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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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cpSecY and psTha4 are integral membrane proteins of the nonappressed membrane regions of thylakoids. Pea thylakoids were treated with import buffer (Mock, lane 1), 0.1 mg/ml thermolysin (lane 2), or 0.2 M Na2CO3 (lane 3). Appressed membranes (lane 4) and nonappressed membranes (lane 5) were prepared from pea thylakoids (see Materials and Methods). Samples, equivalent to 5 μg chlorophyll of thylakoids, were fractionated by SDS-PAGE, blotted to nitrocellulose, and proteins were immunodetected by the ECL method with the antibodies indicated to the left of the panel (top). A Coomassie-stained gel of the samples is shown in the bottom panel. CF1 α/β, the two large subunits of the chloroplasts coupling factor, is a marker for nonappressed membranes. LHCP is a marker for appressed membranes.
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Figure 4: cpSecY and psTha4 are integral membrane proteins of the nonappressed membrane regions of thylakoids. Pea thylakoids were treated with import buffer (Mock, lane 1), 0.1 mg/ml thermolysin (lane 2), or 0.2 M Na2CO3 (lane 3). Appressed membranes (lane 4) and nonappressed membranes (lane 5) were prepared from pea thylakoids (see Materials and Methods). Samples, equivalent to 5 μg chlorophyll of thylakoids, were fractionated by SDS-PAGE, blotted to nitrocellulose, and proteins were immunodetected by the ECL method with the antibodies indicated to the left of the panel (top). A Coomassie-stained gel of the samples is shown in the bottom panel. CF1 α/β, the two large subunits of the chloroplasts coupling factor, is a marker for nonappressed membranes. LHCP is a marker for appressed membranes.

Mentions: Antibodies were used to verify the expected properties of endogenous pea cpSecY and psTha4 (Fig. 4). As with the imported proteins, endogenous psTha4 and cpSecY were recovered primarily in the thylakoid membrane fraction (data not shown). They were resistant to carbonate extraction, but digested by added thermolysin (Fig. 4, lanes 2 and 3). A band corresponding to the 20-kD protease-protected fragment of imported cpSecY was not immunodecorated with anti-cpSecY (data not shown), indicating that the protease protected fragment corresponds to an NH2-terminal or internal fragment. These results verify that cpSecY and psTha4 are integral membrane proteins exposed to the stroma. Thylakoid membranes consist of two structurally and functionally distinct domains, the nonappressed membranes and the appressed membranes. Upon subfractionation of thylakoids with digitonin (see Materials and Methods), cpSecY and psTha4 were recovered with the nonappressed membranes (Fig. 4, lane 5) rather than the appressed membranes (Fig. 4, lane 4). cpSecA, which is peripherally associated with thylakoids (Fig. 4, lanes 1 and 3), was also recovered in the nonappressed membranes (Fig. 4, lane 5). Maize Hcf106 was localized in the nonappressed membranes of maize thylakoids (data not shown). These results are consistent with other studies implicating the nonappressed membranes as the site of protein transport/integration (Kirwin et al. 1988; Kohorn and Yakir 1990; Hashimoto et al. 1997).


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)

cpSecY and psTha4 are integral membrane proteins of the nonappressed membrane regions of thylakoids. Pea thylakoids were treated with import buffer (Mock, lane 1), 0.1 mg/ml thermolysin (lane 2), or 0.2 M Na2CO3 (lane 3). Appressed membranes (lane 4) and nonappressed membranes (lane 5) were prepared from pea thylakoids (see Materials and Methods). Samples, equivalent to 5 μg chlorophyll of thylakoids, were fractionated by SDS-PAGE, blotted to nitrocellulose, and proteins were immunodetected by the ECL method with the antibodies indicated to the left of the panel (top). A Coomassie-stained gel of the samples is shown in the bottom panel. CF1 α/β, the two large subunits of the chloroplasts coupling factor, is a marker for nonappressed membranes. LHCP is a marker for appressed membranes.
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Related In: Results  -  Collection

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

Figure 4: cpSecY and psTha4 are integral membrane proteins of the nonappressed membrane regions of thylakoids. Pea thylakoids were treated with import buffer (Mock, lane 1), 0.1 mg/ml thermolysin (lane 2), or 0.2 M Na2CO3 (lane 3). Appressed membranes (lane 4) and nonappressed membranes (lane 5) were prepared from pea thylakoids (see Materials and Methods). Samples, equivalent to 5 μg chlorophyll of thylakoids, were fractionated by SDS-PAGE, blotted to nitrocellulose, and proteins were immunodetected by the ECL method with the antibodies indicated to the left of the panel (top). A Coomassie-stained gel of the samples is shown in the bottom panel. CF1 α/β, the two large subunits of the chloroplasts coupling factor, is a marker for nonappressed membranes. LHCP is a marker for appressed membranes.
Mentions: Antibodies were used to verify the expected properties of endogenous pea cpSecY and psTha4 (Fig. 4). As with the imported proteins, endogenous psTha4 and cpSecY were recovered primarily in the thylakoid membrane fraction (data not shown). They were resistant to carbonate extraction, but digested by added thermolysin (Fig. 4, lanes 2 and 3). A band corresponding to the 20-kD protease-protected fragment of imported cpSecY was not immunodecorated with anti-cpSecY (data not shown), indicating that the protease protected fragment corresponds to an NH2-terminal or internal fragment. These results verify that cpSecY and psTha4 are integral membrane proteins exposed to the stroma. Thylakoid membranes consist of two structurally and functionally distinct domains, the nonappressed membranes and the appressed membranes. Upon subfractionation of thylakoids with digitonin (see Materials and Methods), cpSecY and psTha4 were recovered with the nonappressed membranes (Fig. 4, lane 5) rather than the appressed membranes (Fig. 4, lane 4). cpSecA, which is peripherally associated with thylakoids (Fig. 4, lanes 1 and 3), was also recovered in the nonappressed membranes (Fig. 4, lane 5). Maize Hcf106 was localized in the nonappressed membranes of maize thylakoids (data not shown). These results are consistent with other studies implicating the nonappressed membranes as the site of protein transport/integration (Kirwin et al. 1988; Kohorn and Yakir 1990; Hashimoto et al. 1997).

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