Skip to main content
SpringerLink
Log in

Import and assembly of the α and β-polypeptides of the light-harvesting complex I (B870) in the membrane system of Rhodobacter capsulatus investigated in an in vitro translation system

  • Regular Paper
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

Transcripts of the genes pufBA, pufB or pufA from Rhodobacter capsulatus were translated in a cell-free system of R. capsulatus. The incorporation of the nascent polypeptides LHIα and β in various types of membranes and the assembly of the light-harvesting (LH) complex I (B870) were investigated. The highest rate of stable incorporation of LHIα and β into the membrane was observed with membranes from the wild type strain grown under chemotrophic conditions. Addition of membranes from cells defective in biosynthesis of pigment-binding proteins resulted in a less efficient or less stable incorporation of LHIαβ. The single polypeptides LHIα or β were synthesized and inserted into the membrane but were extractable to a higher percentage by 6 M urea than the pairwise inserted LHI polypeptides.

If the ribosomes and the S135 extract were depleted of DnaK the rate of synthesis of both polypeptides, LHIα and β, was strongly reduced. Removal of GroEL from the cell-free system did not impair the synthesis and membrane association of both proteins, but affected the stable insertion. A high percentage of the LHIαβ polypeptides became extractable by 6 M urea if the cell-free system was depleted of GroEL. Addition of GroEL to the cell-free system restored the capacity of stable insertion of both proteins into the membrane. GroEL interacted with LHIα and β before membrane targeting as shown by immunological means.

A protein fraction, which can be removed from the membrane with a low-salt buffer, supported the effective and stable incorporation of LHIαβ into the membrane. It is concluded that the assembly of the LHI complex in the membrane system of R. capsulatus is a multistep process guided and supported by polypeptides located in the cytoplasm and in the membrane. In the cell-free in vitro system not only the correct insertion of the LHI polypeptides but also an assembly with bacteriochlorophyll was observed. BChl was synthesized from δ-amino levulinate in the cell free system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BChl:

bacteriochlorophyll

EDTA:

ethylene diamine tetraacetate

LH:

light-harvesting

PMSF:

phenyl methylsulfonyl fluoride

PR:

protein-rich fraction

RC:

reaction center

R. :

Rhodobacter

SDS:

sodium dodecylsulfate

PAGE:

polyacrylamide gel electrophoresis

TCA:

trichloracetic acid

References

  • Bustamante PL and Loach PA (1994) Reconstitution of a functional photosynthetic receptor complex with isolated subunits of core light-harvesting complex and reaction centers. Biochemistry 33: 13329–13339

    PubMed  Google Scholar 

  • Chang MC, Callahan PM, Parkes-Loach PS, Cotton TM and Loach PA (1990) Spectroscopic characterization of the light-harvesting complex of Rhodospirillum rubrum and its structural subunit. Biochemistry 29: 421–429

    PubMed  Google Scholar 

  • Dierstein R and Drews G (1986) Effect of uncoupler on assembly pathway for pigment-binding protein of bacterial photosynthetic membranes. J Bacteriol 168: 167–172

    PubMed  Google Scholar 

  • Dierstein R, Schumacher A and Drews G (1981) On insertion of pigment-associated polypeptides during membrane biogenesis in Rhodopseudomonas capsulata. Arch Microbiol 128: 376–383

    Google Scholar 

  • Dörge B, Klug G, Gad'on N, Cohen SN and Drews G (1990) Effects on the formation of antenna complex B870 of Rhodobacter capsulatus by exchange of charged amino acids in the N-terminal domain of the α and β pigment-binding proteins. Biochemistry 29: 7754–7758

    PubMed  Google Scholar 

  • Drews G (1985) Structure and functional organization of light-harvesting complexes and photochemical reaction centers in membranes of phototrophic bacteria. Microbiol Rev 49: 59–70

    PubMed  Google Scholar 

  • Drews G (1991) Regulated development of the photo synthetic apparatus in anoxygenic bacteria. In: Bogorad L and Vasil IK (eds) The Photosynthetic Apparatus: Molecular Biology and Operation, pp 113–148. Academic Press, New York

