Requirements for a conservative protein translocation pathway in chloroplasts

Vojta, Lea; Soll, Jürgen; Bölter, Bettina

doi:10.1016/j.febslet.2007.05.004

Cited by 34 publications

(31 citation statements)

References 18 publications

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“…Another newly imported protein, the Rieske iron-sulfur protein, was recovered in a stromal Hsp70-containing protein complex (Madueno et al, 1993). More recently, stromal Hsp70 has been observed in contact with importing proteins belonging to a subset of Toc/Tic substrates (Vojta et al, 2007). These latter data suggest that stromal Hsp70s might be involved in protein import.…”

Section: Introductionmentioning

confidence: 54%

A Stromal Heat Shock Protein 70 System Functions in Protein Import into Chloroplasts in the Moss Physcomitrella patens

2010

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Heat shock protein 70s (Hsp70s) are encoded by a multigene family and are located in different cellular compartments. They have broad-ranging functions, including involvement in protein trafficking, prevention of protein aggregation, and assistance in protein folding. Hsp70s work together with their cochaperones, J domain proteins and nucleotide exchange factors (e.g., GrpEs), in a functional cycle of substrate binding and release accompanied by ATP hydrolysis. We have taken advantage of the gene targeting capability of the moss Physcomitrella patens to investigate the functions of chloroplast Hsp70s. We identified four Hsp70 genes and two GrpE cochaperone homolog genes (CGE) in moss that encode chloroplast proteins. Disruption of one of the Hsp70 genes, that for Hsp70-2, caused lethality, and protein import into heat-shocked chloroplasts isolated from temperature-sensitive hsp70-2 mutants was appreciably impaired. Whereas the double cge null mutant was not viable, we recovered a cge1 null/cge2 knock down mutant in which Hsp70-2 was upregulated. Chloroplasts isolated from this mutant demonstrated a defect in protein import. In addition, two different precursors staged as early import intermediates could be immunoprecipitated with an Hsp70-2-specific antibody. This immunoprecipitate also contained Hsp93 and Tic40, indicating that it represents a precursor still in the Toc/Tic translocon. Together, these data indicate that a stromal Hsp70 system plays a crucial role in protein import into chloroplasts.

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Section: Introductionmentioning

confidence: 54%

A Stromal Heat Shock Protein 70 System Functions in Protein Import into Chloroplasts in the Moss Physcomitrella patens

2010

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“…Hsp93 in the stroma is suggested to function primarily as the regulatory chaperone for the ClpP proteases and/or as an independent chaperone for general protein folding/unfolding and assembly (Adam and Clarke, 2002). Hsp93 has also been proposed to be involved in photosystem biogenesis (Sjogren et al, 2004) and in insertion of Tic110 from the stroma (Vojta et al, 2007). The lethality of the hsp93-III hsp93-V and the cphsc70-1 cphsc70-2 double mutants could be caused by failure to carry out these functions.…”

Section: Discussionmentioning

confidence: 99%

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

2010

The Plant Cell

166

185

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Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.

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“…This is somewhat difficult to understand mechanistically because ATP, GTP, UTP, and CTP all produced comparable stimulation of integration. The NTP requirement needs to be investigated rigorously, especially as Vojta et al (2007) reported an ATP requirement for TIC110 insertion during a chloroplast import assay. Nevertheless, all of these studies consistently point to integration machinery involved in TIC40 integration.…”

Section: Discussionmentioning

confidence: 99%

“…In the stop-transfer pathway the substrate's TM stops its transfer at the TOC complex during plastid import (Froehlich and Keegstra, 2011). In the post-import pathway, proteins are initially imported across the TOC/TIC before insertion into the inner envelope membrane from the stromal side (Li and Schnell, 2006;Tripp et al, 2007;Vojta et al, 2007). This latter pathway suggests a conservative sorting mechanism and implies that a conserved traslocase is present in the inner envelope membrane.…”

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confidence: 99%

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase

Martin

Aldama

et al. 2017

Plant Physiol.

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Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40.

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Requirements for a conservative protein translocation pathway in chloroplasts

Cited by 34 publications

References 18 publications

A Stromal Heat Shock Protein 70 System Functions in Protein Import into Chloroplasts in the Moss Physcomitrella patens

A Stromal Heat Shock Protein 70 System Functions in Protein Import into Chloroplasts in the Moss Physcomitrella patens

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase

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