception of bovine subunit tional changes in Fx considered to be essential for the 9, represent that portion of mtATPase specified by the synthesis of ATP are elicited3. mitochondrial genome (mtDNA); the remainder of the The subunit relationships in the Fo sectors of complex is encoded in the nucleus. mitochondrial and bacterial ATP synthase complexes can be summarized as follows. Subunits 6 and 9 of mtATPase Composition and function of the membrane are homologous to E. coli subunits a and c, respectively, Fo sector in terms of amino acid sequence and structural disposition For a useful perspective, I shall first comment briefly across the membranes-u. There is no bacterial equivaon the FoFI ATPase of Escherichia coll. This complex lent of subunits 8 or A6L. Recent analyses of bovine and contains eight subun/ts (Fig. I) and the distinction yeast mtATPase complexes (see Ref. 4) suggest that

Eukaryote membrane genetics: the Fo sector of mitochondrial ATP synthase Phillip Nagley

46

1988,ElsevierPublkations.Cambridge 0168- 9525/88/$02.00

T I G - February 1988, Vol. 4, no. 2

vi

MITOCHONDRIA re they each contain a subunit similar in its predicted BACTERIA structure to that of the b subunit ofE. coli; in yeast this subunits is P25 (Fig. 1). Some components of mtATPase indicated in Fig. I are Often designated, by default, as being part of Fo on the basis that they are not any of the five well-defined FI sector subuuits. Since these additional subunits are probably involved m physical and bioenergetic coupling of Fo and FI activities and in modulation of energytransduction properties of the complex, and they are not necessarily themselves integrated into the membrane, it Yeast Bovine E.coli seems preferable to assign them collectively to a group termed Fa (associated factors). In this review the term Rg. 1. Protein components of FoF1 ATPases of mitoFo is reserved for mtATPase subunits that are demon- chondria and bacteria. The Fo sector, in which the proton strably integral membrane proteins. The proteins channel is constituted, contains hydrophobic integral membrane subunits. F1 can be isolated as a self-contained under detailed consideration here (Fig. 1) dissolve in unit with ATPase activity3. Other proteins associated with cholorofonn-methanol solventsI ° ' ~ I and thus are the FoF1 ATPase complex are assigned to FA. Some of termed proteolipids. these are involved in coupling proton translocation to

FA ro

Conserved structural organization of Fo sector subunits Proposed schemes for the folding of yeast Fo sector subunits across the inner mitochondrial membrane are depicted in Fig. 2. In subunit 9 (Fig. 2) there are wellconserved regions in the glycine-rich N-terminal membrane-spanning hydrophobic domain (hl), in the central hydrophi]ic loop region and in the C-terminal transmembrane stem (h2). There is always an acidic residue in the h2 domain of subunit 9 homolognes (glutamate in mitochondria, aspartate in bacteria), which is the site of the covalent binding of DCCDI°. In subuuit 6, five transmembrane stems have been predicted in broadly equivalent positions in different mitochondrial and bacterial proteins, even in the absence of strong amino acid homologies in all regions of these proteinsII. There are, however, two well-conserved domains (Fig. 2) lying towards the C-terminus, each containing sequences predicted to span the membrane. There is a striking representation of charged and polar residues within these putative transmembrane spans, denoted h4 and h5, whose amino acid sequences are capable of generating amphiphilic C-helices with the charged/polar residues lying along one face of the helix and proposed to be involved in proton translocation1~. The situation regarding homology of structure and function between subunits 8 and A6L is less clear. The yeast and bovine subuuits share a common four-zczidue sequence motif at their N-termini (Fig. 3) but there is little if any direct sequence homology elsewhere in these proteins. Nevertheless, there is a common broad distribution of positively charged amino acids which are concentrated towards the C-termini (Fig. 3). Moreover, secondary structure predictions for these proteins show similarities and one transmembrane stem is predicted in each case 13. The C-terminus of subuuit 8 in S. cerev/.v/ae has been proposed is to face the matrix space (Fig. 2), but no information is yet available on the orientation of subunit A6L. Subunit function revealed through the study of m u t a n t s Molecular genetic analyses on S. cerevisiae have enabled the definition by DNA sequencing16-Is of many mutations in the mitochondrial genes encoding subunits 9, 6 and 8 (olil, oil2 and aapl, respectively), which result in phenotypes of drug resistance, defective mitochondrBl

