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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 Hcf106. (A) Maize thylakoids were preincubated with 0.1 mg/ml anti-Hcf106 or preimmune (PI) IgGs. Thylakoids were then washed and assayed for transport or integration of various precursors in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 0.5 μM nigericin and 1.0 μM valinomycin (Nig/Val). The radiolabeled precursors used in each assay are indicated next to the panel. (B) Maize thylakoids were preincubated with increasing amounts of anti-Hcf106 Fab fragments in the absence or presence of 20 μM hcf106sd or with PI-Fab. After washing, the thylakoids were assayed for protein transport or integration. iOE17 (•, anti-Hcf106; ▴, plus hcf106sd; ▾, PI-Fab), iOE23 (○, anti-Hfc106; ▵, plus hcf106sd; ▿, PI-Fab), iOE33 (□), pPC (⋄), and pLHCP (▪). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
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Figure 5: Inhibition of Delta pH pathway protein transport by antibodies to Hcf106. (A) Maize thylakoids were preincubated with 0.1 mg/ml anti-Hcf106 or preimmune (PI) IgGs. Thylakoids were then washed and assayed for transport or integration of various precursors in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 0.5 μM nigericin and 1.0 μM valinomycin (Nig/Val). The radiolabeled precursors used in each assay are indicated next to the panel. (B) Maize thylakoids were preincubated with increasing amounts of anti-Hcf106 Fab fragments in the absence or presence of 20 μM hcf106sd or with PI-Fab. After washing, the thylakoids were assayed for protein transport or integration. iOE17 (•, anti-Hcf106; ▴, plus hcf106sd; ▾, PI-Fab), iOE23 (○, anti-Hfc106; ▵, plus hcf106sd; ▿, PI-Fab), iOE33 (□), pPC (⋄), and pLHCP (▪). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.

Mentions: Genetic disruption of the maize Hcf106 gene and genes for homologous proteins in E. coli results in mislocalization of precursor proteins (Voelker and Barkan 1995; Sargent et al. 1998; Weiner et al. 1998). Fig. 5 provides biochemical evidence that Hcf106 is directly involved in Delta pH pathway transport. Maize thylakoids were preincubated with or without anti-Hcf106 IgG or with preimmune IgG. The thylakoids were then washed with buffer and used for in vitro transport/integration assays. Transport of Delta pH pathway substrates, iOE17 and iOE23, was inhibited ∼95% by anti-Hcf106 IgG (Fig. 5 A, lane 4), but not by preimmune IgG (Fig. 5 A, lane 6). Anti-Hcf106 had no effect on transport of Sec pathway substrates, iOE33 and pPC, or on integration of the SRP substrate pLHCP.


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 Hcf106. (A) Maize thylakoids were preincubated with 0.1 mg/ml anti-Hcf106 or preimmune (PI) IgGs. Thylakoids were then washed and assayed for transport or integration of various precursors in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 0.5 μM nigericin and 1.0 μM valinomycin (Nig/Val). The radiolabeled precursors used in each assay are indicated next to the panel. (B) Maize thylakoids were preincubated with increasing amounts of anti-Hcf106 Fab fragments in the absence or presence of 20 μM hcf106sd or with PI-Fab. After washing, the thylakoids were assayed for protein transport or integration. iOE17 (•, anti-Hcf106; ▴, plus hcf106sd; ▾, PI-Fab), iOE23 (○, anti-Hfc106; ▵, plus hcf106sd; ▿, PI-Fab), iOE33 (□), pPC (⋄), 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 5: Inhibition of Delta pH pathway protein transport by antibodies to Hcf106. (A) Maize thylakoids were preincubated with 0.1 mg/ml anti-Hcf106 or preimmune (PI) IgGs. Thylakoids were then washed and assayed for transport or integration of various precursors in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 0.5 μM nigericin and 1.0 μM valinomycin (Nig/Val). The radiolabeled precursors used in each assay are indicated next to the panel. (B) Maize thylakoids were preincubated with increasing amounts of anti-Hcf106 Fab fragments in the absence or presence of 20 μM hcf106sd or with PI-Fab. After washing, the thylakoids were assayed for protein transport or integration. iOE17 (•, anti-Hcf106; ▴, plus hcf106sd; ▾, PI-Fab), iOE23 (○, anti-Hfc106; ▵, plus hcf106sd; ▿, PI-Fab), iOE33 (□), pPC (⋄), and pLHCP (▪). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
Mentions: Genetic disruption of the maize Hcf106 gene and genes for homologous proteins in E. coli results in mislocalization of precursor proteins (Voelker and Barkan 1995; Sargent et al. 1998; Weiner et al. 1998). Fig. 5 provides biochemical evidence that Hcf106 is directly involved in Delta pH pathway transport. Maize thylakoids were preincubated with or without anti-Hcf106 IgG or with preimmune IgG. The thylakoids were then washed with buffer and used for in vitro transport/integration assays. Transport of Delta pH pathway substrates, iOE17 and iOE23, was inhibited ∼95% by anti-Hcf106 IgG (Fig. 5 A, lane 4), but not by preimmune IgG (Fig. 5 A, lane 6). Anti-Hcf106 had no effect on transport of Sec pathway substrates, iOE33 and pPC, or on integration of the SRP substrate pLHCP.

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