The ubiquinol-cytochrome c reductase complex, like the other proton-pumping respiratory complexes of mitochondria, is an assembly of many different subunits. However, only a few of these subunits participate directly in the electron transfer and proton translocation. The roles of the other subunits are largely unknown. We discuss here some intriguing features of two of these subunits

Bechmann

Berry

Hanns Weiss

Hatefi

Hauska

Hawlitschek

Jensen

Karlsson

Kevin Leonard

Leonard

Linke

Mitchell

Oudshoorn

Pfanner

Pollock

Schoppink

Schulte

Tzagoloff

Walter Neupert

Weiss

Witte

English

Crossref

Puzzling subunits of mitochondrial cytochrome reductase

Weiss, Hanns

Leonard, Kevin

Neupert, Walter

Open Access LMU

TALKING POINT TIBS 15 - MA Y1990 CYTOCHROME REDUCTASE (also known as cytochrome bc~ complex or complex lid is the second of three energy-transducing respiratory chain complexes of mitochondria. The function of the complex is to link the electron transfer from ubiquinol to cytochrome c with proton translocation across the inner membrane. In doing so a proton- motive force is generated which can subsequently drive ATP synthesisL The actual mechanism ofcytochrome r duc- tase, which is best described by the ubiquinone cycle 2, involves only the three subunits of the enzyme which contain redox centres: the two heme- containing cytochromes, b and c~, and the Rieske-iron-sulfur protein. How- ever, in all organisms studied so far this mitochondrial enzyme is composed of many more subunits. Cytochrome reductase from fungi contains nine sub- units, while the mammalian enzyme was found to have l 1 subunits 3.Since no mechanistic differences between the much simpler prokaryotic and the eukaryotic enzyme have yet been found, the role of the additional sub- units in the mitochondrial enzyme remains mysterious. We discuss here the largest of these subunits, ! and II (traditionally called core proteins) which contribute almost half of the total protein of the mitochondrial cytochrome r ductase 4. Structural properties Electron microscopic studies of the three-dimensional structure of cytochrome reductase from Neurospora crassa suggest that subunits I and II are peripheral membrane proteins. When the enzyme was isolated using the detergent Triton and cleaved by salt treatmenP, two subcomplexes were obtained; a detergent-bound bcl sub- H. Woiss is at the Institut for Biochemie, Universit~t DOsseldorf, Universit~tsstraBe 1,4000 DOsseldorf 1, FRG. K. Leonard is at the European Molecular Biology Laboratory, Meyerhofstrat~e 1, 6900 Heidelberg, FRG. W. Neupert is at the Institut f~r Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Universit~t MOnchen, Goethestra6e 22, 8000 MOnchen 2, FRG. 178 The ubiquinol-cytochrome c reductase complex, like the other proton- pumping respiratory complexes of mitochondria, is an assembly of many different subunits. However, only a few of these subunits participate directly in the electron transfer and proton translocation. The roles of the other subunits are largely unknown. We discuss here some intriguing features of two of these subunits. complex comprising the cytochromes b and c~ and the four smallest subunits without redox centres, and a water-sol- uble subcomplex of the subunits I and II. Membrane crystals of the whole enzyme 6 and the bcl subcomplex 5 were prepared by incorporating the prepar- ations into artificial phospholipid bi- layers. Low resolution three-dimension- ai structures of the two proteins were determined from tilted electron-micro- scope views of the negatively stained membrane crystals (Fig. l). Comparison of the structures clearly showed that the subcomplex of subunits I and II is located peripherally at the matrix side of the mitochondrial inner membrane a,7. The sequences of subunits l and II of Rgure 1 Side view parallel to the plane of the membrane of whole cytochrome reductase (left) and bc: subcomplex (right). Both preparations were obtained as dimers. The white section rep- resents the membrane, and the upper and lower shaded areas the matrix space and the