DOCSLIB.ORG

Biosynthesis of the Chloroplast Cytochrome B6f Complex: Studies In

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Biosynthesis of the Chloroplast Cytochrome B6f Complex: Studies In The Plant Cell, Vol. 3, 203-212, February 1991 O 1991 American Society of Plant Physiologists Biosynthesis of the Chloroplast Cytochrome bsf Complex: Studies in a Photosynthetic Mutant of Lemna Barry D. Bruce’ and Richard Malkin2 Department of Plant Biology, University of California, Berkeley, Berkeley, California 94720 The biosynthesis of the cytochrome b6f complex has been studied in a mutant, no. 1073, of Lemna perpusilla that contained less than 1% of the four protein subunits when compared with a wild-type strain. RNA gel blot analyses of the mutant indicated that the chloroplast genes for cytochrome f, cytochrome b6, and subunit IV (petA, petB, and petD, respectively) are transcribed and that the petB and petD transcripts undergo their normal processing. Analysis of polysomal polyA+ RNA indicated that the leve1 of translationally active mRNA for the nuclear-encoded Rieske Fe-S protein (petC) was reduced by >lOO-fold in the mutant. lmmunoprecipitation of in vivo labeled proteins indicated that both cytochrome f and subunit IV are synthesized and that subunit IV has a 10-fold higher rate of protein turnover in the mutant. These results are discussed in terms of the assembly of the cytochrome complex and the key role of the Rieske Fe-S protein in this process. INTRODUCTION All organisms capable of photosynthetic autotrophic me- has only started to receive attention. Recent studies have tabolism or heterotrophic respiratory metabolism share the begun to investigate the questions of how these proteins ability to couple electron transport to proton translocation are inserted into a membrane, when and where prosthetic across a membrane. In all cases, this coupled electron groups are attached, and what directs the assembly of the transport is carried out in distinct multisubunit membrane individual subunits into a functional complex. complexes. The most widespread and well studied of The present work considers the mechanism of assembly these complexes is the cytochrome bcl (complex 111) and of the chloroplast cytochrome b6f complex using a pho- bsf class of electron transport complexes. These com- tosynthetic mutant of the higher plant Lema perpusilla plexes function as quinol/cytochrome c (plastocyanin),ox- no. 1073. This mutant was originally isolated as a flowering idoreductases. In all known organisms, this complex con- mutant, and subsequent biochemical characterization in- tains a b-type cytochrome containing two hemes of differ- dicated that it was a photosynthetic mutant lacking the ent potentials, a c-type cytochrome, and a high potential thylakoid membrane cytochrome bsf complex (Shahak et 2Fe-2S center bound by a protein known as a Rieske al., 1976; Malkin and Posner, 1978; Lam and Malkin, Fe-S protein (Malkin, 1988). In chloroplasts, there is an 1985). However, this mutant was not defective in heme additional subunit (subunit IV) that has been shown to bind biosynthesis or in heme insertion because its thylakoids plastoquinone (Doyle et al., 1989). In mitochondria and showed normal amounts of another chloroplast cyto- purple bacteria, the protein sequence homologous to sub- chrome, cytochrome b-559. Lam and Malkin (1985) also unit IV has become fused with the cytochrome b protein, used protein gel blotting to show that not only were the yielding a larger protein with a possible added role in prosthetic groups lacking in the mutant but that the apo- quinone binding (Hauska et al., 1988). proteins for cytochrome f, cytochrome bs, Rieske Fe-S Although extensive studies have been carried out on the protein, and subunit IV were absent. mechanism of electron transfer and proton translocation The present work considers the mRNA levels for the catalyzed by cytochrome bcl/b6f complexes (Rich, 1986), cytochrome complex subunits, the transient synthesis of the mechanism by which these complexes are assembled the corresponding polypeptides, and the fate of these proteins in the thylakoid membrane of L. perpusilla no. ’ Current address: Department of Botany, University of Wisconsin, 1073. These results are considered in relation to the Madison, WI 53706. mechanism of assembly of this multimeric complex in To whom correspondence should be addressed. thylakoids. 