r~ Fo

FI~ Fo

ATPase or ATP synthase activity; others, the so-called inhibitor proteins (inh)7, may modulate the enzymic activity. Further characterization of individual FA proteins may lead to their assignment to the Fo sector. Sequence relationships between mitochondrial and bacterial Fo sector subunits are considered in the text; other relationships between non-Fo subunits are summarized elsewhere4. This diagram is intended to classify subunits and should not be interpreted as a strict representation of the topological disposition of the various subunits. The Fo subunits considered in detail in this review are represented within thick lines; all except bovine subunit 9 are encoded in mtDNA.

function (mit=) or temperature sensitivity. At the same time, specific properties of the mtATPase complex, such as its assembly and functions, have been studied using such mutant strainss. The properties of some mutants of particular relevance to this review are summarized in Table 1. Oligomycin is an inhibitor of proton translocation mediated by the Fo sector. The substitution of particular amino acids in olignmycin-resistant mutants identifies them as being involved in interactions with this drug. Such amino acids are likely to lie close to those directly involved in the proton translocation mechanism. The domains in which such substitutions are found include portions of the transmembrane helices hl and h2 of subunit 9, and h4 and h5 of subunit 6 (Table 1). A major domain for binding oligomycin, revealed by chemical ~mnlty lsbelling, lies on the h2 helix of subunit 9 aromld the DCCD binding site (see Ref. 19), and it is possible that this binding may be modulated by amino acid substitutions in the other dlree relevant transmembrane stems z°. The resistance domain for venturicidin~9, another inhibitor of proton channel function, lies only within the two domains hl and h2 of subunit 9 (cf. Table 1). Subuuits a and c inE. coli FoF~ ATPase are considered to be directly involved in proton channel function and its coupling to ATP synthesis on the F1 sectorix. In yeast, certain amino acid substitutions (Table 1, central section) in the corresponding Fo sector subuuits 6 and 9 are found to produce a mit= phenotype, but do not cause gross impairment in the assembly of the Fo sector. Domain hl of subuuit 9 is thought to be involved in proton translocation as such, while the loop domain of subuuit 9 and the portion of domain h5 in subuuit 6 in the vicinity of residue

