On the mode of integration of the thylakoid membrane protein cytochrome b6 into cytoplasmic membrane of Escherichia coli
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In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b6 into the bacterial membrane. Cytochrome b6 naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b6 or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b6 in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b6 into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b6 devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b6 is possible, as long as the B and D helices bearing axial ligands to heme are present.
- Aldridge C, Cain P, Robinson C (2009) Protein transport in organelles: Protein transport into and across the tylakoid membrane. FEBS J 276: 1177-1186.
- Bendtsen JD, Kiemer L, Fausboll A, Brunak S (2005) Non-classical protein secretion in bacteria. BMC Microbiol 5: 58.
- van der Berg B, Clemons WM, Collinson I, Modis Y, Hartman E, Harrison SC, Rapoport TA (2004) X-ray structure of a protein-conducting channel. Nature 427: 36-44.
- Betton JM, Hofnung M (1996) Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. J Biol Chem 271: 8046-8052.
- Boronowsky U, Wenk S, Schneider D, Jager C, Rogner M (2001) Isolation of membrane protein subunits in their native state: evidence for selective binding of chlorophyll and carotenoid to the b(6) subunit of the cytochrome b(6)f complex. Biochim Biophys Acta 1506: 55-66.
- Cassoly R (1978) Evidence against the binding of native hemoglobin to spectrin of human erythrocytes. FEBS Lett 85: 357-360.
- Choquet Y, Stern DB, Wostrikoff K, Kuras R, Girard-Bascou J, Wollman FA (1998) Translation of cytochrome f is autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas chloroplasts. Proc Natl Acad Sci USA 95: 4380-4385.
- Cline K, Ettinger WF, Theg SM (1992) Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP. J Biol Chem 267: 2688-2696.
- Cline K, Dabney-Smith C (2008) Plastid protein import and sorting: different paths to the same compartments. Curr Opin Plant Biol 11: 585-592.
- Dalbey RE, Kuhn A (2004) YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins. J Cell Biol 166: 769-774.
- Dreher C, Prodohl A, Weber M, Schneider D (2007) Heme binding properties of heterologously expressed spinach cytochrome b(6): implications for transmembrane b22 type cytochrome formation. FEBS Lett 581: 2647-2651.
- Emanuelsson O, von Heijne G (2001) Prediction of organellar targeting signals. Biochim Biophys Acta 1541: 114-119.
- Froderberg L, Houben E, Samuelson JC, Chen M, Park SK, Phillips GJ, Dalbey R, Luirink J, De Gier JW (2003) Versatility of inner membrane protein biogenesis in Escherichia coli. Mol Microbiol 47: 1015-1027.
- von Heijne G (1985) Signal sequences. The limits of variation. J Mol Biol 184: 99-105.
- von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14: 4683-4690.
- Heinemeyer W, Alt J, Herrmann RG (1984) Ncleotide sequence of the clustered genes for apocytochrome b6 and subunit 4 of the cytochrome b6f complex in the spinach plastid chromosome. Curr Genet 8: 543-549.
- Herrmann RG (1982) The preparation of circular DNA from plastids. In Methods in Chloroplast Molecular Biology. Edelman M, Hallick RB, Chua N-H eds, pp 259-279. Elsevier, Amsterdam.
- Hynds PJ, Robinson D, Robinson C (1998) The Sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the tylakoid membrane. J Biol Chem 273: 34868-34874.
- Klee EW, Ellis LB (2005) Evaluating eukaryotic secreted protein prediction. BMC Bioinformatics 6: 256.
- Kroliczewski J, Szczepaniak A (2002) In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli. Biochim Biophys Acta 1598: 177-184.
- Kroliczewski J, Hombek-Urban K, Szczepaniak A (2005) Integration of the thylakoid membrane protein cytochrome b6 in the cytoplasmic membrane of Escherichia coli. Biochemistry 44: 7570-7576.
- Kurisu G, Zhang H, Smith JL, Cramer WA (2003) Structure of the cytochrome b6f complex of oxygenic photosynthesis: tuning the cavity. Science 302: 1009-1014.
- Leclerc E, Leclerc L, Cassoly R, der Terrosian E, Wojcman H, Poyart C, Marden MC (1993) Heme binding to calmodulin, troponin C, and parvalbumin, as a probe of calcium-dependent conformational changes. Arch Biochem Biophys 306: 163-168.