intermembrane space, respectively, of mitochondria. In the holoenzyme, cytochrome b with its two heme goups (b,Mr ,,,43000)lies mainly in the membrane; cytochrome c~ (Cl.Mr ,,,28 000) and the iron-sulfur protein (FeS,Mr ,,,22 000) protude into the intermembrane space, and the subcomplex of subunits I and II (Mr ,,,48000 and ,,,44000) is located peripherally on the matrix side of the membrane. The cytochrome bc: subcomplex com- prises the cytochrome b and c~ and the four smallest subunits without redox groups (Mr 16 000-9 000; not shown in the figure) and lacks the subcomplex of subunits I and II and the iron-sulfur protein. The scale bar represents 10 nm. © 1990, Elsevier Science Publishers Ltd,(UK) 0376 - 5067/90/$02.00 TIBS 15-MAY1990 the S. cerevisiae cytochrome reduc- tase ,9 and subunit l of the N. crassa enzyme 4 (see below) contain no hydrophobic stretches long enough to span the membrane and thus would also indicate that the two subunits could be peripheral proteins. Speculetion on the role of subunits I and II In electron transfer For a long time no function could be attributed to subunits l and II. Their equivalents are not found in isolated cytochrome b6f complex from chloro- plasts, nor in bacterial bc complexes ~°,~t although these complexes are closely related to the mitochondriai cyto- chrome reductase both functionally and structurally. This suggested that sub- units I and II are not required for elec- tron transfer and proton translocation per se. On the other hand, reconstitution experiments with isolated N. crassa cytochrome reductase indicated that the enzyme is inactive when it lacks subunits I and I1 ]~. Yeast mutants de- ficient in subunit If exhibit strongly reduced levels of enzymatically active cytochrome reductase 9J3 and mutants deficient in subunit I do not have any activity 8. Furthermore, these mutants contain much less cytochrome b rela- tive to cytochrome c/. This led to the suggestion that the subunits are required for assembly of cytochrome reductase or maturation of cytochrome b. Alternatively, they might function as control subunits regulating the activity of the enzyme. All these ideas remain speculative, however, and await further experiments. A now protein family We have cloned and sequenced cDNA encoding subunit l of the N. crassa cytochrome reductase 4. The deduced amino acid sequence (which was veri- fied by partial protein sequences), ismost surprisingly identical to the sequence of processing enhancing pro- tein (PEP) which stimulates the activity of the matrix processing peptidase (MPP) in N. crassa TM. In yeast, MPP and PEP are the products of the Mas2 (or Mif2) and Masl (or Mill) genes~S-l¢; how- ever, yeast PEP is homologous but not identical to subunit I ~6,]7. Alignment of the sequences of the yeast PEP, MPP, subunits 1 and lI, and subunit l/PEP of N. crassa reveals ignifi- cant similarities. Whenever one mem- ber of this family is compared to the other four members, N. crassa subunit I/PEP shows the highest degree of sequence identity (namely 51%) to yeast PEP, 26% to N. crassa MPP and 23% to yeast MPP 4. Functional Identity of $ul~nit I and PEP In N. ©rasu The ability of whole cytochrome reductase and isolated subunits I and I1 has been compared with that of PEP itself to stimulate the processing ac- tivity 4,~4. For the assay, a PEP-free MPP preparation was used to process the precursor of the ~-subunit of ATP- synthase. MPP alone showed very low activity which was stimulated by either isolated PEP, isolated subunit 1 or whole cytochrome reductase, all having simi- lar specific enhancing activity. Isolated subunit I1 was inactive 4 (Fig. 2). The PEP function of the N. crassa sub- unit I may be located on a hydrophilic N-terminal domain since this domain shows a much higher similarity to the N-terminal half of yeast PEP (65%) than to the N-terminal half of yeast subunit I (23%). A more hydrophobic C-terminal domain might contact subunit II and contribute to the respiratory function. Compared to the N-terminal domain this domain has lower similarity to the C-terminal half