Abstract
A 7.4 kb segment of a Euglena nuclear gene encoding a portion of the polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) has been isolated and sequenced. Nine exons can be assembled into a continuous open reading frame encoding 113 amino acids of the C-terminus of an LHCPII, followed by 4 complete LHCPIIs. The intron-exon junctions required to maintain maximum amino acid sequence homology between the LHCPIIs encoded by the Euglena genomic clone and Arabidopsis LHCPII do not conform to the consensus splice site sequences present in most eukaryotic organisms. Three types of LHCPIIs are contained within the polyprotein precursor. There is greater than 90% sequence identity at both the protein and nucleic acid level within a type while between types, there is less than 65% sequence identity. All Euglena LHCPIIs contain three hydrophobic membrane-spanning domains in the same positions as found in other LHCPIIs. Hybridization of genomic Southern blots with a probe encoding LHCPII suggests that the Euglena genome encodes a large number (30–50) of LHCPIIs. A probe derived from the 3′ end of the sequenced genomic clone hybridizes with equal intensity to 2–3 genomic fragments on Southern blots. The probe encoding LHCPII hybridizes to RNAs of 9.5 and 6.6 kb on northern blots of total RNA while the 3′-end probe hybridizes only to the 6.6 kb RNA. The 6.6 kb LHCPII band detected on northern blots appears to be composed of transcription products derived from 2–3 LHCPII genes. Euglena appears to be unique in that a large number of LHCPIIs are encoded by a small number (3–5) of polyprotein genes.
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Benton WD, Davis RW: Screening λgt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182 (1977).
Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7: 1513–1523 (1979).
Brumbaugh JA, Middendorf LR, Grone DL, Ruth JL: Continuous on-line DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores. Proc Natl Acad Sci USA 85: 5610–5614 (1988).
Buetow DE, Chen H, Erdos G, Yi LSH: Regulation and expression of the multigene family coding light-harvesting chlorophyll a/b-binding proteins of photosystem II. Photosynth Res 18: 61–97 (1988).
Carrington JC, Freed DD, Oh C-S: Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing. EMBO J 9: 1347–1353 (1990).
Chan RL, Keller M, Canaday J, Weil J-H, Imbault P: Eight small subunits of Euglena ribulose 1,5-bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein. EMBO J 9: 333–338 (1990).
Chitnis PR, Thornber JP: The major light-harvesting complex of photosystem II: aspects of its molecular and cell biology. Photosynth Res 16: 41–63 (1988).
Csank C, Taylor FM, Martindale DW. Nuclear pre-mRNA introns: analysis and comparison of intron sequences from Tetrahymena thermophila and other eucaryotes. Nucl Acids Res 18: 5133–5141 (1990).
Cunningham FXJr, Schiff JA: Chlorophyll-protein complexes from Euglena gracilis and mutants deficient in chlorophyll b. I. Pigment composition. Plant Physiol 80: 223–230 (1986).
Cunningham FXJr, Schiff JA: Chlorophyll-protein complexes from Euglena gracilis and mutants deficient in chlorophyll b. II. Polypeptide composition. Plant Physiol 80: 231–238 (1986).
Curtis SE, Rawson JRY: Characterization of the nuclear ribosomal DNA of Euglena gracilis. Gene 15: 237–247 (1981).
Darr SC, Somerville SC, Arntzen CJ: Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II. J Cell Biol 103: 733–740 (1986).
Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).
Douglass J, Civelli O, Herbert E: Polyprotein gene expression: Generation of diversity of neuroendocrine peptides. Annu Rev Biochem 53: 665–715 (1984).
Dubertret G, Lefort-Tran M: Chloroplast molecular structure with particular reference to thylakoids and envelopes. In: Buetow DE (ed) The Biology of Euglena, vol 3, pp. 253–312. Academic Press, New York (1982).
Gibbs SP: The comparative ultrastructure of the algal chloroplast. Ann NY Acad Sci 175: 454–473 (1970).
Hellen CUT, Krausslich H-G, Wimmer E: Proteolytic processing of polyproteins in the replication of RNA viruses. Biochemistry 28: 9881–9890 (1989).
Houlne G, Schantz R: Molecular analysis of the transcripts encoding the light-harvesting chlorophyll a/b proteins in Euglena gracilis: unusual size of the mRNA. Curr Genet 12: 611–616 (1987).
Houlne G, Schantz R: Characterization of cDNA sequences for LHCI apoproteins in Euglena gracilis: The mRNA encodes a large precursor containing several consecutive divergent polypeptides. Mol Gen Genet 213: 479–486 (1988).
Hurtley SM, Helenius A: Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol 5: 277–307 (1989).
Imbault P, Wittemer C, Johanningmeier U, Jacobs JD, Howell SH: Structure of the Chlamydomonas reinhardtii cabII-1 gene encoding a chlorophyll a/b-binding protein. Gene 73: 397–407 (1988).
Jansson S, Gustafsson P: Type I and type II genes for the chlorophyll a/b-binding protein in the gymnosperm Pinus sylvestris (scots pine): cDNA cloning and sequence analysis. Plant Mol Biol 14: 287–296 (1990).