iews ' SUBUNIT

TIG-

February 1988, Vol. 4, no. 2

Mutants synthesizing mmcated versions of subC unit 9 characteristically do C 259 not assemble any of the mitochondrially encoded proteins of the complex, although subunits of the Ft "(~) a 6 ~ sector assemble amongst @4"-------51 : (~) 1 7 0 - 248 themselves in mitochondria. Other consequences 19 of the effective absence of subC ® unit 9 from mitochondria are W ak z I0 that subunit 6 is synthesized Wn" h4 h5 hl h2 in reduced ~nounts and that ZCB q ZX J= cytochrome c oxidase is --W ee ® o ®: not assembled so respiration is severely impaired. The latter phenomenon suggests a major role for sub10 194 224 75 13__ ( unit 9 in the internal organi! = ® zation of the inner z I mitochondrial membrane. n'l&I The oapl mit- mutants so 1 N far characterized TM contain only truncated subunit 8 pro1 N teins (cf. Table 1) and are Fig. 2• Proposer; folding of Fo sector subunits of S. cerevisiae across the inner defective in the assembly of mitochondrial membrane. The probable dispositions '''15'21 across the inner mito- the Fo sector• While subunit chondrial membrane of subunits 9 and 8, and the highly conserved portion of subunit 6, 9 is assembled with F1, are drawn to show the relative positions Of N- and C-termini, the locations of charged neither subunit 6 nor subunit residues and the inferred positions of transmembrane stems (hl, h2, h4, h5). (Note that 8 appears in the immunoother schemes for subunit a incorporate six or seven transmembrane spansS'9.) Residue precipitate, although subnumbers are indicated. No representation of secondary structure is made here. The unit 6 continues to be S.l~ 9positions of conserved residues are indicated as follows: open circles, residues thesizedin the organelle, . common to '3. cerevisiae and B. bovis (cow); filled circles, residues common to Examination of a series of S. cerevisiae, B. bovis and E. coli. oli2 mit- mutants producing truncated derivatives 17 of Thr248 may play roles in energy coupling to the F1 subunit 6 showed that wherever the shortened version of sector 5'17'z~. In this regard the loop mutation in subunit 9 subunlt 6 is detected amongst mitochondrial translation (Arg39---~Met)is especially interesting, because amongst products by gel electrophoresis (at an apparent size of several classes of revertants that have been 11 kDa or greater), it is also detected in the immunocharacterized zl is a recessive nuclear suppressor of this precipitate. This implies that although the C-terminal mit- mutation• This suppressor possibly lies within a regions of subunit 6 are essential for energy-transduction gene coding for an FI or FA subunit of the complex which functions, they are not required for its assembly with other Fo subunits. interacts with the loop region of subunit 9. The A6L protein in rat liver mitochondria has been suggested2z (under the name chargerin II) to be directly Molecular aspects ofgene expression involved m energy transduction. The direct bioenergetic and protein topogenesis An outstanding feature of Fo sector biogenesis is the role of subunit 8 in the yeast mtATPase complex remains an open question, but this subunlt is required for the diversity of gene expression contexts in which the production of closely related sets of subunits takes place• assembly of the Fo sector (see below)• In Table 2 are summarized genetic, transcriptional and Use of mutants to derive Fo sector translational features relevant to the biosynthesis of Fo assembly pathway sector subunits in E• coil, S• cerevisiae and Bos bovis Many mutations affecting the Fo sector are frameshift (cow). Genes for mitochondrial Fo sector subunits have or nonsense mutations 1e-is leading to the production of become dispersed both within the mtDNA genome itself tnmcated proteins (Table 1). Such mutants have been and into the nucleus, in contrast to the tightly clustered very useful in dissecting the assembly pathway of the Fo array of genes in the unc operon of E. coli ~. sector, which has been analysed by using a monoclonal The mitochondrial genes in S. cerevisiae that encode antibody specific for subunit [3 of the F1 sector23. The subunits 9, 8 and 6 are represented in long primary antibody is immobilizedon Sepharose beads; subunits are transcripts which cover several adjacent genes and which defined as being assembled if they are co-precipitated by are processed by endonucleolytic cleavage to produce this antibody. On the basis of the datas summarized in the mature mRNA molecules with long 5'- and 3'bottom portion of Table 1, the order of assembly of Fo untranslated sequences z. The mature olii mRNA is sector subunits in yeast is inferred to be: first subunit 9, monocistronic TM. The aapl and oli2 genes, physically then subunit 8, followed by subunit 6. remote from the olii gene in mtDNA of S. cerevisiae, are

