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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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Both the intermediate and mature forms of GFP and 23K accumulate in the stroma. (A) Controls for thylakoid intactness and fractionation efficiency. Intact chloroplasts were isolated from tobacco protoplasts expressing pre-GFP and fractionated into stromal and thylakoid samples. The panel shows immunoblots of chloroplast (C), stromal (S), and thylakoid (T) samples using antibodies to the lumenal 33-kD photosystem II protein (33K) and to 23K. Asterisk denotes a polypeptide nonspecifically recognized by 23K antibodies in all fractions. Also shown is a Coomassie-stained gel of the same fractions, with the mobilities of molecular mass markers (kD) indicated on the left. The Rubisco large and small subunit bands (LSU and SSU) are indicated on the right together with the major component of the thylakoid light-harvesting complex (LHC). (B) A pulse-chase analysis of the transport of endogenous 33K in protoplasts that were mock transfected with empty vector. The protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were then isolated and processed to yield stromal, thylakoid, and protease-treated thylakoid (T+) samples, as detailed in Materials and methods, and subjected to immunoprecipitation using antibodies to wheat 33K protein. (C) Pre-GFP and pre-GFPΔTPP were expressed in protoplasts for 18 h as detailed in Fig. 3, after which the protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were isolated and fractionated as in B. GFP, mobility of mature GFP marker. (D) Protoplasts expressing pre-23K and pre-23KΔTPP for 18 h were then pulse labeled for 3 h, after which the chloroplasts were isolated and subsequently fractionated exactly as for GFP constructs in C. Samples were then immunoprecipitated using antibodies to 23K. i23K, intermediate form of 23K.
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fig4: Both the intermediate and mature forms of GFP and 23K accumulate in the stroma. (A) Controls for thylakoid intactness and fractionation efficiency. Intact chloroplasts were isolated from tobacco protoplasts expressing pre-GFP and fractionated into stromal and thylakoid samples. The panel shows immunoblots of chloroplast (C), stromal (S), and thylakoid (T) samples using antibodies to the lumenal 33-kD photosystem II protein (33K) and to 23K. Asterisk denotes a polypeptide nonspecifically recognized by 23K antibodies in all fractions. Also shown is a Coomassie-stained gel of the same fractions, with the mobilities of molecular mass markers (kD) indicated on the left. The Rubisco large and small subunit bands (LSU and SSU) are indicated on the right together with the major component of the thylakoid light-harvesting complex (LHC). (B) A pulse-chase analysis of the transport of endogenous 33K in protoplasts that were mock transfected with empty vector. The protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were then isolated and processed to yield stromal, thylakoid, and protease-treated thylakoid (T+) samples, as detailed in Materials and methods, and subjected to immunoprecipitation using antibodies to wheat 33K protein. (C) Pre-GFP and pre-GFPΔTPP were expressed in protoplasts for 18 h as detailed in Fig. 3, after which the protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were isolated and fractionated as in B. GFP, mobility of mature GFP marker. (D) Protoplasts expressing pre-23K and pre-23KΔTPP for 18 h were then pulse labeled for 3 h, after which the chloroplasts were isolated and subsequently fractionated exactly as for GFP constructs in C. Samples were then immunoprecipitated using antibodies to 23K. i23K, intermediate form of 23K.

Mentions: To determine the locations of the GFP forms, intact chloroplasts were isolated from transfected protoplasts and fractionated into stroma and thylakoids, after which the GFP polypeptides were again detected by immunoprecipitation. Control experiments for the effectiveness of the fractionation procedure and intactness of the thylakoid fraction are shown in Fig. 4 A. First, we immunoblotted samples of the intact chloroplasts and the stromal and thylakoid fractions obtained after lysis of the chloroplasts (Fig. 4 A, left, lanes C, S, and T) using antibodies to 23K and the lumenal 33-kD photosystem II subunit (33K, a Sec substrate). These data show that the majority of 33K and 23K are found in the thylakoid fraction, confirming that minimal breakage of thylakoids occurs during fractionation. The efficiency of the fractionation protocol is confirmed by the stained gel in this panel, which shows that the abundant large and small subunits of the stromal enzyme Rubisco are present in the stromal fraction as expected, whereas the abundant 26-kD polypeptide of the light-harvesting complex is only found in the thylakoid fraction.


