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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 Hcf106 and psTha4 inhibit the entire range of known Delta pH pathway substrates. Pea thylakoids were preincubated with anti-Hcf106 IgG (1 mg/ml) or anti-psTha4 IgG (0.5 mg/ml) in the absence or presence of 20 μM hcf106sd or tha4sd as shown above the panel. Thylakoids were incubated with the corresponding preimmune IgGs at the same concentrations. After washing, thylakoids were assayed for transport of Delta pH pathway substrates (depicted to the left of each panel). Assay conditions were as shown above the panels.
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Figure 7: Antibodies to Hcf106 and psTha4 inhibit the entire range of known Delta pH pathway substrates. Pea thylakoids were preincubated with anti-Hcf106 IgG (1 mg/ml) or anti-psTha4 IgG (0.5 mg/ml) in the absence or presence of 20 μM hcf106sd or tha4sd as shown above the panel. Thylakoids were incubated with the corresponding preimmune IgGs at the same concentrations. After washing, thylakoids were assayed for transport of Delta pH pathway substrates (depicted to the left of each panel). Assay conditions were as shown above the panels.

Mentions: E. coli has three genes that are related to Hcf106: tatA, tatB, and tatE. Disruption of each of these genes singly causes partial inhibition of the translocation of overlapping sets of substrates (Sargent et al. 1998; Weiner et al. 1998), whereas double mutations result in a more complete inhibition (Sargent et al. 1998). One interpretation of these genetic results is that individual Hcf106 homologues have preferences for certain substrates. To assess whether Hcf106 or Tha4 show any selectivity for substrate, antibody inhibition experiments were conducted with four well-documented Delta pH pathway substrates: OE23, OE17, PSI-N, and PSII-T. We observed that antibodies to maize Hcf106 inhibit the Delta pH pathway, but not the Sec or SRP pathways, of pea thylakoid membranes (data not shown). This allowed us to simultaneously test the effects of anti-Hcf106 and anti-Tha4 with one membrane system. As shown in Fig. 7, either anti-Hcf106 or anti-psTha4 IgGs virtually eliminated transport of all Delta pH pathway substrates with pea thylakoids. In the experiment shown in Fig. 7, inhibition of PSII-T transport was not complete. However, in other experiments anti-Tha4 inhibited PSII-T transport to the same extent as that of the other substrates. Importantly, the inhibition by anti-Hcf106 was suppressed by hcf106sd, but not tha4sd (Fig. 7, lanes 2–4), and the inhibition by anti-Tha4 was suppressed by tha4sd, but not by hcf106sd (Fig. 7, lanes 6–8). This suggests that anti-Hcf106 binds to the pea Hcf106 orthologue. Neither antibody inhibited the integration of LHCP (data not shown), which was conducted as a control. We interpret these results to mean that antibody binding to either Hcf106 or Tha4 in vitro can disable the entire 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)

Antibodies to Hcf106 and psTha4 inhibit the entire range of known Delta pH pathway substrates. Pea thylakoids were preincubated with anti-Hcf106 IgG (1 mg/ml) or anti-psTha4 IgG (0.5 mg/ml) in the absence or presence of 20 μM hcf106sd or tha4sd as shown above the panel. Thylakoids were incubated with the corresponding preimmune IgGs at the same concentrations. After washing, thylakoids were assayed for transport of Delta pH pathway substrates (depicted to the left of each panel). Assay conditions were as shown above the panels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199744&req=5

Figure 7: Antibodies to Hcf106 and psTha4 inhibit the entire range of known Delta pH pathway substrates. Pea thylakoids were preincubated with anti-Hcf106 IgG (1 mg/ml) or anti-psTha4 IgG (0.5 mg/ml) in the absence or presence of 20 μM hcf106sd or tha4sd as shown above the panel. Thylakoids were incubated with the corresponding preimmune IgGs at the same concentrations. After washing, thylakoids were assayed for transport of Delta pH pathway substrates (depicted to the left of each panel). Assay conditions were as shown above the panels.
Mentions: E. coli has three genes that are related to Hcf106: tatA, tatB, and tatE. Disruption of each of these genes singly causes partial inhibition of the translocation of overlapping sets of substrates (Sargent et al. 1998; Weiner et al. 1998), whereas double mutations result in a more complete inhibition (Sargent et al. 1998). One interpretation of these genetic results is that individual Hcf106 homologues have preferences for certain substrates. To assess whether Hcf106 or Tha4 show any selectivity for substrate, antibody inhibition experiments were conducted with four well-documented Delta pH pathway substrates: OE23, OE17, PSI-N, and PSII-T. We observed that antibodies to maize Hcf106 inhibit the Delta pH pathway, but not the Sec or SRP pathways, of pea thylakoid membranes (data not shown). This allowed us to simultaneously test the effects of anti-Hcf106 and anti-Tha4 with one membrane system. As shown in Fig. 7, either anti-Hcf106 or anti-psTha4 IgGs virtually eliminated transport of all Delta pH pathway substrates with pea thylakoids. In the experiment shown in Fig. 7, inhibition of PSII-T transport was not complete. However, in other experiments anti-Tha4 inhibited PSII-T transport to the same extent as that of the other substrates. Importantly, the inhibition by anti-Hcf106 was suppressed by hcf106sd, but not tha4sd (Fig. 7, lanes 2–4), and the inhibition by anti-Tha4 was suppressed by tha4sd, but not by hcf106sd (Fig. 7, lanes 6–8). This suggests that anti-Hcf106 binds to the pea Hcf106 orthologue. Neither antibody inhibited the integration of LHCP (data not shown), which was conducted as a control. We interpret these results to mean that antibody binding to either Hcf106 or Tha4 in vitro can disable the entire 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