    Google Scholar 

  • Fujiki Y, Hubbard AL, Fowler S and Lazarow PB (1982) Isolation of intracellular membranes by means of sodium carbonate treatment. J Cell Biol 93: 97–102

    Article  PubMed  Google Scholar 

  • Garcia AF, Drews G and Reidl HH (1981) Comparative studies of two membrane fractions isolated from chemotrophically and phototrophically grown cells of Rhodoseudomonas capsulata. J Bacteriol 145: 1121–1128

    PubMed  Google Scholar 

  • Garcia AF, Meryandini A, Brand M, Tadros MH and Drews G (1994) Phosphorylation of the α and β polypeptides of the light-haruesting complex I (B870) of Rhodobacter capsulatus. FEMS Microbiol Lett 124: 87–92

    Article  Google Scholar 

  • Ghosh R, Hauser H and Bachofen R (1988) Reversible dissociation of the B873 light-harvesting complex from Rhodospirillum rubrum G9. Biochemistry 27: 1004–1014

    Google Scholar 

  • Gilmore R and Blobel G (1985) Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell 42: 497–505

    Article  PubMed  Google Scholar 

  • Gragerov A, Zeng L, Zhao X, Burkholder W and Gottesman ME (1994) Specifity of DnaK-peptide binding. J Mol Biol 235: 848–854

    Article  PubMed  Google Scholar 

  • Hartl FU, Hlodan R and Langer T (1994) Molecular chaperones in protein folding: the art of avoiding sticky situations. Trends Biochem Sci 19: 20–25

    Article  PubMed  Google Scholar 

  • Hendrick JP, Langer T, Davis TA, Hartl FU and Wudmann M (1993) Control of folding and membrane translocation by binding of the chaperone DnaJ to nascent polypeptides. Proc Natl Acad Sci USA 90: 10216–10220

    PubMed  Google Scholar 

  • Hornberger U, Liebetanz R, Tichy HV and Drews G (1990) Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain. Mol Gen Genet 221: 371–378

    Article  PubMed  Google Scholar 

  • Hunter CN, Pennoyer JD, Sturgis JN, Farrelly D, Niederman RA (1988) Oligomerization states and association of light-harvesting pigment-protein complexes of Rhodobacter sphaeroides as analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry 27: 3459–3467

    Google Scholar 

  • Hübner P, Dame G, Sandmeier U, Vandekerkchove J, Beyer P and Tadros MH (1995) Molecular analysis of the Rhodobacter capsulatus chaperonin (cpn) operon, purification and characterization of CPN6O. Arch Microbiol (in press)

  • Karrasch S, Bullough PA and Ghosh R (1995) The 8.5 Å projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMO J 14: 631–638

    Google Scholar 

  • Langer T, Lu C, Echols H, Flanagan HK and Hartl FU (1992) Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature 356: 683–689

    Article  PubMed  Google Scholar 

  • Inamine GS, vanHouten J and Neiderman RA (1984) Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides. J Bacteriol 158: 425–429

    PubMed  Google Scholar 

  • Kaufmann N, Reidl HH, Golecki JR, Garcia AF and Drews G (1982) Differentiation of the membrane system in cells of Rhodopseudomonas capsulata after transition from the chemotrophic to phototrophic growth conditions. Arch Microbiol 131: 313–323

    Google Scholar 

  • Kaplan S, Cain BD, Donohue TJ, Shepherd WD and Yen GSL (1983) Biosynthesis of the photosynthetic membranes of Rhodopseu-domonas sphaeroides. J Cell Biochem 22: 15–29

    PubMed  Google Scholar 

  • Klug G (1993) Regulation of expression of photosynthesis genes in anoxygenic photosynthetic bacteria. Arch Microbiol 159: 397–404

    Article  PubMed  Google Scholar 

  • Klug G, Liebetanz R and Drews G (1986) The influence of bacteriochlorophyll biosynthesis on formation of pigment-binding proteins and assembly of pigment protein complexes in Rhodopseu-domonas capsulata. Arch Microbiol 146: 284–291

    Google Scholar 

  • Meadow KA, Iida K, Tsuda K, Recchia PA, Heller BA, Antonio B, Nango M and Loach PA (1995) Enzymatic and chemical cleavage of the core light-harvesting polypeptides of photosynthetic bacteria: determination of the minimal polypeptide size and structure required for subunit and light-harvesting complex formation. Biochemistry 34: 1559–1574

