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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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Inhibition of Delta pH pathway protein transport by antibodies to psTha4. Pea thylakoids were preincubated with increasing amounts of anti-psTha4 IgG in the absence or presence of 20 μM tha4sd or with preimmune (PI) IgG. After washing, the thylakoids were assayed for protein transport or integration. (A) Fluorogram of transport assays. Assay conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE17 (•, anti-psTha4; ▴, plus tha4sd; ▾, PI-IgG), iOE23 (○, anti-psTha4; ▵, plus tha4sd; ▿, PI-IgG), iOE33 (□), and pLHCP (⋄). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
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Figure 6: Inhibition of Delta pH pathway protein transport by antibodies to psTha4. Pea thylakoids were preincubated with increasing amounts of anti-psTha4 IgG in the absence or presence of 20 μM tha4sd or with preimmune (PI) IgG. After washing, the thylakoids were assayed for protein transport or integration. (A) Fluorogram of transport assays. Assay conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE17 (•, anti-psTha4; ▴, plus tha4sd; ▾, PI-IgG), iOE23 (○, anti-psTha4; ▵, plus tha4sd; ▿, PI-IgG), iOE33 (□), and pLHCP (⋄). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.

Mentions: A similar analysis was conducted with pea thylakoid membranes and antibodies to psTha4 (Fig. 6). Preincubation of pea thylakoids with increasing amounts of anti-psTha4 IgG inhibited protein transport of the Delta pH pathway substrates iOE17 and iOE23. Preimmune IgG had no effect on transport, and the inhibition by anti-Tha4 was suppressed by including antigen tha4sd during the preincubation step. Transport of the Sec pathway substrate iOE33 and integration of the SRP pathway substrate pLHCP were unaffected by anti-psTha4 IgG. Assays conducted in the absence and presence of ionophores confirmed that anti-psTha4 IgG binding did not compromise the thylakoidal ΔpH (data not shown). These results indicate that psTha4 also directly functions in protein transport on the Delta pH pathway.


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)

Inhibition of Delta pH pathway protein transport by antibodies to psTha4. Pea thylakoids were preincubated with increasing amounts of anti-psTha4 IgG in the absence or presence of 20 μM tha4sd or with preimmune (PI) IgG. After washing, the thylakoids were assayed for protein transport or integration. (A) Fluorogram of transport assays. Assay conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE17 (•, anti-psTha4; ▴, plus tha4sd; ▾, PI-IgG), iOE23 (○, anti-psTha4; ▵, plus tha4sd; ▿, PI-IgG), iOE33 (□), and pLHCP (⋄). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
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Related In: Results  -  Collection

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Figure 6: Inhibition of Delta pH pathway protein transport by antibodies to psTha4. Pea thylakoids were preincubated with increasing amounts of anti-psTha4 IgG in the absence or presence of 20 μM tha4sd or with preimmune (PI) IgG. After washing, the thylakoids were assayed for protein transport or integration. (A) Fluorogram of transport assays. Assay conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE17 (•, anti-psTha4; ▴, plus tha4sd; ▾, PI-IgG), iOE23 (○, anti-psTha4; ▵, plus tha4sd; ▿, PI-IgG), iOE33 (□), and pLHCP (⋄). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
Mentions: A similar analysis was conducted with pea thylakoid membranes and antibodies to psTha4 (Fig. 6). Preincubation of pea thylakoids with increasing amounts of anti-psTha4 IgG inhibited protein transport of the Delta pH pathway substrates iOE17 and iOE23. Preimmune IgG had no effect on transport, and the inhibition by anti-Tha4 was suppressed by including antigen tha4sd during the preincubation step. Transport of the Sec pathway substrate iOE33 and integration of the SRP pathway substrate pLHCP were unaffected by anti-psTha4 IgG. Assays conducted in the absence and presence of ionophores confirmed that anti-psTha4 IgG binding did not compromise the thylakoidal ΔpH (data not shown). These results indicate that psTha4 also directly functions in protein transport on the Delta pH pathway.

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
Related in: MedlinePlus