204 The Plant Cell LemnaperpusilloWT Lemnaperpusilla 1073 accumulate Rieske <3%th do t d e an ,protei n accumulated to <2% of the levels of these subunits relative to wild-type 125 25 10 5 1 125 25 10 5 1 membranes. The amount of another photosynthetic mem- brane protein, light-harvesting chlorophyll-protein complex I; II (LHCP II), that accumulated in wild-type and mutant thylakoids was identical (data not shown), although there a sligh s t wa decreas e amounth n i ef subuni o t f o I I t photosystem (thepsaI D gene product mutane th n )i t (see Figure 2). RN l BloAGe t Analysi Cytochrome th f so e b6f Genes To determine whether the lack of protein accumulation in the mutan failura t o membranet mRNn ei e du As wa s accumulation l bloge t A analyseRN , s were don totan eo l RNA and a polysomal polyA+ RNA fraction. Figure 3 shows the results of an analysis of the chloroplast-encoded sub- cytochrome unitth f so f complexe eb . This blot contained equal amounts of total RNA that was ethanol precipitated before loading ont gele oth . Therefore slighe th , t differ- ences in intensity cannot necessarily be interpreted as a CYTOCHROMEf difference in the absolute RNA levels. It is clear from this Figur . Quantitative1 e Protein Blot Analysi perpusilla. L f so Wild- figure that all plants, including the closely related Type (WT Mutand an ) t (1073) Thylakoid r Cytochromsfo e. f L. gibba, accumulated a single predominant mRNA tran- scrip f approximatelo t r peffo Ab k (cytochrom 1 y4. ef) Proteins were resolve SDS-PAGy db electroblotted Ean d onto nitrocellulose. The blot was probed with antiserum raised against spinach cytochrome r. The numbers represent the amount of chlorophyl microgramn i l s loade eacdn i h laneantibode Th . s ywa visualized with goat anti-rabbit horseradish peroxidase. VvT 1073 0 8.01.2 0 4 6 0 8.01.0 2 6 0 4 6 0 6 Cytochrome RESULTS Subunit IV Protei l BlonGe t Analysi Thylakoif so d Protein Lemnasn i To determine accurately the level of the subunits of the cytochrome comple . perpusillaL n xi . 107no 3 versue sth PSI-subunit II wild-type/., perpusilla, quantitative protein gel blotting was done varyiny B . amoune gth f proteio t n loaded onte oth gel before blotting, it was possible to compare the signals Rieske Fe-S of the wild type and mutant and, thus, provide an estimate relative th n o e amount f proteiso n that accumulatee th dn i respective thylakoids. These blots were normalizee th n 6 do 1. 0 8. 0 2 0 4 0 6 6 1. 0 8. 0 2 0 4 0 6 basis of the amount of chlorophyll loaded. Figure 1 shows Figur . eQuantitativ2 e Protein Blot Analysi . perpusillaL f so Wild- a quantitative protein gel blot using a cytochrome f anti- Type (WT) and Mutant (1073) Thylakoids for Subunits of the body from spinach. By comparing the signal intensity from Cytochrom f Complex6 eb . the 1 HQ lane of the wild type versus the 125 n<3 lane for Analyses were carried out as in Figure 1 using spinach antibodies the mutant, it was possible to conclude that cytochrome f raised against cytochrome £>6, subunit IV, the Rieske Fe-S protein, accumulated in the mutant thylakoids to <1% of the level controlanda s a , , subuni I (thI t e psaD gene product f spinaco ) h that it occurred in the wild-type thylakoids. Extending this chloroplast photosyste mI (PSI) . Becaus f contaminatioeo y nb typ f analysieo othee th ro st subunit cytochrome th f so e nonspecific reactions with the Rieske antibody, the presence of complex s showa , n Figuri n , indicate2 e de th tha n i t the cross-reacting produc indicates i t arrobottoe e th th wy n di mb mutant, cytochrome t>6 accumulated to <1%, subunit IV panel. Cytochrome £> 6f Comple x Assembl5 20 y ro ro ro resula gene s f theio tswa r cotranscription int singloa e i>- ro- large polycistronic transcript that subsequently underwent o o o o a comple post-transcriptiof o t xse n processin splicind gan g 7> '55 steps. This cotranscriptio post-transcriptionad nan l proc- o => ^ essin bees ha gn observe maizen di , a spinachpe d an , 3. .O O. O. 5 S a s i fc (Heinemeye t al.e r , 1984; Berend t al.se , 1986; Roct ke o> a. a. O> Q. Q. L C . Q > 0 al., 1987) interestins i t I . noto gt e thamRNe th t A accu- mulatio r botnfo f thesho e genemutane th n si t appearo st aboue b t twic wilee thath d f typeo t . Whethee r thidu s si increasn a o t transcriptionan ei l activit r mRNyo A stability t knownisno . From these conclude e studiesb n ca t i , d that transcripts for all three chloroplast cytochrome complex genes accumulate in the L. perpusil/a mutant. Although thers accumulatiowa e f mRNAo e n th r fo s chloroplast genes in the preceding experiment, it was still 3.94kb possible thamutane th tdefectivs wa t transcriptioe th en i n or accumulation of the mRNA for the nuclear-encoded 1.97kb subunit, the Rieske Fe-S protein. In fact, RNA gel blot analysis of total RNA for the Rieske Fe-S transcript indi- cated that the mRNA level in the mutant was <10% of that founwile th d dn typi e (dat t shown)ano . However, this signal was quite low and required hybridizations under low-stringency conditions.