9

6

8

review

TIG m F e b r ~ y 1988, Vol. 4, no. 2

4. + + represented by mature **** 10 **20 30 40 mRNAmoleculesthatcuntain S U 8 Ir.J~ ~LVPFYflW(II.NYGFLLNITLL!LFSQFFLPRILRLYVSRLFISKL protein-coding regions for both subunits 8 and 6 sepmated by 706 nucleotides of 4. 4. 4. + - ÷ 4intergenic sequence24. The **** lO *'20 30 40 50 60 biological significance of the J ~ 6 L wvlICE IlP(ILDTS~L~ILSRFLTLFI IFQLKVSKHNFYHNPELTPTKNLKi;NTPWETKIfl'KIYLPLLLPI physical proximity and cotranscription of the cap/and Fig. 3. Features of the sequences of subunits 8 and A6L of the mtATPase complexes oli2 genes is not clear from S. cerevisiae and B. bovis, respectively. Residues common to both sequences la,2s because in various mutant are indicated by asterisks and the positions of charged residues are shown. Putative transmembrane stems ~3 are indicated by horizontal bars; the proposed orientation of forms of S. ceredsiae sub- subunit 8 across the inner mitochondrial membrane is shown in Fig. 2. units 8 and 6 can be synthesized independently of one another. In yeast, subunits 9 and 8 do not undergo any post- cDNA clone, denoted P1 and P2, capable of encoding translational cleavage of amino acids; each retains at its subunit 9 were encountered in bovine liver~. The N-terminns the N-formyl methionine with which trans- predicted gene products consist of identical subunit 9 lation was initiatedl°'~. These subunits are delivered proteolipids each with a different N-terminal leader that directly from the mitochondrial ribosomes into the inner is presumed to function as a cleavable transit peptide mitochondrlal membrane (Fig. 4, A and 13), where they targeting the subunit 9 to ndtochondria (see below). assemble together with subunit 6 to produce a functional Interestingly, the mRNAs represented by bovine P1 and Fo sector. Subunit 6 folds several times across the 1)2 are expressed in a tissue-specific manner~. Although there are little data on the import of mammembrane (cf. Fig. 2); this subunit is suspected also not to undergo post-translational cleavage, but definitive malian subunit 9 proteins into mitochondria, the events occurring during the import of subunit 9 ofN. crassa have proof is so far lacking. In manmmlian cells the protein-coding regions of the been extensively characterized27. This 81-amino acid A6L and subunit 6 genes are present in the same mRNA proteolipid is synthesized in the cytosol with a hydr?molecule. The sequences of these two genes overlap one philic, positivelY, charged leader 66 amino acids long~. another by 40 base~ in bovine mtDNAz~. The primary The import process may be summarized as follows~ (see transcript is processed endonucleolytically to generate Fig. 4, C). The leader binds to a receptor on the outside mature mRNA with little or no 5'- or 3'-untranslated of mitochondria; the precursor is imported in an energysequences save the poly(A) taiL The A6L and subunit 6 dependent manner and a proteolytic enzyme located in proteins are translated independently and both retain the matrix space of the organelle cleaves the leader at specific sites to generate the mature proteolipid which N-formyl methionine at the~ N-termini14. Genes encoding subunit 9 in bovine cells lie in the then integrates into the Fo sector. It seems clear that some reon'entation of transnucleus, a feature common to all metazoa and to filamentous fungi such as Newo@ora crassa. Two types of membrane segments of imported subunit 9 must occur

Table 1. Some mutations affecting Fo sector subunits in S. cerevisiae a Phenotypic c!~_ssification

Gene affected

Subunit affected

Domain affected

Exampleof amino acid change

Comments

Oligomycin-resistant (normalfunction in absence of drug)

olil

g

hl h2

Gly23--) Ala Leu53--) Phe Phe64--) Leu

Venturicidin resistant Venturicidin sensitive Venturicidin resistant

oli2

6

h4 h5

Ser175--) Cys Leu232--) Phe

Venturicidin sensitive Venturicidin sensitive

olil

9

hl Loop

Gly23-,Asp Arg39--) Met

Defective proton channel? Defective couplingto F17

oli2

6

h5

"rhr248-) Lys

mit(non-functional; but assembly of Fosector not grossly impaired)

Defective couplingto F1?qh

mitolil (non-functional.with abnormal assemblyof Fosector)

9

hl h2 All

Gly18--, Aspb Set68--, Stop Frameshift; deletes all but first 4 residues

No assemblyof subunits 9. 8 and6

aap 1

8

All

Frameshift; deletes all but first 2 residues

Assemblyof subunit 9; no assemblyof subunits 8 and 6

oli2

6

C-terminus Ser250--, stop All

Frameshift; deletes all but first 37 residues

Assembly of subunits 8. 9 and truncated subunit 6 Assemblyof subunitsS. 9 (no truncated subunit 6 is visible)

aDetailsof mutations, from the author's and from other laboratories,are compiled in the following references: olil. Ref. 19; oli2. Refs 17 and 20; aaplo Ref. 18. Seealso Ref. 5. bThismutant subunit 9 is presumed to be unableto integrate into the membrane.

