Partial purification and properties of enzymes involved in the processing of a chloroplast import protein from <i>Chlamydomonas reinhardii</i>

Su, Qingxiang; Boschetti, Arminio

doi:10.1111/j.1432-1033.1993.tb18335.x

Cited by 28 publications

(24 citation statements)

References 37 publications

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“…EUKARYOT. CELL have previously been described in plants and algae (20,39,48), but a consensus cleavage motif remains elusive, possibly because several chloroplast peptidases may recognize different cleavage sites (12,22). The role of TP cleavage in plastid import is not clearly understood, but the down-regulation of a chloroplast-processing enzyme by antisense technology has been correlated with an inhibition of protein import into the chloroplast (55), consistent with our observation that the abolition of processing is correlated with protein localization to the apicoplast periphery.…”

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confidence: 99%

Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

et al. 2004

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Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle-the apicoplastacquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP ؉ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.

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confidence: 99%

Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

et al. 2004

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“…The 0.8 kb PvuII-Pstl fragment of the resulting plasmid was ligated with the 2.9 kb PvuW-Pstl fragment of plasmid pSSpSP64, finally leading to plasmid pSSHispSP64. The in vitro transcript obtained from this plasmid served to synthesize in vitro precursor proteins of which the last amino acid (vai) was replaced by the hexahistidyl tail [17]. All operations were essentially performed according to Maniatis et al [18].…”

Section: Construction Of Plasmicimentioning

confidence: 99%

“…This neglect is probably due to the tedious isolation procedure for chloroplasts and to the fact that precursor proteins from higher plants are not imported or not correctly processed by Chlamydomonas chloroplasts, and vice versa [14].…”

Section: Introductionmentioning

confidence: 99%

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions

et al. 1997

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The precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and a modified pSS containing a C-terminal hexahistidyl tail (pSS(His) 6 ) were imported into isolated Chlamydomonas chloroplasts with comparable efficiency. In the presence of Ni 2+ ions the import of pSS(His) 6 was inhibited and the precursor bound to the envelope remained protease sensitive, while import of pSS was not affected. Addition of an excess of i -histidine suppressed the inhibition demonstrating that the hexahistidyl-Ni 2+ complex was responsible for import inhibition. Inhibition could be observed between about 0.5 and 10 mM Ni 2+ , depending on the total protein content in the assay. Import incompetent Ni 2+ -precursor complexes can be used to study early events in chloroplast protein import.© 1997 Federation of European Biochemical Societies.

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“…The plasmid pQE-12 (Qiagen GmbH) which contained the hexahistidyl-coding sequence was cut with BglII, blunted with Klenow enzyme and cut with EcoRI. amino acid (val) was replaced by the hexahistidyl tail [17]. All operations were essentially done according to Maniatis et al [18].…”

Section: Construction Of Plasmidmentioning

confidence: 99%

“…Not much attention has so far been paid to the protein import into chloroplasts of Chlamydomonas reinhardii, although this unicellular green alga is widely used as a model organism to investigate various cell biological problems of green plants [13]. This neglect is probably due to the tedious isolation procedure for chloroplasts and to the fact that precursor proteins from higher plants are not imported or not correctly processed by Chlamydomonas chloroplasts, and vice versa [14].…”

Section: Introductionmentioning

confidence: 99%

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions

Rothen¹,

Thiess²,

Schumann³

et al. 1996

FEBS Letters

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The precursor of the small subunit of ribulose-l,5-bisphosphate carboxylase (pSS) and a modified pSS containing a C-terminal hexahistidyl tail (pSS(His)6) were imported into isolated Chlamydomonas chloroplasts with comparable efficiency. In the presence of Ni 2+ ions the import of pSS(His)6 was inhibited and the precursor bound to the envelope remained protease sensitive, while import of pSS was not affected. Addition of an excess of L-histidine suppressed the inhibition demonstrating that the hexahistidyl-Ni 2+ complex was responsible for import inhibition. Inhibition could be observed between about 0.5 and l0 mM Ni ~+, depending on the total protein content in the assay. Import incompetent Ni2+-precursor complexes can be used to study early events in chloroplast protein import.

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Partial purification and properties of enzymes involved in the processing of a chloroplast import protein from Chlamydomonas reinhardii

Cited by 28 publications

References 37 publications

Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions

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Partial purification and properties of enzymes involved in the processing of a chloroplast import protein from Chlamydomonas reinhardii

Cited by 28 publications

References 37 publications

Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni2+ ions

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni2+ ions

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Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions

Import inhibition of poly(His) containing chloroplast precursor proteins by Ni²⁺ ions