A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

Tranel, Patrick J.; Froehlich, John E.; Goyal, Arun; Keegstra, Kenneth

doi:10.1002/j.1460-2075.1995.tb07241.x

Cited by 208 publications

(244 citation statements)

References 40 publications

(53 reference statements)

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“…Import with wheat germ-translated 35 S-prSS was performed as described by Tranel et al (14). The imported proteins were analyzed by SDS-PAGE and fluorography.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Translocation Intermediate Occupying Functional Protein Import Sites in the Chloroplastic Envelope Membrane

Froehlich

Keegstra

1997

Journal of Biological Chemistry

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We used complexes of avidin and biotinylated precursors to generate translocation intermediates that occupy functional transport sites and thereby block the transport of other precursor proteins into pea chloroplasts. Cysteine residues of purified precursor to the small subunit of rubisco (prSS) were modified with the biotinylation reagent biotin-1-biotinamido-4-[-4-(maleimidomethyl)-cyclohexane-carboxamido]butane. Chemically biotinylated prSS was readily imported into chloroplasts. The addition of avidin, however, resulted in the formation of an avidin-biotinylated precursor complex that could not be imported into chloroplasts even when precursors had already engaged the transport apparatus before avidin was added. On fractionation, the avidin-biotinylated precursor complex associated with envelope membranes. Titration of transport sites with avidin-biotinylated precursor complexes revealed that saturation was reached at 2,000 molecules/chloroplast. Even with less than saturating levels of complexes, a sufficient number of translocation sites could be occupied with avidin-precursor complexes so that the import rate of freshly added radiolabeled prSS was reduced by 35%. From these observations we conclude that the trapped intermediates were blocking functional translocation sites. These biotinylated translocation intermediates should be useful in future efforts to purify and characterize the chloroplastic protein import machinery.

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“…Import with wheat germ-translated 35 S-prSS was performed as described by Tranel et al (14). The imported proteins were analyzed by SDS-PAGE and fluorography.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Translocation Intermediate Occupying Functional Protein Import Sites in the Chloroplastic Envelope Membrane

Froehlich

Keegstra

1997

Journal of Biological Chemistry

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“…For control assays, an equal number of chloroplasts were resuspended in resuspension buffer without ATP and the ATP regeneration system and incubated at 22°C in the dark. An equal amount of chloroplasts were taken at 0, 1.5, and 3 h and analyzed by SDS-PAGE and immunoblotting using antibodies against Toc75 (Tranel et al, 1995) and GS2 (Agrisera; AS08 296). The primary antibodies were used at 1:4,000 and 1:5,000 dilutions for Toc75 and GS2, respectively.…”

Section: In Organello Proteolytic Assaymentioning

confidence: 99%

The Amino-Terminal Domain of Chloroplast Hsp93 Is Important for Its Membrane Association and Functions in Vivo

Chu

2012

Plant Physiology

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Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-DN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-DN proteins did not associate with membranes and atHsp93V-DN failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-DN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-DN transgenic plants. Hsp93-DN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.Chloroplasts are structurally complex organelles that perform diverse functions (Leister, 2003;Block et al., 2007). They are composed of three membranes, the outer and inner envelope membranes and the thylakoid membranes, and these membranes enclose three aqueous compartments, the intermembrane space, stroma, and thylakoid lumen. Although chloroplasts have their own genome, most chloroplast proteins are encoded by the nuclear genome and are translated in the cytosol as a precursor protein with an N-terminal extension called the transit peptide. Transit peptides direct the import of proteins into chloroplasts through the translocon complex located in the chloroplast envelope. Translocon components of the outer membrane are called Toc (for translocon at the outer envelope membrane of chloroplasts) proteins and those in the inner envelope membrane are called Tic (for translocon at the inner envelope membrane of chloroplasts) proteins. Three Toc components, Toc159, Toc34, and Toc75, form the Toc core complex. Toc159 and Toc34 function as receptors that recognize the precursors targeting to chloroplasts. Toc75 forms a protein-conducting channel across the outer membrane. Three proteins, Tic20, Tic21, and Tic110, have been suggested to function as the channel for precursor translocation across the inner membrane. Tic110 has also been shown to function as the stromal-side receptor for transit peptides and as a scaffold for assembling other stromal translocon components. Tic40 is a cochaperone that coordinates the actions of...

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“…Import reactions were performed as described by Bruce et al (1994). Import and extraction experiments were performed as described previously by Tranel et al (1995).…”

Section: Immunoblot Analysismentioning

confidence: 99%

Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor CYO1 Is a Protein Disulfide Isomerase

et al. 2007

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Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the albino cotyledon phenotype by complementation. CYO1 has a C 4 -type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons.

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A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

Cited by 208 publications

References 40 publications

Identification of a Translocation Intermediate Occupying Functional Protein Import Sites in the Chloroplastic Envelope Membrane

Identification of a Translocation Intermediate Occupying Functional Protein Import Sites in the Chloroplastic Envelope Membrane

The Amino-Terminal Domain of Chloroplast Hsp93 Is Important for Its Membrane Association and Functions in Vivo

Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor CYO1 Is a Protein Disulfide Isomerase

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