Load more

Recommended publications
  • Structure of the Lumen-Side Domain of the Chloroplast Rieske Protein Christopher J Carrell, Huamin Zhang, William a Cramer and Janet L Smith*
    Research Article 1613 Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Rieske protein Christopher J Carrell, Huamin Zhang, William A Cramer and Janet L Smith* Background: The cytochrome b6f complex functions in oxygenic Address: Department of Biological Sciences, photosynthesis as an integral membrane protein complex that mediates coupled Purdue University, West Lafayette, IN 47907-1392, electron transfer and proton translocation. The Rieske [2Fe–2S] protein subunit USA. of the complex functions at the electropositive (p) membrane interface as the *Corresponding author. electron acceptor for plastoquinol and donor for the cytochrome f subunit, and E-mail: [email protected] may have a dynamic role in catalyzing electron and proton transfer at the Key words: membrane interface. There are significant structure/function similarities to the cytochrome b6f complex, cytochrome f, energy transduction, plastoquinone, proton cytochrome bc1 complex of the respiratory chain. translocation Results: The 1.83 Å crystal structure of a 139-residue C-terminal fragment of Received: 19 August 1997 the Rieske [2Fe–2S] protein, derived from the cytochrome b f complex of Revisions requested: 29 September 1997 6 Revisions received: 15 October 1997 spinach chloroplasts, has been solved by multiwavelength anomalous Accepted: 16 October 1997 diffraction. The structure of the fragment comprises two domains: a small ‘cluster-binding’ subdomain and a large subdomain. The [2Fe–2S] cluster- Structure 15 December 1997, 5:1613–1625 binding subdomains of the chloroplast and mitochondrial Rieske proteins are http://biomednet.com/elecref/0969212600501613 virtually identical, whereas the large subdomains are strikingly different despite © Current Biology Ltd ISSN 0969-2126 a common folding topology.
    [Show full text]
  • Electron Transfer from the Rieske Iron-Sulfur Protein to Cytochrome F In
    THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 44, Issue of November 1, pp. 41865–41871, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Electron Transfer from the Rieske Iron-Sulfur Protein (ISP) to Cytochrome f in Vitro IS A GUIDED TRAJECTORY OF THE ISP NECESSARY FOR COMPETENT DOCKING?* Received for publication, June 11, 2002, and in revised form, August 23, 2002 Published, JBC Papers in Press, August 30, 2002, DOI 10.1074/jbc.M205772200 Glenda M. Soriano‡§, Lian-Wang Guo¶**, Catherine de Vitryʈ, Toivo Kallas¶, and William A. Cramer‡§ From the ‡Department of Biological Sciences and Program in Biochemistry/Molecular Biology, Purdue University, West Lafayette, Indiana 47907-1392, the ¶Department of Biology and Microbiology, University of Wisconsin, Oshkosh, Wisconsin 54901, and ʈPhysiologie Membranaire et Mole´culaire du Chloroplaste, CNRS UPR 1261, Institute de Biologie Physico-Chimique, 75005 Paris, France 3 3 The time course of electron transfer in vitro between tochrome f plastocyanin or cytochrome c6 photosystem I soluble domains of the Rieske iron-sulfur protein (ISP) on the lumen (p-side) of the membrane, comprises the high and cytochrome f subunits of the cytochrome b6f com- potential electron transport chain of the plastoquinol oxidase, 3 plex of oxygenic photosynthesis was measured by whereas electron transfer between the two b-hemes, heme bp stopped-flow mixing. The domains were derived from heme bn, to a putative n-side bound quinone defines the low Chlamydomonas reinhardtii and expressed in Esche- potential chain. Absorption of a photon and charge separation richia coli.
