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Large-scale translocation reversal within the thylakoid Tat system in vivo.

Di Cola A, Robinson C - J. Cell Biol. (2005)

Bottom Line: However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma.Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus.Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England, UK.

ABSTRACT
In vitro import assays have shown that the thylakoid twin-arginine translocase (Tat) system transports folded proteins in a unidirectional manner. Here, we expressed a natural substrate, pre-23K, and a 23K presequence-green fluorescent protein (GFP) chimera in vivo in tobacco protoplasts. Both are imported into chloroplasts, targeted to the thylakoids, and processed to the mature size by the lumen-facing processing peptidase. However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma. Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus. Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery. We conclude that, in vivo, the Tat system can reject substrates at a late stage in translocation and on a very large scale, indicating the influence of factors that are absent in reconstitution assays.

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Related in: MedlinePlus

Import of pre-GFP and pre-GFPΔTPP into intact chloroplasts. Pre-GFP and pre-GFPΔTPP were synthesized in vitro in the presence of [35S]-Met, and the translation products (lanes Tr1 and 2) were incubated with intact pea chloroplasts. After incubation, we analyzed samples of the chloroplast (C), the chloroplasts after treatment with thermolysin (C+), and the stromal (S) and thylakoid (T) fractions after lysis of the organelles. Lanes T+, trypsin-treated thylakoids; GFP, mature GFP; arrow, iGFP; pre-GFP, full precursor form.
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fig2: Import of pre-GFP and pre-GFPΔTPP into intact chloroplasts. Pre-GFP and pre-GFPΔTPP were synthesized in vitro in the presence of [35S]-Met, and the translation products (lanes Tr1 and 2) were incubated with intact pea chloroplasts. After incubation, we analyzed samples of the chloroplast (C), the chloroplasts after treatment with thermolysin (C+), and the stromal (S) and thylakoid (T) fractions after lysis of the organelles. Lanes T+, trypsin-treated thylakoids; GFP, mature GFP; arrow, iGFP; pre-GFP, full precursor form.

Mentions: Before expression in vivo, we tested whether the GFP constructs behave as typical Tat substrates in standard in vitro chloroplast protein import assays. Pre-GFP and pre-GFPΔTPP were synthesized in vitro and imported into chloroplasts, and the organelles were fractionated to assess the locations of the polypeptides. Fig. 2 shows that both the pre-GFP and pre-GFPΔTPP translation products (lanes Tr1 and 2) are imported into the chloroplasts and processed to smaller forms that are resistant to proteolysis of the organelles (lanes C and C+). Pre-GFP is processed primarily to the mature form, which is found in the thylakoid fraction (Fig. 2, lane T), where it is resistant to protease treatment (T+), confirming a lumenal location. When the mutant lacking the consensus site for TPP (pre-GFPΔTPP) is imported, the precursor is targeted with equal efficiency to the thylakoid lumen, where it accumulates exclusively as the intermediate GFP (iGFP). This result shows that the mutation completely blocks processing to the mature size, as expected based on a previous study (Shackleton and Robinson, 1991), but does not affect translocation. It is known that Tat-mediated translocation into the thylakoid lumen is completely blocked when the ΔpH across the thylakoid membrane is dissipated in vitro (Mould and Robinson, 1991; Cline et al., 1992), and the same applies to these GFP constructs (unpublished data). Pre-GFP and pre-GFPΔTPP thus behave as absolutely typical Tat substrates in these in vitro import assays. As with every Tat substrate analyzed in vitro to date, translocation is unidirectional, and the mature 23K and GFP are found exclusively in the thylakoid lumen.


Large-scale translocation reversal within the thylakoid Tat system in vivo.

Di Cola A, Robinson C - J. Cell Biol. (2005)

Import of pre-GFP and pre-GFPΔTPP into intact chloroplasts. Pre-GFP and pre-GFPΔTPP were synthesized in vitro in the presence of [35S]-Met, and the translation products (lanes Tr1 and 2) were incubated with intact pea chloroplasts. After incubation, we analyzed samples of the chloroplast (C), the chloroplasts after treatment with thermolysin (C+), and the stromal (S) and thylakoid (T) fractions after lysis of the organelles. Lanes T+, trypsin-treated thylakoids; GFP, mature GFP; arrow, iGFP; pre-GFP, full precursor form.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171186&req=5

fig2: Import of pre-GFP and pre-GFPΔTPP into intact chloroplasts. Pre-GFP and pre-GFPΔTPP were synthesized in vitro in the presence of [35S]-Met, and the translation products (lanes Tr1 and 2) were incubated with intact pea chloroplasts. After incubation, we analyzed samples of the chloroplast (C), the chloroplasts after treatment with thermolysin (C+), and the stromal (S) and thylakoid (T) fractions after lysis of the organelles. Lanes T+, trypsin-treated thylakoids; GFP, mature GFP; arrow, iGFP; pre-GFP, full precursor form.
Mentions: Before expression in vivo, we tested whether the GFP constructs behave as typical Tat substrates in standard in vitro chloroplast protein import assays. Pre-GFP and pre-GFPΔTPP were synthesized in vitro and imported into chloroplasts, and the organelles were fractionated to assess the locations of the polypeptides. Fig. 2 shows that both the pre-GFP and pre-GFPΔTPP translation products (lanes Tr1 and 2) are imported into the chloroplasts and processed to smaller forms that are resistant to proteolysis of the organelles (lanes C and C+). Pre-GFP is processed primarily to the mature form, which is found in the thylakoid fraction (Fig. 2, lane T), where it is resistant to protease treatment (T+), confirming a lumenal location. When the mutant lacking the consensus site for TPP (pre-GFPΔTPP) is imported, the precursor is targeted with equal efficiency to the thylakoid lumen, where it accumulates exclusively as the intermediate GFP (iGFP). This result shows that the mutation completely blocks processing to the mature size, as expected based on a previous study (Shackleton and Robinson, 1991), but does not affect translocation. It is known that Tat-mediated translocation into the thylakoid lumen is completely blocked when the ΔpH across the thylakoid membrane is dissipated in vitro (Mould and Robinson, 1991; Cline et al., 1992), and the same applies to these GFP constructs (unpublished data). Pre-GFP and pre-GFPΔTPP thus behave as absolutely typical Tat substrates in these in vitro import assays. As with every Tat substrate analyzed in vitro to date, translocation is unidirectional, and the mature 23K and GFP are found exclusively in the thylakoid lumen.

Bottom Line: However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma.Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus.Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England, UK.

ABSTRACT
In vitro import assays have shown that the thylakoid twin-arginine translocase (Tat) system transports folded proteins in a unidirectional manner. Here, we expressed a natural substrate, pre-23K, and a 23K presequence-green fluorescent protein (GFP) chimera in vivo in tobacco protoplasts. Both are imported into chloroplasts, targeted to the thylakoids, and processed to the mature size by the lumen-facing processing peptidase. However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma. Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus. Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery. We conclude that, in vivo, the Tat system can reject substrates at a late stage in translocation and on a very large scale, indicating the influence of factors that are absent in reconstitution assays.

Show MeSH
Related in: MedlinePlus