! eWS

~IG - - F e b r ~ , j 1988, VoL 4, ,o. 2

Table 2. Genes, transcripts and protein precursors of Fo sector subunits in different organisms Organism Bacterium (E. coh) Yeast (S. cerevisiae) Mammal (B. bovis)

Fosector subunit

Coding site

Gene or c D N A

Mature mRNA characteristics

Cleavageof protein precursor

a b c 6 8 9 6 A6L 9

Chromosome Chromosome Chromosome Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria Nucleus

uncB ] uncF ~ uncE J oli2 ~ aapl ~ olil ATPase-6~ URF-A6L ] ATPase-9-P1 ], ATPase-9-P2~

Polycistronic, singleoperon(9 unc genes)

No No No Not known No No No No Yes Yes

because the precursor enters the matrix space Nterminus first, while the final disposition of subur, it 9 is such that its N-terminus faces outwards (Fig. 4, C). For both subunit 9 in mitochondria of S. cerevisiae (Fig. 4, A) 10 12 and subunit c in the cytoplasmic membrane ofE. coli" A . SUBUNIT 9

Dicistronic, multigenicprecursor Monocistronic, multigenicprecursor Dicistronic,overlapping ORFs, multigenic precursor Eachmonocistronic, spliced, poly(A)+, tissue-specific

the mature homologous proteolipid is delivered from the ribosomes directly into the membrane, N-terminus first. The proposal has been made 3° that where a gene coding for a protein such as subunit 9 has been transferred to the nucleus from the organelle, which can be considered as a

(YEAST M I T O C H O N D R I A L GENE)

C . SUBUNIT 9

( N E U R O S P O R A N U C L E A R GENE) N ~ B L

Mitochondricl ribosomes 1

MATRIX

MATRIX

(~*** m-N9

,

.... "

c

Nil(; N6

m-N

""'"' " IMS

,,li

iu "-"-~'":...::~ sis

i~i'~'i

i~i~i.i

OM ~i~'~ CYTOSOL

i~i~i~i

i"-;:'-;"

•("~ ,,"~ ,~

OM

.:..'i~!~!iu IMS

::::-'.': :'::':

. . . . . N............ ,........ c

i ~ OM

.

.

.

.

:':i:i:OM

-.:-:.:.:."

.

.

p-Nil [

CYTOSOL

Cytosolic ribosomes

B. SUBUNIT 8

(YEAST M I T O C H O N D R I A L GENE)

Mltochondrlal rlbosomoc

MATRIX

1 l

O. SUBUNIT 8 MATRIX

IU

~.:~i:,!

Ye

..

"'? C

Yi

i8

~i.;~i-

".:.:_:.:,'i

";';':" :.;.:.:

~i~i~i

y6

"1~

o. !~:.~iii iM

~

Y8

\,TI

Y6

\... i I IM$ OM i'i~i OYTO801.

( A R T I F I C I A L YEAST NUCLEAR GENE)

... ... .......... : ....

