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Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways.

Mori H, Summer EJ, Ma X, Cline K - J. Cell Biol. (1999)

Bottom Line: Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates.This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates.These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611, USA.

ABSTRACT
Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in approximately 10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

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cDNAs for pea cpSecY and psTha4 encode precursor proteins that are imported into chloroplasts and localized to thylakoids. (A) In vitro synthesized pea cpSecY and psTha4 were incubated with intact chloroplasts in the presence of ATP for 10 min. Chloroplasts were repurified without (C, lane 2) or with (CP, lane 3) posttreatment with thermolysin. Repurified chloroplasts were also fractionated into stroma (S, lane 4) and total membranes (M, lane 5) and the membranes treated with thermolysin (MP, lane 6), or extracted with 0.2 M Na2CO3 (MC, lane 7). Lane 1 contains the equivalent of 1 μl of translation product and lanes 2–7 contain samples representing 5 μl of the amount of translation product added to the import reaction. SDS-PAGE/fluorograms are depicted. (B) Radiolabeled pea cpSecY translation product (lane 1) or chloroplasts recovered from an import reaction with precursor to cpSecY (lane 4) were immunoprecipitated with antibody directed against the COOH terminus of pea cpSecY (see Materials and Methods) (lanes 2 and 5) or an irrelevant antibody (anti-psTha4; lanes 3 and 6) and the samples were analyzed by SDS-PAGE/fluorography.
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Figure 2: cDNAs for pea cpSecY and psTha4 encode precursor proteins that are imported into chloroplasts and localized to thylakoids. (A) In vitro synthesized pea cpSecY and psTha4 were incubated with intact chloroplasts in the presence of ATP for 10 min. Chloroplasts were repurified without (C, lane 2) or with (CP, lane 3) posttreatment with thermolysin. Repurified chloroplasts were also fractionated into stroma (S, lane 4) and total membranes (M, lane 5) and the membranes treated with thermolysin (MP, lane 6), or extracted with 0.2 M Na2CO3 (MC, lane 7). Lane 1 contains the equivalent of 1 μl of translation product and lanes 2–7 contain samples representing 5 μl of the amount of translation product added to the import reaction. SDS-PAGE/fluorograms are depicted. (B) Radiolabeled pea cpSecY translation product (lane 1) or chloroplasts recovered from an import reaction with precursor to cpSecY (lane 4) were immunoprecipitated with antibody directed against the COOH terminus of pea cpSecY (see Materials and Methods) (lanes 2 and 5) or an irrelevant antibody (anti-psTha4; lanes 3 and 6) and the samples were analyzed by SDS-PAGE/fluorography.

Mentions: The psTha4 cDNA encodes a protein with 137 residues. The amino terminus has characteristics of a chloroplast transit peptide. This was experimentally verified with an in vitro chloroplast import assay. The psTha4 translation product migrated at 19 kD (Fig. 2 A, lane 1). Upon incubation with chloroplasts, a faster migrating 16-kD band was produced that copurified with intact chloroplasts and was protected from exogenous protease (Fig. 2 A, lanes 2 and 3). This indicates that the precursor was imported into chloroplasts and processed to mature size. The imported psTha4 protein fractionated predominantly with the thylakoid membranes (Fig. 2 A, lane 5) and was integrated into the membrane as assessed by resistance to alkaline extraction (Fig. 2 A, lane 7). Similar to Hcf106, the hydrophilic domain of imported psTha4 was exposed to the stromal compartment as it was degraded by exogenous protease (Fig. 2 A, lane 6).


Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways.

Mori H, Summer EJ, Ma X, Cline K - J. Cell Biol. (1999)

cDNAs for pea cpSecY and psTha4 encode precursor proteins that are imported into chloroplasts and localized to thylakoids. (A) In vitro synthesized pea cpSecY and psTha4 were incubated with intact chloroplasts in the presence of ATP for 10 min. Chloroplasts were repurified without (C, lane 2) or with (CP, lane 3) posttreatment with thermolysin. Repurified chloroplasts were also fractionated into stroma (S, lane 4) and total membranes (M, lane 5) and the membranes treated with thermolysin (MP, lane 6), or extracted with 0.2 M Na2CO3 (MC, lane 7). Lane 1 contains the equivalent of 1 μl of translation product and lanes 2–7 contain samples representing 5 μl of the amount of translation product added to the import reaction. SDS-PAGE/fluorograms are depicted. (B) Radiolabeled pea cpSecY translation product (lane 1) or chloroplasts recovered from an import reaction with precursor to cpSecY (lane 4) were immunoprecipitated with antibody directed against the COOH terminus of pea cpSecY (see Materials and Methods) (lanes 2 and 5) or an irrelevant antibody (anti-psTha4; lanes 3 and 6) and the samples were analyzed by SDS-PAGE/fluorography.
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Related In: Results  -  Collection

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Figure 2: cDNAs for pea cpSecY and psTha4 encode precursor proteins that are imported into chloroplasts and localized to thylakoids. (A) In vitro synthesized pea cpSecY and psTha4 were incubated with intact chloroplasts in the presence of ATP for 10 min. Chloroplasts were repurified without (C, lane 2) or with (CP, lane 3) posttreatment with thermolysin. Repurified chloroplasts were also fractionated into stroma (S, lane 4) and total membranes (M, lane 5) and the membranes treated with thermolysin (MP, lane 6), or extracted with 0.2 M Na2CO3 (MC, lane 7). Lane 1 contains the equivalent of 1 μl of translation product and lanes 2–7 contain samples representing 5 μl of the amount of translation product added to the import reaction. SDS-PAGE/fluorograms are depicted. (B) Radiolabeled pea cpSecY translation product (lane 1) or chloroplasts recovered from an import reaction with precursor to cpSecY (lane 4) were immunoprecipitated with antibody directed against the COOH terminus of pea cpSecY (see Materials and Methods) (lanes 2 and 5) or an irrelevant antibody (anti-psTha4; lanes 3 and 6) and the samples were analyzed by SDS-PAGE/fluorography.
Mentions: The psTha4 cDNA encodes a protein with 137 residues. The amino terminus has characteristics of a chloroplast transit peptide. This was experimentally verified with an in vitro chloroplast import assay. The psTha4 translation product migrated at 19 kD (Fig. 2 A, lane 1). Upon incubation with chloroplasts, a faster migrating 16-kD band was produced that copurified with intact chloroplasts and was protected from exogenous protease (Fig. 2 A, lanes 2 and 3). This indicates that the precursor was imported into chloroplasts and processed to mature size. The imported psTha4 protein fractionated predominantly with the thylakoid membranes (Fig. 2 A, lane 5) and was integrated into the membrane as assessed by resistance to alkaline extraction (Fig. 2 A, lane 7). Similar to Hcf106, the hydrophilic domain of imported psTha4 was exposed to the stromal compartment as it was degraded by exogenous protease (Fig. 2 A, lane 6).

Bottom Line: Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates.This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates.These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611, USA.

ABSTRACT
Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in approximately 10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.

Show MeSH