of yeast PEP (43%) and a higher one to the C-terminal half of yeast subunit I (40%) 4. New questions adse Why should proteins with such differ- ent functions belong to a single family? So far the roles of the various members of this protein family are poorly under- stood. It seems that subunits I and I! function by interacting with other sub- units of the cytochrome reductase and thereby contribute to the assembly and stabilization of the complex. MPP has a proteolytic activity, and PEP is required for this reaction by interacting with MPP and/or the precursor proteins. The common denominator with all these functions might be that protein-protein interaction must occur. Thus, one essential function of all the members of the family may be to make contact o certain active epitopes on other pro- teins and thereby facilitate an ordered sequence of reactions. However, these proteins may have other functions. For instance, subunits I and lI might function as a control unit, sensing what is going on inside the mitochondria. Differential affects of the membrane potential on redox activity and/or H+/e - stoichiometry of cyto- chrome reductase might imply that the reaction pathway in the enzyme could Figure 2 Stimulation of the activity of mitochon- drial processing peptidase (MPP) by the processing enhancing protein (PEP), subunit I, subunit II, and whole cyto- chrome reductase. (A) PEP, (o) cytochrome reductase, (m) subunit I, (V) subunit I1. be affected ~9. PEP in yeast may have a role in translocation, in addition to enhancing the processing of precursor proteins. In PEP-deficient mutants the rate of the protein import was found to be reduced (F-U. Hartl, unpublished). It will be interesting to learn whether there are other organisms in which subunit 1 and PEP are identical. Re- cently the MPP from rat liver has been purified and described as a tight com- plex of two components 2°.Sequencing of this protein family in mammals might provide clues to their function(s). The existence of two different genes in yeast for subunit I and PEP may be related to the exceptional bility of this fungus to repress respiration under aerobic con- ditions. The promitochondria of fer- menting cells lack the respiratory chain complexes, but still perform protein import and processing. Thus, require- ment of PEP in repressed yeast might have led to divergence of PEP and sub- unit I. More questions than answers Could there be any function for PEP in N. crassa in association with cyto- chrome reductase? Membrane trans- location of precursor proteins happens via contact sites between the outer and inner membranes, involves ATP- dependent unfolding of the preproteins and requires an electrical potential across the inner membrane. During or after translocation the presequences are cleaved off by the processing enzyme TM. Does this imply that in N. crassa processing can only occur in vicinity of cytochrome r ductase? 179 Although the puzzling findings on the structure and function of the subunits Iand II of cytochrome reductase raise more questions than we can answer, they may contribute to an understand- ing of why the mitochondrial respirat- ory chain complexes contain so many additional subunits, which are not di- rectly involved in energy transduction. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. References 1 Hatefi, Y. (1985) Annu. Rev. Biochem. 54, 1016-1065 2 Mitchell, P. (1976) J. Theor. Biol. 62, 327-367 3 Weiss, H. (1987) Curr. Top. Bioenergetics 15, 67-90 4 Schulte, U., Arretz, M., Schneider, H., Tropschug, M., Wachter, E., Neupert, W. and Weiss, H. (1989) Nature 339, 147-149 5 Karlsson, B., Hovm611er, S., Weiss, H. and Leonard, K. (1983) J. Mol. Biol. 165, 287-302 6 Leonard, K., Wingfield, P., Arad, T. and Weiss, H. (1981) J. Mol. Biol. 149, 259-274 7 Weiss, H. and Leonard, K. (1987) Chemica Scripta 27B, 73-81 8 Tzagoloff, A., Wu, M. and Crivellone, M. (1986) J. Biol. Chem. 261, 17163-17169 90udshoom, P., van Steeg, H., Swinkels, B. W., Schoppink, P. and Grivell, L. A. (1987) Eur. J. Biochem. 