    [Show full text]
  • (LCMS ) of an Oligomeric Membrane Protein
    Research Full Subunit Coverage Liquid Chromatography Electrospray Ionization Mass Spectrometry LCMS؉) of an Oligomeric Membrane Protein) □ CYTOCHROME b6f COMPLEX FROM SPINACH AND THE CYANOBACTERIUM MASTIGOCLADUS LAMINOSUS* S Julian P. Whitelegge‡§, Huamin Zhang¶, Rodrigo Aguilera‡, Ross M. Taylorʈ, and William A. Cramer¶ Highly active cytochrome b6f complexes from spinach ing the presence of a DNA sequencing error or a previ- and the cyanobacterium Mastigocladus laminosus have ously undiscovered RNA editing event. Clearly, complete been analyzed by liquid chromatography with electros- annotation of genomic data requires detailed expression -pray ionization mass spectrometry (LCMS؉). Both size- measurements of primary structure by mass spectrome exclusion and reverse-phase separations were used to try. Full subunit coverage of an oligomeric intrinsic mem- separate protein subunits allowing measurement of their brane protein complex by LCMS؉ presents a new facet to -molecular masses to an accuracy exceeding 0.01% (؎3 intact mass proteomics. Molecular & Cellular Proteom Da at 30,000 Da). The products of petA, petB, petC, petD, ics 1:816–827, 2002. petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach ؉ complex also contained ferredoxin-NADP oxidoreduc- Mass spectrometry (MS)1 has revolutionized the biological tase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. sciences since the development of matrix-assisted laser de- (2001) Flavonucleotide:ferredoxin reductase is a subunit sorption ionization (MALDI) (1) and electrospray ionization of the plant cytochrome b f complex. J. Biol. Chem. 276, 6 (ESI) (2) in the late eighties (3), fertilizing the emergence of a 38159–38165). While the measured masses of PetC and new discipline called proteomics.
    [Show full text]
  • Metalloproteins in the Biology of Heterocysts
    life Review Metalloproteins in the Biology of Heterocysts Rafael Pernil 1,* and Enrico Schleiff 1,2,3 1 Institute for Molecular Biosciences, Goethe University Frankfurt, Max-von-Laue-Straβe 9, 60438 Frankfurt am Main, Germany; schleiff@bio.uni-frankfurt.de 2 Frankfurt Institute for Advanced Studies, Ruth-Moufang-Straße 1, 60438 Frankfurt am Main, Germany 3 Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Straβe 15, 60438 Frankfurt am Main, Germany * Correspondence: [email protected]; Tel.: +49-69-798-29285 Received: 30 January 2019; Accepted: 28 March 2019; Published: 3 April 2019 Abstract: Cyanobacteria are photoautotrophic microorganisms present in almost all ecologically niches on Earth. They exist as single-cell or filamentous forms and the latter often contain specialized cells for N2 fixation known as heterocysts. Heterocysts arise from photosynthetic active vegetative cells by multiple morphological and physiological rearrangements including the absence of O2 evolution and CO2 fixation. The key function of this cell type is carried out by the metalloprotein complex known as nitrogenase. Additionally, many other important processes in heterocysts also depend on metalloproteins. This leads to a high metal demand exceeding the one of other bacteria in content and concentration during heterocyst development and in mature heterocysts. This review provides an overview on the current knowledge of the transition metals and metalloproteins required by heterocysts in heterocyst-forming cyanobacteria. It discusses the molecular, physiological, and physicochemical properties of metalloproteins involved in N2 fixation, H2 metabolism, electron transport chains, oxidative stress management, storage, energy metabolism, and metabolic networks in the diazotrophic filament. This provides a detailed and comprehensive picture on the heterocyst demands for Fe, Cu, Mo, Ni, Mn, V, and Zn as cofactors for metalloproteins and highlights the importance of such metalloproteins for the biology of cyanobacterial heterocysts.