    PubMed  Google Scholar 

  • Mc Dermott G, Prince SM, Freer AA, Hawthornwaite-Lawless AM, Papiz MZ, Cogdell RJ and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374: 517–521

    Article  Google Scholar 

  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K and Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl Acids Res 12: 7035–7056

    PubMed  Google Scholar 

  • Miller JF, Hinchigere SB, Parkes-Loach PS, Callahan PM, Sprinkle JR, Riccobono JR and Loach PA (1987) Isolation and characterization of a subunit form of the light-harvesting complex ofRhodospirillum rubrum. Biochemistry 26: 5055–5062

    PubMed  Google Scholar 

  • Müller M and Mac Farlane J (1994) Membrane assembly in bacteria. In: Maddy AH and Harris JR (eds) Membrane Biogenesis, Subcellular Biochemistry, Vol 22, pp 327–359. Plenum Press, New York

    Google Scholar 

  • Richter P and Drews G (1991) Incorporation of light-harvesting complex I α and β polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus. J Bacteriol 173: 5336–5345

    PubMed  Google Scholar 

  • Richter P, Cortez N and Drews G (1991) Possible role of the highly conserved amino acids Trp-8 and Pro-13 in the N-terminal segment of the pigment-binding polypeptide LHIα of Rhodobacter capsulatus. FEBS Lett 285: 80–84

    Article  PubMed  Google Scholar 

  • Richter P, Brand M and Drews G (1992) Characterization of LHI− and LHI+ Rhodobacter capsulatus pufA mutants. J Bacteriol 174: 3030–3041

    PubMed  Google Scholar 

  • Schrägger H and vonJagow G (1987) Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379

    PubMed  Google Scholar 

  • Stiehle H, Cortez N, Klug G and Drews G (1990) A negative charged N-terminus in the α polypeptide inhibited formation of the light-harvesting complex I in Rhodobacter capsulatus. J Bacteriol 172: 7121–7137

    Google Scholar 

  • Szabo A, Langer T, Schröder H, Flanagan J, Bukau B and Hartl FU (1994) The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp 70 system-DnaK, DnaJ, and GrpE. Proc Natl Acad Sci USA 91: 10345–10349

    PubMed  Google Scholar 

  • Tadros MH, Frank R, Dörge B, Gad'on N, Takemoto JY and Drews G (1987) Orientation of the B800–850, B870 and reaction center polypeptides on the cytoplasmic and periplasmic surfaces of Rhodobacter capsulatus membranes. Biochemistry 26: 7680–7687

    Google Scholar 

  • Troschel D and Müller M (1990) Development of a cell-free system to study the membrane assembly of photosynthetic proteins of Rhodobacter capsulatus. J Cell Biol 111: 87–94

    Article  PubMed  Google Scholar 

  • vonHeijne G (1994) Membrane proteins: from sequence to structure. Annu Rev Biophys Biomol Struct 23: 167–192

    Article  PubMed  Google Scholar 

  • Yang Z and Bauer CE (1990) Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway. J Bacteriol 172: 5001–5010

    PubMed  Google Scholar 

  • Youvan DC, Ismail S and Bylina EJ (1985) Chromosomal deletion and plasmid complementation of the photosynthetic reaction center and light-harvesting genes from Rhodopseudomonas capsulata. Gene 38: 19–30

    Article  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Anja Meryandini

    Present address: IPB, Raya Pajajaran 41, Bogor, Indonesia

Authors and Affiliations

  1. Institute of Biology 2, Microbiology, Albert-Ludwigs-University, Schaenzlestrasse 1, D-79104, Freiburg, Germany

    Anja Meryandini & Gerhart Drews

Authors
  1. Anja Meryandini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerhart Drews
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meryandini, A., Drews, G. Import and assembly of the α and β-polypeptides of the light-harvesting complex I (B870) in the membrane system of Rhodobacter capsulatus investigated in an in vitro translation system. Photosynth Res 47, 21–31 (1996). https://doi.org/10.1007/BF00017750

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00017750

Key words

Search

Navigation