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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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23K is not highly overexpressed in transfected protoplasts. (A) Tobacco protoplasts (106) were transfected with 40 μg of empty vector (Control) or 1 μg/40 μg of vector encoding pre-23K exactly as in Fig 6. After incubation for 24 h, the indicated number of cells from each incubation were analyzed by immunoblotting using antisera to spinach 23K, after which the 23K band signal intensities were calculated using a densitometer. All values are shown plotted relative to the 23K signal obtained with 250,000 cells from the sample transfected with empty vector. (B) Immunoblot of samples from control (empty vector) transfections and protoplasts transfected with 40 μg of pre-23K vector. Lanes 1–5 represent sample loadings of 250,000 cells down to 15,600 cells as in A.
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fig7: 23K is not highly overexpressed in transfected protoplasts. (A) Tobacco protoplasts (106) were transfected with 40 μg of empty vector (Control) or 1 μg/40 μg of vector encoding pre-23K exactly as in Fig 6. After incubation for 24 h, the indicated number of cells from each incubation were analyzed by immunoblotting using antisera to spinach 23K, after which the 23K band signal intensities were calculated using a densitometer. All values are shown plotted relative to the 23K signal obtained with 250,000 cells from the sample transfected with empty vector. (B) Immunoblot of samples from control (empty vector) transfections and protoplasts transfected with 40 μg of pre-23K vector. Lanes 1–5 represent sample loadings of 250,000 cells down to 15,600 cells as in A.

Mentions: A quantitative assessment of the overexpression levels is shown in Fig. 7. Here, we transfected the standard protoplasts (106) with 40 μg of empty vector (control) with 1 μg of vector encoding pre-23K + 39 μg of empty vector, or with 40 μg of pre-23K vector as used in Fig. 6. An identical number of protoplasts was simultaneously transfected with the same vector expressing pre-GFP, and after expression for 24 h, confocal microscopy revealed that 3.4 or 11% of cells were transfected with 1 or 40 μg of vector, respectively (unpublished data). We analyzed varying numbers of the protoplasts expressing pre-23K by immunoblotting with 23K antibodies, and the calculated signal intensities are shown plotted against number of protoplasts in Fig. 7, together with the immunoblot of samples from protoplasts transfected with empty vector or 40 μg of pre-23K vector. The steady decrease in signal intensity with dilution shows that the signals are within the linear range, and the plotted data show that none of the pre-23K overexpressing samples yields a signal that is significantly higher than the equivalent control sample. Even allowing for the facts that only 11% of the cells were transfected and that the stromal 23K is slightly less stable than the thylakoid-localized protein, this finding clearly indicates that the protein is not grossly overexpressed relative to the wild-type endogenous protein. An increased signal intensity would be clearly evident if the pre-23K were overexpressed by 10-fold or greater.


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

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

23K is not highly overexpressed in transfected protoplasts. (A) Tobacco protoplasts (106) were transfected with 40 μg of empty vector (Control) or 1 μg/40 μg of vector encoding pre-23K exactly as in Fig 6. After incubation for 24 h, the indicated number of cells from each incubation were analyzed by immunoblotting using antisera to spinach 23K, after which the 23K band signal intensities were calculated using a densitometer. All values are shown plotted relative to the 23K signal obtained with 250,000 cells from the sample transfected with empty vector. (B) Immunoblot of samples from control (empty vector) transfections and protoplasts transfected with 40 μg of pre-23K vector. Lanes 1–5 represent sample loadings of 250,000 cells down to 15,600 cells as in A.
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Related In: Results  -  Collection

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fig7: 23K is not highly overexpressed in transfected protoplasts. (A) Tobacco protoplasts (106) were transfected with 40 μg of empty vector (Control) or 1 μg/40 μg of vector encoding pre-23K exactly as in Fig 6. After incubation for 24 h, the indicated number of cells from each incubation were analyzed by immunoblotting using antisera to spinach 23K, after which the 23K band signal intensities were calculated using a densitometer. All values are shown plotted relative to the 23K signal obtained with 250,000 cells from the sample transfected with empty vector. (B) Immunoblot of samples from control (empty vector) transfections and protoplasts transfected with 40 μg of pre-23K vector. Lanes 1–5 represent sample loadings of 250,000 cells down to 15,600 cells as in A.
Mentions: A quantitative assessment of the overexpression levels is shown in Fig. 7. Here, we transfected the standard protoplasts (106) with 40 μg of empty vector (control) with 1 μg of vector encoding pre-23K + 39 μg of empty vector, or with 40 μg of pre-23K vector as used in Fig. 6. An identical number of protoplasts was simultaneously transfected with the same vector expressing pre-GFP, and after expression for 24 h, confocal microscopy revealed that 3.4 or 11% of cells were transfected with 1 or 40 μg of vector, respectively (unpublished data). We analyzed varying numbers of the protoplasts expressing pre-23K by immunoblotting with 23K antibodies, and the calculated signal intensities are shown plotted against number of protoplasts in Fig. 7, together with the immunoblot of samples from protoplasts transfected with empty vector or 40 μg of pre-23K vector. The steady decrease in signal intensity with dilution shows that the signals are within the linear range, and the plotted data show that none of the pre-23K overexpressing samples yields a signal that is significantly higher than the equivalent control sample. Even allowing for the facts that only 11% of the cells were transfected and that the stromal 23K is slightly less stable than the thylakoid-localized protein, this finding clearly indicates that the protein is not grossly overexpressed relative to the wild-type endogenous protein. An increased signal intensity would be clearly evident if the pre-23K were overexpressed by 10-fold or greater.

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