Jansson S, Selstam E, Gustafsson P: The rapidly phosphorylated 25 kDa polypeptide of the light-harvesting complex of photosystem II is encoded by the type 2 cab-II genes. Biochim Biophys Acta 1019: 110–114 (1990).
Karlin-Neumann GA, Kohorn BD, Thornber JP, Tobin EM: A chlorophyll a/b-protein encoded by a gene containing an intron with characteristics of a transposable element. J Mol Appl Genet 3: 45–61 (1985).
Keegstra K, Olsen LJ, Theg SM: Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Physiol Plant Mol Biol 40: 471–501 (1989).
Ko K, Cashmore AR: Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 Kd oxygen-evolving protein. EMBO J 8: 3187–3194 (1989).
Kohorn BD, Harel E, Chitnis PR, Thornber JP, Tobin EM: Functional and mutational analysis of the light-harvesting chlorophyll a/b protein of thylakoid membranes. J Cell Biol 102: 972–981 (1986).
Krausslich HG, Wimmer E: Viral proteinases. Annu Rev Biochem 57: 701–754 (1988).
Kuhlbrandt W, Wang DN: Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature 350: 130–134 (1991).
LaRoche, Bennet J, Falkowski PG: Characterization of cDNA encoding for the 28.5 kDa LHCII apoprotein from the unicellular marine chlorophyte Dunaliella tertiolecta. Gene 95: 165–171 (1990).
Leutwiler LS, Meyerowitz EM, Tobin EM: Structure and expression of three light-harvesting chlorophyll a/b-binding protein genes in Arabidopsis thaliana. Nucl Acids Res 14: 4051–4064 (1986).
Liljestrom P, Garoff H: Internally located cleavable signal sequences direct the formation of Semliki Forest Virus membrane proteins from a polyprotein precursor. J Virol 65: 147–154 (1991).
Long Z, Wang SY, Nelson N: Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina. Gene 76: 299–312 (1989).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
McCarthy SA, Schwartzbach SD: Absence of photoregulation of abundant mRNA levels in Euglena. Plant Sci Lett 35: 61–66 (1984).
Morishige DT, Thornber JP: The major light-harvesting chlorophyll a/b protein (LHC IIb): The smallest subunit is a novel cab gene product. In: Baltscheffsky M (ed) Current Research in Photosynthesis, vol. 2, pp. 261–264. Kluwer, Dordrecht (1990).
Muchhal US, Schwartzbach SD: Characterization of a Euglena chlorophyll a/b binding protein (LHCPII) gene encoding a polyprotein precursor. Plant Physiol 93: S244 (1990).
Mullet JE: The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplast. J Biol Chem 258: 9941–9948 (1983).
Olson TS, Lane MD: Post-translational acquisition of insulin binding activity by the insulin proreceptor. J Biol Chem 262: 6816–6822 (1987).
Osafune T, Schiff JA, Hase E: Immunogold localization of LHCPII apoprotein in the golgi of Euglena. Cell Struc Funct 15: 99–105 (1990).
Payan LA, Cline K: A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein. J Cell Biol 112: 603–613 (1991).
Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448 (1988).
Perez LG, Hunter E: Mutations within the proteolytic cleavage site of the Rous Sarcoma virus glycoprotein that block processing to gp85 and gp37. J Virol 61: 1609–1614 (1987).
Pichersky E, Hoffman NE, Malik VS, Bernatzky R, Tanksley SD, Szabo L, Cashmore AR: The tomato cab-4 and cab-5 genes encode a second type of cab polypeptides located in photosystem II. Plant Mol Biol 9: 109–120 (1987).
Rikin A, Schwartzbach SD: Extremely large and slowly processed precursors to the Euglena light-harvesting chlorophyll a/b binding proteins of photosystem II. Proc Natl Acad Sci USA 85: 5117–5121 (1988).
Schiff JA, Schwartzbach SD, Osafune T, Hase E: Photocontrol and processing of LHCPII apoprotein in Euglena: Possible role of golgi and other cytoplasmic sites. J Photochem Photobiol Biol Sect (in press).
Schmidt GW, Mishkind ML: The transport of proteins into chloroplasts. Annu Rev Biochem 55: 879–891 (1986).
Smeekens S, Bauerle C, Hageman J, Keegstra K, Weisbeek P: The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell 46: 365–375 (1986).
Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517 (1975).
Stayton MM, Black M, Bedbrook J, Dunsmuir P: A novel chlorophyll a/b binding (Cab) protein gene from petunia which encodes the lower molecular weight Cab precursor protein. Nucl Acids Res 14: 9781–9796 (1986).
von Heijne G: Signal sequences. The limits of variation. J Mol Biol 184: 99–105 (1985).
von Heijne G: A new method for predicting signal sequence cleavage sites. Nucl Acids Res 14: 4683–4690 (1986).
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Muchhal, U.S., Schwartzbach, S.D. Characterization of a Euglena gene encoding a polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II. Plant Mol Biol 18, 287–299 (1992). https://doi.org/10.1007/BF00034956
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DOI: https://doi.org/10.1007/BF00034956