163, 97-103 10 Hauska, G., Hurt, E., Gabellini, N. and Lockau, W. (1983) Biochim. Biophys. Acta 726, 97-133 11 Berry, E. and Trumpower, B. L. (1985) J. Biol. TIBS 15-MAY1990 Chem. 260, 2458-2467 12 Linke, P. and Weiss, H. (1986) Methods Enzymoh 126, 201-210 13 Schoppink, P. J., Hemrika, W. and Berden, J. A. (1989) Biochim. Biophys. Acta 974, 192-201 14 Hawlitschek, G., Schneider, H., Schmid, B., Tropschug, M., Hartl, F. U. and Neupert, W. (1988) Ce/153, 795-806 15 Witte, C., Jensen, R. E., Yaffe, M. P. and Schatz, G. (1988) EMBO J. 7,143-144 16 Jensen, R. E. and Yaffe, M. P. (1988) EMBO J. 7, 3863-3871 17 Pollock, R. A., Hartl, F. U., Cheng, M. Y., Ostermann, J., Horwick, A. and Neupert, W. (1988) EMBO J. 7, 3493-3500 18 Planner, N., Hartl, F-U. and Neupert, W. (1988) Eur. J. Biochem. 175, 205-212 19 Bechmann, G., Zweck, A. and Weiss, H. (1989) Biol. Chem. Hoppe-Seyler 370, 875 20 Ou, W-J., Ito, A., Okazaki, H. and Omura, T. (1989) EMBO J. 8, 2605-2612 LETTERS Evolution of transport ATPases In the recent discussion about the origins of transport ATPases 1-3, an essential physiological requirement has been ignored, i.e. cell-volume regulation 4.Any cell surrounded by a semipermeable membrane and containing both permeable ions (e.g. H ÷, Na +) and impermeable ions (e.g. proteins) will swell infinitely unless: (1) there is a rigid cell wall that can sustain the elevated osmotic pressure resulting from the Donnan equilibrium distribution of ions; or (2) the membrane is impermeable to water; or (3) ion pumps (such as active transport ATPases) prevent he Donnan distribution of ions. Obvious functional problems for primitive cells result from solutions (1) and (2), which are, moreover, difficult to attain absolutely. For solution (3) an efficient approach to maintaining a non-equilibrium steady- state asymmetry of permeable ions is for the pump to match the leak s. This is most easily achieved if the major ion leaking in is the major ion pumped out 4. Consequently, if the primitive cells arose in acidic environments (caused either by inorganic 2 or biological s processes), then a primordial H+-ATPase could solve the problem of cell-volume maintenance; alternatively, if the primitive cells arose in saline environments, then a primordial Na÷-ATPase would be expected 4'6. References 1 Nelson, N. and Taiz, L. (1989) Trends Biochem. Sci. 14, 113-116 2 Silverstein, T. (1989) Trends Biochem. Sci. 14, 48O 3 Nelson, N. (1989) Trends Biochem. Sci. 14, 480 4 Robinson, J. D. (1968) J. Theoret. Biol. 19, 90-96 5 Tosteson, D. C. and Hoffman, J. F. (1960) J. Gen. Physiol. 44, 169-194 6 Skulachev, V. P. (1989) FEBS Lett. 250, 106-114 JOSEPH D. ROBINSON Department of Pharmacology, State University of New York Health Science Center, Syracuse, NY 13210, USA. Lipoxgenase specificity We read with great interest Tim Hunt's recent article on lipoxygenase and the maturation of reticulocytes (TIBS 14, 393-394). However, Fig. 1 of the article was misleading, since it showed only the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from arachidonic acid. In fact, lipoxygenase exhibits dual positional specificity; the major product formed from arachidonic acid is 15-HETE (see Figure), with 12-HETE formed in a side reaction. HARMUT KLIHN AND TANKRED SCHEWE Bereich Medizin (Chafit(~) der Humboldt-Universit~t zu Berlin, Institut f~r Biochemie, Berlin, GDR. 180 Arachidonic acid COOH l o%/  J HO0 _ ~ ~ C O O H  COOH I OOH 

Lipoxgenase specificity We read with great interest Tim Hunt's recent article on lipoxygenase and the maturation of reticulocytes

of the article was misleading, since it showed only the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from arachidonic acid. In fact, lipoxygenase exhibits dual positional specificity; the major product formed from arachidonic acid is 15-HETE (see

Universität München: Elektronischen Publikationen

https://epub.ub.uni-muenchen.de/7552/1/Neupert_Walter_7552.pdf

Puzzling subunits of mitochondrial cytochrome reductase

Abstract

Similar works

Full text

Available Versions

Crossref

Open Access LMU

Universität München: Elektronischen Publikationen