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

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

Both the intermediate and mature forms of GFP and 23K accumulate in the stroma. (A) Controls for thylakoid intactness and fractionation efficiency. Intact chloroplasts were isolated from tobacco protoplasts expressing pre-GFP and fractionated into stromal and thylakoid samples. The panel shows immunoblots of chloroplast (C), stromal (S), and thylakoid (T) samples using antibodies to the lumenal 33-kD photosystem II protein (33K) and to 23K. Asterisk denotes a polypeptide nonspecifically recognized by 23K antibodies in all fractions. Also shown is a Coomassie-stained gel of the same fractions, with the mobilities of molecular mass markers (kD) indicated on the left. The Rubisco large and small subunit bands (LSU and SSU) are indicated on the right together with the major component of the thylakoid light-harvesting complex (LHC). (B) A pulse-chase analysis of the transport of endogenous 33K in protoplasts that were mock transfected with empty vector. The protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were then isolated and processed to yield stromal, thylakoid, and protease-treated thylakoid (T+) samples, as detailed in Materials and methods, and subjected to immunoprecipitation using antibodies to wheat 33K protein. (C) Pre-GFP and pre-GFPΔTPP were expressed in protoplasts for 18 h as detailed in Fig. 3, after which the protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were isolated and fractionated as in B. GFP, mobility of mature GFP marker. (D) Protoplasts expressing pre-23K and pre-23KΔTPP for 18 h were then pulse labeled for 3 h, after which the chloroplasts were isolated and subsequently fractionated exactly as for GFP constructs in C. Samples were then immunoprecipitated using antibodies to 23K. i23K, intermediate form of 23K.
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Related In: Results  -  Collection

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fig4: Both the intermediate and mature forms of GFP and 23K accumulate in the stroma. (A) Controls for thylakoid intactness and fractionation efficiency. Intact chloroplasts were isolated from tobacco protoplasts expressing pre-GFP and fractionated into stromal and thylakoid samples. The panel shows immunoblots of chloroplast (C), stromal (S), and thylakoid (T) samples using antibodies to the lumenal 33-kD photosystem II protein (33K) and to 23K. Asterisk denotes a polypeptide nonspecifically recognized by 23K antibodies in all fractions. Also shown is a Coomassie-stained gel of the same fractions, with the mobilities of molecular mass markers (kD) indicated on the left. The Rubisco large and small subunit bands (LSU and SSU) are indicated on the right together with the major component of the thylakoid light-harvesting complex (LHC). (B) A pulse-chase analysis of the transport of endogenous 33K in protoplasts that were mock transfected with empty vector. The protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were then isolated and processed to yield stromal, thylakoid, and protease-treated thylakoid (T+) samples, as detailed in Materials and methods, and subjected to immunoprecipitation using antibodies to wheat 33K protein. (C) Pre-GFP and pre-GFPΔTPP were expressed in protoplasts for 18 h as detailed in Fig. 3, after which the protoplasts were pulsed with 35S-Met and 35S-Cys for 1 h and chased for 2 h. The chloroplasts were isolated and fractionated as in B. GFP, mobility of mature GFP marker. (D) Protoplasts expressing pre-23K and pre-23KΔTPP for 18 h were then pulse labeled for 3 h, after which the chloroplasts were isolated and subsequently fractionated exactly as for GFP constructs in C. Samples were then immunoprecipitated using antibodies to 23K. i23K, intermediate form of 23K.
Mentions: To determine the locations of the GFP forms, intact chloroplasts were isolated from transfected protoplasts and fractionated into stroma and thylakoids, after which the GFP polypeptides were again detected by immunoprecipitation. Control experiments for the effectiveness of the fractionation procedure and intactness of the thylakoid fraction are shown in Fig. 4 A. First, we immunoblotted samples of the intact chloroplasts and the stromal and thylakoid fractions obtained after lysis of the chloroplasts (Fig. 4 A, left, lanes C, S, and T) using antibodies to 23K and the lumenal 33-kD photosystem II subunit (33K, a Sec substrate). These data show that the majority of 33K and 23K are found in the thylakoid fraction, confirming that minimal breakage of thylakoids occurs during fractionation. The efficiency of the fractionation protocol is confirmed by the stained gel in this panel, which shows that the abundant large and small subunits of the stromal enzyme Rubisco are present in the stromal fraction as expected, whereas the abundant 26-kD polypeptide of the light-harvesting complex is only found in the thylakoid fraction.

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