Abstract
Abstract We have begun to take a genetic approach to study chloroplast protein import inChlamydomonas reinhardtii by creating deletions in the transit peptide of the γ-subunit of chloroplast ATPase-coupling factor 1 (CF1-γ, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into anatpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-γ accumulation in vivo. The CF1-γ transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-γ.
References
53
Referenced
23
10.1007/BF00042345
/ Plant Mol Biol / Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region. by Archer (1993)10.1016/S0021-9258(17)33973-X
/ J Biol Chem / Mutations at the stromal processing peptidase cleavage site of a thylakoid lumen protein precursor affect the rate of processing, but not the fidelity. by Bassham (1994)10.1104/pp.72.4.1130
/ Plant Physiol / Partial purification of intact chloroplasts from Chlamydomonas reinhardtii. by Belknap (1983)10.1073/pnas.81.7.1991
/ Proc Natl Acad Sci USA / Genomic sequencing. by Church (1984)10.1083/jcb.114.4.681
/ J Cell Biol / Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins. by Clark (1991)10.1146/annurev.cellbio.12.1.1
/ Annu Rev Cell Dev Biol / Import and routing of nucleus-encoded proteins. by Cline (1996)10.1002/j.1460-2075.1991.tb07825.x
/ EMBO J / Protein targeting towards the thylakoid lumen of chloroplasts. Proper localization of fusion proteins is only observed in vivo. by de Boer (1991)10.1016/0304-4157(91)90015-O
/ Biochim Biophys Acta / Chloroplast protein topogenesis: import, sorting, and assembly. by de Boer (1991)10.1002/j.1460-2075.1989.tb08426.x
/ EMBO J / The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. by Debuchy (1989)10.2307/3869540
/ Plant Cell / Evidence for nuclear control of the expression of the atpA and atpB chloroplast genes in Chlamydomonas. by Drapier (1992)10.1002/j.1460-2075.1994.tb06535.x
/ EMBO J / Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering. by Dumas (1994)10.1038/346297a0
/ Nature / Light-based detection of biomolecules. by Durrant (1990)10.1016/0003-2697(83)90418-9
/ Anal Biochem / A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. by Feinberg (1983)10.1073/pnas.86.17.6449
/ Proc Natl Acad Sci USA / Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. by Fernández (1989)10.1016/0014-5793(90)80094-Y
/ FEBS Lett / Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences. by Franzén (1990)10.1146/annurev.bi.58.070189.000551
/ Annu Rev Biochem / ATP synthase (H+-ATPase): results by combined biochemical and molecular biological approaches. by Futai (1989)10.1104/pp.89.1.15
/ Plant Physiol / Preparation of Chlamydomonas chloroplasts for the in vitro import of polypeptide precursors. by Goldschmidt-Clermont (1989)10.1016/S0962-8924(00)89018-2
/ Trends Cell Biol / Protein translocation across chloroplast envelope membranes. by Gray (1995){'key': '2021050315522656800_R19', 'article-title': 'The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use.', 'author': 'Harris', 'year': '1989'}
/ The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. by Harris (1989)10.1111/j.1749-6632.1989.tb25164.x
/ Ann NY Acad Sci / Protons, the thylakoid membrane, and the chloroplast ATP synthase. by Junge (1989)10.1016/0092-8674(89)90898-2
/ Cell / Transport and routing of proteins into chloroplasts. by Keegstra (1989)10.1146/annurev.pp.40.060189.002351
/ Annu Rev Plant Physiol Plant Mol Biol / Chloroplastic precursors and their transport across envelope membranes. by Keegstra (1989)10.1073/pnas.87.3.1228
/ Proc Natl Acad Sci USA / High-frequency nuclear transformation of Chlamydomonas reinhardtii. by Kindle (1990)10.1083/jcb.109.6.2589
/ J Cell Biol / Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. by Kindle (1989)10.1016/0076-6879(91)04008-C
/ Methods Enzymol / Efficient site-directed mutagenesis using uracil-containing DNA. by Kunkel (1991)10.1007/BF00338082
/ Mol Gen Genet / Targeting of proteins to chloroplasts in transgenic tobacco by fusion to mutated transit peptides. by Kuntz (1986)10.1074/jbc.272.33.20357
/ J Biol Chem / Alterations in the Chlamydomonas plastocyanin transit peptide have distinct effects on in vitro import and in vivo protein accumulation. by Lawrence (1997){'key': '2021050315522656800_R28'}
10.1016/S0021-9258(19)38356-5
/ J Biol Chem / Isolation and characterization of a complementary DNA clone for algal pre-apoplastocyanin. by Merchant (1990)10.1104/pp.75.3.781
/ Plant Physiol / Synthesis and turnover of the chloroplast coupling factor 1 in Chlamydomonas reinhardtii. by Merchant (1984)10.1016/S0021-9258(19)84901-3
/ J Biol Chem / Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose bisphosphate caryboxylase. by Ostrem (1989)10.1074/jbc.270.8.3882
