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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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Pre-GFP and pre-GFPΔTPP are correctly targeted to chloroplasts in transfected tobacco protoplasts. Tobacco protoplasts were transfected with constructs expressing pre-GFP and pre-GFPΔTPP. (A) Confocal microscopy data after expression of pre-GFP for 24 h; the individual images were obtained using the red channel (shows pigment autofluorescence) or green channel for GFP fluorescence. The merged images are shown on the right. Bar, 16 μm. (B) Protoplasts expressing pre-GFP or pre-GFPΔTPP were pulse labeled with 35S-Met and 35S-Cys for 1 h and then chased with a mixture of cold Met and cold Cys for the times indicated above the lanes. The protoplasts were then lysed and subjected to immunoprecipitation using antibodies to GFP. The mobility of mature GFP is indicated (GFP) together with iGFP. Mobility of a 30-kD marker protein is indicated on the left of the autoradiograph.
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fig3: Pre-GFP and pre-GFPΔTPP are correctly targeted to chloroplasts in transfected tobacco protoplasts. Tobacco protoplasts were transfected with constructs expressing pre-GFP and pre-GFPΔTPP. (A) Confocal microscopy data after expression of pre-GFP for 24 h; the individual images were obtained using the red channel (shows pigment autofluorescence) or green channel for GFP fluorescence. The merged images are shown on the right. Bar, 16 μm. (B) Protoplasts expressing pre-GFP or pre-GFPΔTPP were pulse labeled with 35S-Met and 35S-Cys for 1 h and then chased with a mixture of cold Met and cold Cys for the times indicated above the lanes. The protoplasts were then lysed and subjected to immunoprecipitation using antibodies to GFP. The mobility of mature GFP is indicated (GFP) together with iGFP. Mobility of a 30-kD marker protein is indicated on the left of the autoradiograph.

Mentions: To study their targeting characteristics in vivo, we transiently expressed the GFP chimeras in tobacco protoplasts. 18 h after transfection, cells expressing pre-GFP and pre-GFPΔTPP constructs were analyzed by fluorescence confocal microscopy. A typical transfected protoplast expressing pre-GFP is shown in Fig. 3 A, with the GFP localized almost exclusively within the chloroplasts. The natural autofluorescence of the chlorophyll is a good visual marker for the thylakoids within the chloroplasts (Fig. 3 A, right, red channel). Perhaps surprisingly, the merged images show that GFP is present in the regions of low red fluorescence that correspond to the stroma. GFP is also visible in smaller punctate structures outside the chloroplasts; these are “stromules,” which protrude from, and form links with, other chloroplasts, as observed in a previous study (Kwok and Hanson, 2004).


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

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

Pre-GFP and pre-GFPΔTPP are correctly targeted to chloroplasts in transfected tobacco protoplasts. Tobacco protoplasts were transfected with constructs expressing pre-GFP and pre-GFPΔTPP. (A) Confocal microscopy data after expression of pre-GFP for 24 h; the individual images were obtained using the red channel (shows pigment autofluorescence) or green channel for GFP fluorescence. The merged images are shown on the right. Bar, 16 μm. (B) Protoplasts expressing pre-GFP or pre-GFPΔTPP were pulse labeled with 35S-Met and 35S-Cys for 1 h and then chased with a mixture of cold Met and cold Cys for the times indicated above the lanes. The protoplasts were then lysed and subjected to immunoprecipitation using antibodies to GFP. The mobility of mature GFP is indicated (GFP) together with iGFP. Mobility of a 30-kD marker protein is indicated on the left of the autoradiograph.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Pre-GFP and pre-GFPΔTPP are correctly targeted to chloroplasts in transfected tobacco protoplasts. Tobacco protoplasts were transfected with constructs expressing pre-GFP and pre-GFPΔTPP. (A) Confocal microscopy data after expression of pre-GFP for 24 h; the individual images were obtained using the red channel (shows pigment autofluorescence) or green channel for GFP fluorescence. The merged images are shown on the right. Bar, 16 μm. (B) Protoplasts expressing pre-GFP or pre-GFPΔTPP were pulse labeled with 35S-Met and 35S-Cys for 1 h and then chased with a mixture of cold Met and cold Cys for the times indicated above the lanes. The protoplasts were then lysed and subjected to immunoprecipitation using antibodies to GFP. The mobility of mature GFP is indicated (GFP) together with iGFP. Mobility of a 30-kD marker protein is indicated on the left of the autoradiograph.
Mentions: To study their targeting characteristics in vivo, we transiently expressed the GFP chimeras in tobacco protoplasts. 18 h after transfection, cells expressing pre-GFP and pre-GFPΔTPP constructs were analyzed by fluorescence confocal microscopy. A typical transfected protoplast expressing pre-GFP is shown in Fig. 3 A, with the GFP localized almost exclusively within the chloroplasts. The natural autofluorescence of the chlorophyll is a good visual marker for the thylakoids within the chloroplasts (Fig. 3 A, right, red channel). Perhaps surprisingly, the merged images show that GFP is present in the regions of low red fluorescence that correspond to the stroma. GFP is also visible in smaller punctate structures outside the chloroplasts; these are “stromules,” which protrude from, and form links with, other chloroplasts, as observed in a previous study (Kwok and Hanson, 2004).

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