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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 against pea cpSecY, psTha4, and maize Hcf106. (A) Coomassie brilliant blue–stained gel of E. coli–expressed and purified pea tha4sd (500 ng, lane 1) and maize hcf106sd (500 ng, lane 2). (B) Immunodetection of psTha4 and maize Hcf106. Pea thylakoid proteins (equivalent to 5 μg chlorophyll, lanes 1 and 3), pea tha4sd (10 ng, lanes 2 and 4), maize thylakoid proteins (equivalent to 2 μg chlorophyll, lanes 5 and 7), and maize hcf106sd (10 ng, lanes 6 and 8) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with anti-Hcf106 or anti-psTha4. Antibodies were preincubated without or with ∼1 μg/ml of each antigen protein for 1 h on ice before immunodecoration. (C) Immunodetection of pea cpSecY. Pea thylakoid protein (equivalent to 5 μg chlorophyll) was separated by SDS-PAGE and blotted to nitrocellulose membrane. The blots were immunodetected with anti-cpSecY that was preincubated without (lane 1) or with 1 μg/μl antigen peptide (lane 2) for 1 h on ice.
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Figure 3: Antibodies against pea cpSecY, psTha4, and maize Hcf106. (A) Coomassie brilliant blue–stained gel of E. coli–expressed and purified pea tha4sd (500 ng, lane 1) and maize hcf106sd (500 ng, lane 2). (B) Immunodetection of psTha4 and maize Hcf106. Pea thylakoid proteins (equivalent to 5 μg chlorophyll, lanes 1 and 3), pea tha4sd (10 ng, lanes 2 and 4), maize thylakoid proteins (equivalent to 2 μg chlorophyll, lanes 5 and 7), and maize hcf106sd (10 ng, lanes 6 and 8) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with anti-Hcf106 or anti-psTha4. Antibodies were preincubated without or with ∼1 μg/ml of each antigen protein for 1 h on ice before immunodecoration. (C) Immunodetection of pea cpSecY. Pea thylakoid protein (equivalent to 5 μg chlorophyll) was separated by SDS-PAGE and blotted to nitrocellulose membrane. The blots were immunodetected with anti-cpSecY that was preincubated without (lane 1) or with 1 μg/μl antigen peptide (lane 2) for 1 h on ice.

Mentions: The stromal domains of Hcf106 and psTha4 were expressed in E. coli with NH2-terminal His tags for purification. Both proteins accumulated in the soluble fraction of E. coli, presumably in a native conformation. Purified pea tha4sd and maize hcf106sd migrated on SDS-PAGE with molecular masses of ∼16 kD and ∼35 kD, respectively (Fig. 3 A, lanes 1 and 2). Both proteins were used as antigens without further treatment.


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 against pea cpSecY, psTha4, and maize Hcf106. (A) Coomassie brilliant blue–stained gel of E. coli–expressed and purified pea tha4sd (500 ng, lane 1) and maize hcf106sd (500 ng, lane 2). (B) Immunodetection of psTha4 and maize Hcf106. Pea thylakoid proteins (equivalent to 5 μg chlorophyll, lanes 1 and 3), pea tha4sd (10 ng, lanes 2 and 4), maize thylakoid proteins (equivalent to 2 μg chlorophyll, lanes 5 and 7), and maize hcf106sd (10 ng, lanes 6 and 8) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with anti-Hcf106 or anti-psTha4. Antibodies were preincubated without or with ∼1 μg/ml of each antigen protein for 1 h on ice before immunodecoration. (C) Immunodetection of pea cpSecY. Pea thylakoid protein (equivalent to 5 μg chlorophyll) was separated by SDS-PAGE and blotted to nitrocellulose membrane. The blots were immunodetected with anti-cpSecY that was preincubated without (lane 1) or with 1 μg/μl antigen peptide (lane 2) for 1 h on ice.
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Related In: Results  -  Collection

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Figure 3: Antibodies against pea cpSecY, psTha4, and maize Hcf106. (A) Coomassie brilliant blue–stained gel of E. coli–expressed and purified pea tha4sd (500 ng, lane 1) and maize hcf106sd (500 ng, lane 2). (B) Immunodetection of psTha4 and maize Hcf106. Pea thylakoid proteins (equivalent to 5 μg chlorophyll, lanes 1 and 3), pea tha4sd (10 ng, lanes 2 and 4), maize thylakoid proteins (equivalent to 2 μg chlorophyll, lanes 5 and 7), and maize hcf106sd (10 ng, lanes 6 and 8) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with anti-Hcf106 or anti-psTha4. Antibodies were preincubated without or with ∼1 μg/ml of each antigen protein for 1 h on ice before immunodecoration. (C) Immunodetection of pea cpSecY. Pea thylakoid protein (equivalent to 5 μg chlorophyll) was separated by SDS-PAGE and blotted to nitrocellulose membrane. The blots were immunodetected with anti-cpSecY that was preincubated without (lane 1) or with 1 μg/μl antigen peptide (lane 2) for 1 h on ice.
Mentions: The stromal domains of Hcf106 and psTha4 were expressed in E. coli with NH2-terminal His tags for purification. Both proteins accumulated in the soluble fraction of E. coli, presumably in a native conformation. Purified pea tha4sd and maize hcf106sd migrated on SDS-PAGE with molecular masses of ∼16 kD and ∼35 kD, respectively (Fig. 3 A, lanes 1 and 2). Both proteins were used as antigens without further treatment.

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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