/ J Biol Chem / Functional domains of the ferredoxin transit sequence involved in chloroplast import. by Pilon (1995)10.1016/S0021-9258(18)53033-7
/ J Biol Chem / The plastocyanin-deficient phenotype of Chlamydomonas ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin. by Quinn (1993){'key': '2021050315522656800_R34', 'first-page': '623', 'article-title': 'Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators.', 'volume': '7', 'author': 'Quinn', 'year': '1995', 'journal-title': 'Plant Cell'}
/ Plant Cell / Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. by Quinn (1995)10.1007/BF00329845
/ Mol Gen Genet / Regions in the transit peptide of SSU essential for transport into chloroplasts. by Reiss (1987)10.1007/BF00039516
/ Plant Mol Biol / Targeting of proteins into and across the thylakoid membrane—a multitude of mechanisms. by Robinson (1994)10.1016/0092-8674(95)90090-X
/ Cell / Shedding light on the chloroplast protein import machinery. by Schnell (1995)10.1074/jbc.272.24.15264
/ J Biol Chem / Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. by Silva-Filho (1997)10.1128/MCB.11.10.5053
/ Mol Cell Biol / Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the γ-subunit of chloroplast coupling factor 1 (CF1). by Smart (1991)10.1104/pp.97.4.1596
/ Plant Physiol / Nucleotide sequence for the genomic DNA encoding the chloroplast ATPase γ-subunit gene (atpC) from Chlamydomonas reinhardtii. by Smart (1991)10.1007/BF00762588
/ J Bioenerg Biomembr / Complementation of a Chlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor 1 (CF1) γ-subunit (atpC). by Smart (1992)10.1016/0092-8674(86)90657-4
/ Cell / The role of the transit peptide in the routing of precursors toward different chloroplast compartments. by Smeekens (1986)10.1073/pnas.90.19.9199
/ Proc Natl Acad Sci USA / Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. by Sodeinde (1993){'key': '2021050315522656800_R44', 'first-page': '285', 'article-title': 'A 3′ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo.', 'volume': '3', 'author': 'Stern', 'year': '1991', 'journal-title': 'Plant Cell'}
/ Plant Cell / A 3′ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. by Stern (1991){'key': '2021050315522656800_R45', 'first-page': '23', 'article-title': 'The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas.', 'volume': '251', 'author': 'Stevens', 'year': '1996', 'journal-title': 'Mol Gen Genet'}
/ Mol Gen Genet / The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. by Stevens (1996)10.1093/genetics/135.2.375
/ Genetics / The use of DNA insertional mutagenesis to clone genes in Chlamydomonas. by Tam (1993)10.1021/bi00136a018
/ Biochemistry / Biophysical characterization of a transit peptide directing chloroplast protein import. by Theg (1992)10.1016/0962-8924(93)90212-J
/ Trends Cell Biol / Protein import into chloroplasts. by Theg (1993)10.1111/j.1432-1033.1989.tb14679.x
/ Eur J Biochem / Domain structure of mitochondrial and chloroplast targeting peptides. by von Heijne (1989)10.1007/BF00338081
/ Mol Gen Genet / The importance of the transit peptide and the transported protein for protein import into chloroplasts. by Wasmann (1986)10.1016/S0021-9258(19)35396-7
/ J Biol Chem / Complete processing of a small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase from pea requires the amino acid sequence ile-thr-ser. by Wasmann (1988)10.1073/pnas.85.5.1369
/ Proc Natl Acad Sci USA / Isolation of a complementary DNA clone for the gamma subunit of the chloroplast ATPase of Chlamydomonas reinhardtii. Import and cleavage of the precursor protein. by Yu (1988)10.1016/S0021-9258(19)77639-X
/ J Biol Chem / cDNA sequence and predicted primary structure of the γ subunit from the ATP synthase from Chlamydomonas reinhardtii. by Yu (1988)
Dates
Type | When |
---|---|
Created | 23 years, 1 month ago (July 27, 2002, 1 a.m.) |
Deposited | 4 years, 3 months ago (May 3, 2021, 11:53 a.m.) |
Indexed | 3 weeks, 2 days ago (Aug. 5, 2025, 8:23 a.m.) |
Issued | 27 years, 5 months ago (March 1, 1998) |
Published | 27 years, 5 months ago (March 1, 1998) |
Published Online | 27 years, 5 months ago (March 1, 1998) |
Published Print | 27 years, 5 months ago (March 1, 1998) |
@article{Kindle_1998, title={Transit Peptide Mutations That Impair in Vitro and in Vivo Chloroplast Protein Import Do Not Affect Accumulation of the γ-Subunit of Chloroplast ATPase1}, volume={116}, ISSN={0032-0889}, url={http://dx.doi.org/10.1104/pp.116.3.1179}, DOI={10.1104/pp.116.3.1179}, number={3}, journal={Plant Physiology}, publisher={Oxford University Press (OUP)}, author={Kindle, Karen L. and Lawrence, Susan D.}, year={1998}, month=mar, pages={1179–1190} }