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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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Antibodies to cpSecY inhibit protein transport of cpSecA-dependent substrates, but not those of the Delta pH or cpSRP pathways. (A) Pea thylakoids were preincubated and assayed for transport or integration as in Fig. 6 except that anti-cpSecY IgG and 20 μM antigen peptide were used. (A) A fluorogram of the transport assays. Precursors used and other conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE33 (•, anti-cpSecY; ▴, plus antigen peptide; ▾, PI-IgG), pPC (○, anti-cpSecY; ▵, plus antigen peptide; ▿, PI-IgG), iOE17 (□, anti-cpSecY), iOE23 (⋄, anti-cpSecY), and pLHCP (▪, anti-cpSecY). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
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Figure 8: Antibodies to cpSecY inhibit protein transport of cpSecA-dependent substrates, but not those of the Delta pH or cpSRP pathways. (A) Pea thylakoids were preincubated and assayed for transport or integration as in Fig. 6 except that anti-cpSecY IgG and 20 μM antigen peptide were used. (A) A fluorogram of the transport assays. Precursors used and other conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE33 (•, anti-cpSecY; ▴, plus antigen peptide; ▾, PI-IgG), pPC (○, anti-cpSecY; ▵, plus antigen peptide; ▿, PI-IgG), iOE17 (□, anti-cpSecY), iOE23 (⋄, anti-cpSecY), and pLHCP (▪, anti-cpSecY). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.

Mentions: To explore the function of cpSecY in thylakoid protein transport, the effect of anti-cpSecY IgG on the three pathways was examined (Fig. 8). Preincubation of pea thylakoids with increasing amounts of anti-cpSecY inhibited transport of Sec pathway precursors, iOE33 and pPC (Fig. 8 A, lanes 1–5). Inclusion of 20 μM antigen peptide during the antibody preincubation step suppressed the inhibition (Fig. 8 A, lanes 6 and 7). No inhibition was observed for control thylakoids preincubated with preimmune IgG (Fig. 8 A, lanes 8 and 9). These results demonstrate that cpSecY operates in conjunction with cpSecA for thylakoid Sec pathway transport. Anti-cpSecY IgG had no effect on transport of the Delta pH pathway precursors, iOE23 and iOE17, or on integration of the SRP pathway substrate pLHCP. Even at higher concentrations of cpSecY IgGs (2.0 mg/ml), there was no inhibition of OE23 transport or LHCP integration (data not shown). Of interest is that Fab fragments prepared from anti-cpSecY IgG were ineffective inhibitors of Sec pathway transport (data not shown). This contrasts with the situation of anti-Hcf106 inhibition and suggests either that aggregation of the Sec translocons is required for inhibition or that IgG inhibits Sec pathway transport by steric hindrance.


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)

Antibodies to cpSecY inhibit protein transport of cpSecA-dependent substrates, but not those of the Delta pH or cpSRP pathways. (A) Pea thylakoids were preincubated and assayed for transport or integration as in Fig. 6 except that anti-cpSecY IgG and 20 μM antigen peptide were used. (A) A fluorogram of the transport assays. Precursors used and other conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE33 (•, anti-cpSecY; ▴, plus antigen peptide; ▾, PI-IgG), pPC (○, anti-cpSecY; ▵, plus antigen peptide; ▿, PI-IgG), iOE17 (□, anti-cpSecY), iOE23 (⋄, anti-cpSecY), and pLHCP (▪, anti-cpSecY). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
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Figure 8: Antibodies to cpSecY inhibit protein transport of cpSecA-dependent substrates, but not those of the Delta pH or cpSRP pathways. (A) Pea thylakoids were preincubated and assayed for transport or integration as in Fig. 6 except that anti-cpSecY IgG and 20 μM antigen peptide were used. (A) A fluorogram of the transport assays. Precursors used and other conditions were as shown in the figure. (B) Quantification of transport assays shown in A. iOE33 (•, anti-cpSecY; ▴, plus antigen peptide; ▾, PI-IgG), pPC (○, anti-cpSecY; ▵, plus antigen peptide; ▿, PI-IgG), iOE17 (□, anti-cpSecY), iOE23 (⋄, anti-cpSecY), and pLHCP (▪, anti-cpSecY). Radiolabeled bands were extracted from excised gel bands and quantified by scintillation counting.
Mentions: To explore the function of cpSecY in thylakoid protein transport, the effect of anti-cpSecY IgG on the three pathways was examined (Fig. 8). Preincubation of pea thylakoids with increasing amounts of anti-cpSecY inhibited transport of Sec pathway precursors, iOE33 and pPC (Fig. 8 A, lanes 1–5). Inclusion of 20 μM antigen peptide during the antibody preincubation step suppressed the inhibition (Fig. 8 A, lanes 6 and 7). No inhibition was observed for control thylakoids preincubated with preimmune IgG (Fig. 8 A, lanes 8 and 9). These results demonstrate that cpSecY operates in conjunction with cpSecA for thylakoid Sec pathway transport. Anti-cpSecY IgG had no effect on transport of the Delta pH pathway precursors, iOE23 and iOE17, or on integration of the SRP pathway substrate pLHCP. Even at higher concentrations of cpSecY IgGs (2.0 mg/ml), there was no inhibition of OE23 transport or LHCP integration (data not shown). Of interest is that Fab fragments prepared from anti-cpSecY IgG were ineffective inhibitors of Sec pathway transport (data not shown). This contrasts with the situation of anti-Hcf106 inhibition and suggests either that aggregation of the Sec translocons is required for inhibition or that IgG inhibits Sec pathway transport by steric hindrance.

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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