IMS i!~OM

:.:.+:-:

lib

.... _:i;-:i:oe

!:i:~:~ ~!".:.:"

c

NIIL/Y6 t

CYTO$OL

/

Cytololic ribosomes 1. BOosynthldls of protein

3.Orientation in inner membrane

2. Binding to inner membrane

4. Assembly into FO-sector

t. Biosynthesis of precursor

3. Import

S. Orientation in inner

2. Binding to o u t e r membrane

4. Processinil 6. Assembly into Po-sOctOr

membrane

i

Fig. 4. Two pathways for delivery of Fo sector subunits to the mitochondrial inner membrane. Panels A and B represent the mitochondrial route, illustrated by subunits 9 and 8 encoded in S. cerevisiae on mitochondrial genes and denoted here Y9 and YS, respectively. Panels C and D illustrate the nucleocytosolic route, used by proteins encoded by nuclear genes, translated in the cytosolic compartment and imported into the organelle 2s.3°. Some important steps in each pathway are indicated in the box below each pair of panels. In panel C is represented the naturally occurring, nuclearly encoded precursor (p-N9) of mature subunit 9 (m-N9) in N. crassa 27.2s. In panel D is drawn the chimaeric precursor (N9L/Y8) specified by an artificial composite nuclear gene encoding the N-terminal leader segment of p-N9 (N9L) fused to Y8. The locations of charged residues are indicated. The negative charge towards the centre of N91_/Y8 arises from the sequence at the fusion point where several N-terminal residues of m-N9 are maintained in the chimaeric precursor 31.3s. Other notations are: Y6, yeast subunit 6; N8 and N6, N. crassa subunits 8 and 6, respectively; IM0 inner membrane; IMS, inter-membrane space; OM, outer membrane; MP, matrix protease. In panels C and D° protein import is represented as occurring at points of fusion between inner and outer membranes3°. It is likely that imported proteolipids, such as subunit 9, maintain an association with the inner membrane following import 27. but the precise topological changes remain to be established.

TIG - - February 1988, Vol. 4, no. 2

descendant of an ancient bacterium, the mode of delivery of the protein into its final membrane orientation remains the same in topological terms. The role of the more recently evolved import process is thus to facilitate the delivery of such a protein into the interior of the organelle where it can now encounter the membrane according to the more ancient pathway. Imported versions of mitochondrially encoded s u b u n i t s The dispersion and recoding of genes such as those specifying Fo sector subunlts took many millions of years during evolution. The deliberate relocation to the nucleus of a yeast mitochondrial gene encoding a functional Fo sector subunit has recently been achieved in the laboratory sx. In undertaking thi_'stask we were motivated not only by curiosity, but also by the imperative of developing a suitable technology for systematic manipulation of mitochondrially encoded subunits of the Fo sector in the absence of a satis~ctory system for the direct transformation of DNA into mitochondria 32. The strategy that has been implemented using subunit 8 in S. cerevisiae is outlined in Fig. 4, D. A new gene specifying subunit 8, the smallest of the mitochondrially encoded mtATPase components, was constructed by !total chemical synthesis ~. The design of this artificial gene, now denoted N A P 8 (Ref. 34), took into account differences in codon dictionary and preferred codon usage between the yeast nucleocytosolic and mitochondrial translation systems. By making an in-frame fusionss between a DNA segment encoding the Nterminal leader (N9L) of N . crassa mtATPase subunit 9 (Fig. 4, C) and the N A P 8 sequence, a chimaeric precursor protein (termed N9L/Y8; see Fig. 4, D) was produced. When expressed by in vitro transcription and translation, N9L/Y8 was imported into isolated yeast mitochondria where it was proteolytically cleaved at the natural cleavage site o f ' t h e N . crassa subunit 9 precursor as. The functional incorporation of the imported subunit 8 into the mtATPase complex was demonstrated in vivo 3~ using a mnlticopy plasmid expression vector to express N9L/Y8 in yeast host cells carrying a mutant aapl gene which prevents the natural production of subunit 8 in mitochondria. Transformants of the m i t - host cells grew vigorously on non-fermentable substrate and contained a functional mtATPase complex"at assembled from a cytosolically synthesized version of subunit 8. The successful import and functional assembly in the inner mitochondrial membrane of a protein that is normally encoded within the organelle has broad implications from several standpoints. First is the opportunity for detailed analyses of the molecular genetics of subunit 8 and its interactions with other mtATPase subunlts. For example, site-specific mutagenesis will allow study of the structure-function relationships in subunit 8. A partivdar hypothesis to be addressed is whether subunit 8 has a direct role in energy transduction beyond its demonstrated role in the assembly of the Fo sector. Second, it now becomes possible to transfer further genes encoding mitochondrial membrane proteins from the organelle to the nucleus with a view to assembling a protein complex such as mtATPase entirely from nuclear gene products. An artificial nuclear gene encoding yeast subunit 9 has been constructed and subunit 9 shown to be imported into mitochondfia in vitro 34. Investigations of