- Marden MC, Hazard ES, Leclerc L, Gibson QH (1989) Flash photolysis of the serum albumin-heme-carbon monoxide complex. Biochemistry 28: 4422-4426.
- Marques JP, Schattat MH, Hause G, Dudeck I, Klosgen RB (2004) In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana. J Exp Bot 55: 1697-1706.
- Nielsen H, Krogh A (1998) Prediction of signal peptides and signal anchors by a hidden Markov model. Proc Int Conf Intell Syst Mol Biol 6: 122-130.
- Nielsen H, Brunak S, von Heijne G (1999) Machine learning approaches for the prediction of signal peptides and other protein sorting signals. Protein Eng 12: 3-9.
- du Plessis DJ, Nouwen N, Driessen AJ (2006) Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion. J Biol Chem 281: 12248-12252.
- Rapoport TA, Jungnickel B, Kutay U (1996) Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu Rev Biochem 65: 271-303.
- Ridge KD, Lee SSJ, Yao LL (1995) In vivo assembly of rhodopsin from expressed polypeptide fragments. Proc Natl Acad Sci USA 92: 3204-3208.
- Ridge KD, Lee SSJ, Abdulaev NG (1996) Examining rhodopsin folding and assembly through expression of polypeptide fragments. J Biol Chem 271: 7860-7867.
- Rochaix JD (1996) Post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Plant Mol Biol 32: 327-341.
- Robinson C, Thompson SJ, Woolhead C (2001) Multiple pathways used for the targeting of thylakoid proteins in chloroplasts. Traffic 2: 245-251.
- Rothstein R, Gatenby AA, Willey DL, Gray JC (1985) Binding of pea cytochrome f to inner membrane of Escherichia coli requires the bacterial secA gene product. Proc Natl Acad Sci USA 82: 7955-7959.
- Roy LM, Barkan A (1998) A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression. J Cell Biol 141: 385-395.
- Saaf A, Wallin E, von Heijne G (1998) Stop-transfer function of pseudo-random amino acid segments during translocation across prokaryotic and eukaryotic membranes. Eur J Biochem 251: 821-829.
- Schatz G, Dobberstein B (1996) Common principles of protein translocation across membranes. Science 271: 1519-1526.
- Schuenemann D (2007) Mechanisms of protein import into tylakoids of chloroplasts. Biol Chem 388: 907-915.
- Schuenemann D, Amin P, Hartman E, Hoffman NE (1999) Chloroplast SecY is complexed to SecE and involved in the translocation of the 33-kDa but not the 23-kDa subunit of the oxygene-evolving complex. J Biol Chem 274: 12177-12182.
- Solar I, Muller-Eberhard U, Shaklai N (1989) Serum proteins as mediators of hemin efflux from red cell membranes: specificity of homeopexin. FEBS Lett 256: 225-229.
- Solar I, Shaklai N (1989) Association of hemin with protein 4.1 as compared to spectrin and actin. Biochim Biophys Acta 983: 199-204.
- Stuart AL, Wasserman AR (1973) Purification of cytochrome b6 a tightly bound protein in chloroplast membranes. Biochim Biophys Acta 314: 284-297.
- Szczepaniak A, Cramer WA (1990) Thylakoid membrane protein topography. Location of the termini of the chloroplast cytochrome b6 on the stromal side of the membrane. J Biol Chem 265: 17720-17726.
- Traxler B, Murphy C (1996) Insertion of the polytopic membrane protein MalF is dependent on the bacterial secterion machinery. J Biol Chem 271: 12394-12400.
- Wallin E, von Heijne G (1998) Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci 7: 1029-1038.
- Woolhead CA, Mant A, Kim SJ, Robinson C, Rodger A (2001) Conformation of a purified 'spontaneously' inserting thylakoid membrane protein precursor in aqueous solvent and detergent micelles. J Biol Chem 276: 14607-14613.
- Yi L, Dalbey RE (2005) Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review). Mol Membr Biol 22: 101-111.
- Yuan J, Henry R, McCaffery M, Cline K (1994) SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting. Science 266: 796-798.
- Zhang L, Paakkarinen V, Suorsa M, Aro EM (2001) A SecY homologue is involved in chloroplast-encoded D1 protein biogenesis. J Biol Chem 276: 37809-37814.
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