    [Show full text]
  • The Complex Architecture of Oxygenic Photosynthesis
    REVIEWS THE COMPLEX ARCHITECTURE OF OXYGENIC PHOTOSYNTHESIS Nathan Nelson and Adam Ben-Shem Abstract | Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on earth. The primary step in this process — the conversion of sunlight into chemical energy — is driven by four, multisubunit, membrane-protein complexes that are known as photosystem I, photosystem II, cytochrome b6f and F-ATPase. Structural insights into these complexes are now providing a framework for the exploration not only of energy and electron transfer, but also of the evolutionary forces that shaped the photosynthetic apparatus. 8,9 PROTONMOTIVE FORCE Oxygenic photosynthesis — the conversion of sunlight After the invention of SDS–PAGE ,the biochemical (pmf). A special case of an into chemical energy by plants, green algae and composition of the four multisubunit protein com- electrochemical potential. It is cyanobacteria — underpins the survival of virtually all plexes began to be elucidated10–14.According to the par- the force that is created by the higher life forms. The production of oxygen and the tial reactions that they catalyse, PSII is defined as a accumulation of protons on one assimilation of carbon dioxide into organic matter water–plastoquinone oxidoreductase, the cytochrome-b f side of a cell membrane. This 6 concentration gradient is determines, to a large extent, the composition of our complex as a plastoquinone–plastocyanin oxidoreduc- generated using energy sources atmosphere and provides all life forms with essential tase, PSI as a plastocyanin–ferredoxin oxidoreductase such as redox potential or ATP. food and fuel. The study of the photosynthetic appara- and the F-ATPase as a pmf-driven ATP synthase15 (FIG.
    [Show full text]
  • Structure and Function of Cytochrome Containing Electron Transport
    Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2015 Structure and Function of Cytochrome Containing Electron Transport Chain Proteins from Anoxygenic Photosynthetic Bacteria Erica Lois-Wunderlich Majumder Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Chemistry Commons Recommended Citation Majumder, Erica Lois-Wunderlich, "Structure and Function of Cytochrome Containing Electron Transport Chain Proteins from Anoxygenic Photosynthetic Bacteria" (2015). Arts & Sciences Electronic Theses and Dissertations. 567. https://openscholarship.wustl.edu/art_sci_etds/567 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Department of Chemistry Dissertation Examination Committee: Robert E. Blankenship, Chair Michael Gross Dewey Holten Robert Kranz Liviu Mirica Structure and Function of Cytochrome Containing Electron Transport Chain Proteins from Anoxygenic Photosynthetic Bacteria by Erica Lois-Wunderlich Majumder A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of
    [Show full text]
  • Mitochondrial Cytochrome C1 Is a Collapsed Di-Heme Cytochrome
    Mitochondrial cytochrome c1 is a collapsed di-heme cytochrome Frauke Baymann*, Evelyne Lebrun, and Wolfgang Nitschke Bioe´nerge´tique et Inge´nierie des Prote´ines͞Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, Marseille, 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France Communicated by Pierre A. Joliot, Institut de Biologie Physico-Chimique, Paris, France, October 7, 2004 (received for review March 26, 2004) Cytochrome c1 from mitochondrial complex III and the di-heme cytochromes c in the corresponding enzyme from ␧-proteobacteria have so far been considered to represent unrelated cytochromes. A missing link protein discovered in the genome of the hyperther- mophilic bacterium Aquifex aeolicus, however, provides evidence for a close evolutionary relationship between these two cyto- chromes. The mono-heme cytochrome c1 from A. aeolicus contains stretches of strong sequence homology toward the ␧-proteobac- terial di-heme cytochromes. These di-heme cytochromes are shown to belong to the cytochrome c4 family. Mapping cytochrome c1 onto the di-heme sequences and structures demonstrates that cytochrome c1 results from a mutation-induced collapse of the di-heme cytochrome structure and provides an explanation for its uncommon structural features. The appearance of cytochrome c1 thus represents an extension of the biological protein repertoire quite different from the widespread innovation by gene duplica- EVOLUTION tion and subsequent diversification. cytochrome bc1 complex ͉ protein repertoire extension ͉ bioenergetic electron transfer ͉ lateral gene transfer rotein-based enzymes in extant organisms catalyze a virtually Pboundless multitude of metabolic reactions. In the early days of life on Earth, only a limited number of polypeptide modules probably evolved to take over specific catalytic roles previously fulfilled by bioinorganic catalysts (1, 2) and͞or ribozymes (3).
    [Show full text]