review

the functional properties in vivo of a nuclearly encoded and imported version of subunit 9, and similar ventures with subunit 6, will certainly provide a range of new insights into the mtATPase complex. A third implication, of relevance to membrane biogenesis in general, conce~s the delivery of proteins into their target membrane from the non-natural side. An important goal will be to establish the limits, if any, on the size and complexity of proteins that can become functionally oriented under these conditions. A special feature of the inner membrane of mitochondria, highlighted in this review, is that its various integral membrane proteins are naturally delivered into it from both sides, The exploitation of this system by n~olecular genetic manipulation promises to open new vistas in the area of eukaryote membrane genetics. Acknowledgements I thank Professor A. W. Linnane and Drs D. Nero, R. J. Devenish, H. B. Lukins and S. Meltzer for useful discussions, and Drs J. E. Walker and T. Higuchi for communicating data prior to publication. References 1 Yo,u~van,D. C. and Daldal, F. (19~) M~cr~bial E ~ "

Transdi~,'f~n: Genetics, Structure a~'mlFunction of Membrane Proteins,

C~d SpringHarbor Laboratory 2 Tz~goloff,A. and Myers, A. M. (1986)Annu. Rev. Biocb~,m. 55, 249-285 3 Pecterse-n,P. L, and Carsfoli, E. (1987) Trends Biochem. Sci. 12, 146-15~ and 186-189 4 Walker, J. E., Runswick, M.J. and Puulter, L. (1987)J. Mol. Biol. 197, 89-100 5 Linnane, A. W. et al. (1985) in Achievements and Perspectives in Mitochondrial Research Vol. L" Bioenergetics (Quag~riello, E. et al., eds), pp. 211-222, Elsevier Science Publishers

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Papa, S., eds), pp. 191-204, Japan ScientificSocieties.Press and Springer-Vedag 20 John, U. P. and Nagley, P. 0986) FEB$ Lett. 267, 79-83 21 W'fllson,1". A. and Nagley, P. (1987)Eur. ]. Biochem. 167, 291297 22 Uclfida, J. et aL (1987) Biochem. Biophys. Res. Commun. 146, 953-958 23 Hadikusumo, R. G., Hertzog, P.J. and Maranki, S. (1984) Biochim. Biophys. Ada. ?65, 258-267 24 Novitski, C. E. et al. (1984) Curt. Genct. 8, 135-146 25 Anderson, S. et al. (lb82)/I. MoL Biol. 156, 683-717 26 Gay, N. J. and Walker,J. E. (1985)EMBO ]. 4, 3519-3524 27 Zwizinski,C. and Neupert, W. (1983)]. Biol. Chem. 258, 1334013346 28 Viebrock, A., Perz, A. and Sebald, W'. (1982)EMBO J. 1, 565571

netics and society

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--February 1988, Vol. 4, no. 2

33 Gearing, D. P., McMuIIen, G. L. and Nagley, P. (1985)Biockem. InL 10, 907-915 34 FarreII, L. B., Gearing, D. P. and Nagley, P. Eur. J'. Biockem. (in press) 35 Gearing, D. P. and Nagley, P. (1986)EMBO]. 5, 3651-3655

P. Nagley is at the Department of Biochemisby and Centre for Molecular Biology and Medicine, Monash University, Clayton, Victoria 3168.. Australia.

