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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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Translocation reversal is not strictly linked to expression levels. Protoplasts were transfected with DNA encoding pre-23K under standard conditions (40 μg DNA per incubation) or with smaller amounts of DNA as indicated. Cells were labeled for 3 h with 35S-Met and 35S-Cys and then fractionated as in Fig. 5. Samples were subjected to immunoprecipitation with antibodies to 23K and the i23K, and 23K bands were quantitated using a phosphorimager; the combined radiolabeled contents (arbitrary units) are shown under the autoradiogram, with 100% representing the expression level obtained with 40 μg DNA.
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fig6: Translocation reversal is not strictly linked to expression levels. Protoplasts were transfected with DNA encoding pre-23K under standard conditions (40 μg DNA per incubation) or with smaller amounts of DNA as indicated. Cells were labeled for 3 h with 35S-Met and 35S-Cys and then fractionated as in Fig. 5. Samples were subjected to immunoprecipitation with antibodies to 23K and the i23K, and 23K bands were quantitated using a phosphorimager; the combined radiolabeled contents (arbitrary units) are shown under the autoradiogram, with 100% representing the expression level obtained with 40 μg DNA.

Mentions: The extensive repartitioning of mature GFP and 23K to the stroma is unexpected, and an important question is whether this process occurs at normal expression levels. The promoter used is known to drive high-level expression, and the translocation reversal may stem from overloading of the system (but see Discussion). We cannot analyze the targeting of the endogenous Tat proteins by similar pulse-chase methods, but we have tested whether the extent of translocation reversal is linked to expression level by systematically reducing the amount of DNA used in the transfection process (Fig. 6). Previous experiments involved transfection of 106 protoplasts with 40 μg DNA, and Fig. 6 (left) shows an identical experiment in which the i23K and 23K polypeptides were immunoprecipitated. The i23K and 23K bands were quantitated by phosphorimager analysis, and the combined 23K expression level is indicated as 100%. Reducing the quantity of DNA used for transfection results in a gradual reduction in 23K expression levels (indicated under the autoradiograph), and with the smallest amount used (0.4 μg), expression is reduced to 13% of the control value. Nevertheless, mature 23K is still found primarily in the stroma, showing that translocation reversal is not significantly affected. Translocation reversal is thus not a simple response to the levels of Tat substrate present.


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

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

Translocation reversal is not strictly linked to expression levels. Protoplasts were transfected with DNA encoding pre-23K under standard conditions (40 μg DNA per incubation) or with smaller amounts of DNA as indicated. Cells were labeled for 3 h with 35S-Met and 35S-Cys and then fractionated as in Fig. 5. Samples were subjected to immunoprecipitation with antibodies to 23K and the i23K, and 23K bands were quantitated using a phosphorimager; the combined radiolabeled contents (arbitrary units) are shown under the autoradiogram, with 100% representing the expression level obtained with 40 μg DNA.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Translocation reversal is not strictly linked to expression levels. Protoplasts were transfected with DNA encoding pre-23K under standard conditions (40 μg DNA per incubation) or with smaller amounts of DNA as indicated. Cells were labeled for 3 h with 35S-Met and 35S-Cys and then fractionated as in Fig. 5. Samples were subjected to immunoprecipitation with antibodies to 23K and the i23K, and 23K bands were quantitated using a phosphorimager; the combined radiolabeled contents (arbitrary units) are shown under the autoradiogram, with 100% representing the expression level obtained with 40 μg DNA.
Mentions: The extensive repartitioning of mature GFP and 23K to the stroma is unexpected, and an important question is whether this process occurs at normal expression levels. The promoter used is known to drive high-level expression, and the translocation reversal may stem from overloading of the system (but see Discussion). We cannot analyze the targeting of the endogenous Tat proteins by similar pulse-chase methods, but we have tested whether the extent of translocation reversal is linked to expression level by systematically reducing the amount of DNA used in the transfection process (Fig. 6). Previous experiments involved transfection of 106 protoplasts with 40 μg DNA, and Fig. 6 (left) shows an identical experiment in which the i23K and 23K polypeptides were immunoprecipitated. The i23K and 23K bands were quantitated by phosphorimager analysis, and the combined 23K expression level is indicated as 100%. Reducing the quantity of DNA used for transfection results in a gradual reduction in 23K expression levels (indicated under the autoradiograph), and with the smallest amount used (0.4 μg), expression is reduced to 13% of the control value. Nevertheless, mature 23K is still found primarily in the stroma, showing that translocation reversal is not significantly affected. Translocation reversal is thus not a simple response to the levels of Tat substrate present.

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