Genetic counselling and the new genetics

Fundamental social and ethical issues are raised by the rapidly advancing genetic revolution and the many new opportunities for preventive intervention that it brings both to individuals and to families. Underlying godney Harris these issues are strongly held ethical principles. Foremost among them The mapping of most important human diseasegenes has already been accomplished, are reverence for human life and its promising epochalchanges in health care and diseasepreventio~. This article esplores extension into prenatal life, respect thepotential benefits and dangers, emphasizing the highprion'ty that must begiven to for individual autonomy and the right non-directive genetic counselling. to informed choices, privacy and confidentiality. Their application to one individual may be in conflict with the concerns of other ease, bowel cancer, familial Aizheimer's pre-senile dementia and manic depressive psychosis. Recombinant individuals and of the wider public. The need to safeguard those at risk of developing or DNA technology used in this way will define genetic transmitting genetic disease, by ensuring that they are heterogeneity and clarify the interaction of gene and properly informed, must be matched by non-directive environment. When individual genetic susceptibilities are counselling. There is a real danger that this practice may known, selective modification of environmental factors be threatened if it is judged incompatible with the aims of may offer a way to prevention. mass genetic screening. The object of this review is to examine some of the implications of the use of the new From families to populations The incidence of individual genetic and congenital genetics for carrier detection, prenatal diagnosis and prevention. disorders can be reduced by measures involving whole population groupss. This has been accomplished with The human gene map conventional biochemical or cytogenetic techniques for Most important mendelian disorders have now been Down's syndrome, Tay--Sachs disease, neural tube demapped to their chromosomal locations and powerful fect, haemoglobinopathies, phenylketonmia, congenital recombinant DNA methods are available for carrier hypothyroidism and erythroblastosis fetalis (see below). detection and prenatal diagnosis in high risk families*'2. Progress towards identifying the genes makes cystic Recombinant DHA tests on chorion villus samples (CVS) fibrosis an early candidate for application of the new allow termination of an affected fetus during the first recombinant DNA technology to screening at the populatrimester - a far more acceptable procedure than late tion level, with 5% of the British population known to be termination following anmiocentesis. However, there carriers of this disease. Similarly the fragile X syndrome, are many more adults and fetuses who can be considered the commonest form of hereditary mental defect, is of 'at risk' than those who eventually develop genetic great potential importance if, as has been recently disease, and in cases where recombinant DNA methods estimated, as many as one in 600 girls may be carriers'. demonstrate that significant risk can be excluded, such tests bring relief to many patients. The net effect of these Genetics and medical education discriminating tests in families is to avoid unnecessary Considerable difficulties were encountered in sickle abortions while still reducing to some extent the in- cell screening programmes because some individuals cidence of handicapping conditions. found to be carriers erroneously believed themselves to The list of diseases for which clinically useful probes be affected and their families were ostracizeds. This has are available increases almost daily (Box 1), and diagnosis emphasized the importance of education of the relevant becomes more reliable as new polymorphisms within or population groups, and of skilled counselling for carriers. adjoining mutant genes make most families informative Unfortunately, genetic knowledge amongst clinicians and recombinational errors rare. Sometimes ;t is even may not always be adequate for this to be achieved. A possible to help families in which there is an isolated case recent survey of British medical schools, as well as of a disease that has a high mutation rate. For example, it reports from the United States, suggest that genetics has already been shown that more than 50% of boys with may not always be adequately taughff. Duchenne muscular dystrophy are likely to have deletions in the Duchenne gene~, allowing direct diagnosis of The genetic team the disease without the need for an informative pedigree. The specialist DNA, cytogenetic and biochemical Gene mapping has also begun to localize major genes laboratories are of course key elements, but the geneticimplicated in common disorders4 including vascular dis- ally trained clinician, who has the responsibility for con~) 1988,ElsevierPul~kations,Cambridge 0168- 952~.881502.00