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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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Complete maturation of pre-GFP and pre-23K is totally dependent on an intact RR motif. Pre-GFP, pre-GFPΔTPP, pre–KK-GFP, pre–KK-GFPΔTPP, and pre–KK-23K were expressed in protoplasts, followed by pulse and chase treatments as described in Fig. 4. The chloroplasts were isolated and samples prepared of chloroplasts (C), protease-treated chloroplasts (C+), stroma (S), thylakoids (T), and thermolysin-treated thylakoids (T+) as for chloroplast import experiments in Fig. 2. Samples were immunoprecipitated using antibodies to GFP (A) or 23K (B).
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fig5: Complete maturation of pre-GFP and pre-23K is totally dependent on an intact RR motif. Pre-GFP, pre-GFPΔTPP, pre–KK-GFP, pre–KK-GFPΔTPP, and pre–KK-23K were expressed in protoplasts, followed by pulse and chase treatments as described in Fig. 4. The chloroplasts were isolated and samples prepared of chloroplasts (C), protease-treated chloroplasts (C+), stroma (S), thylakoids (T), and thermolysin-treated thylakoids (T+) as for chloroplast import experiments in Fig. 2. Samples were immunoprecipitated using antibodies to GFP (A) or 23K (B).

Mentions: The GFP observed in Fig. 4 is mature, which strongly suggests that it is generated by TPP, but we cannot exclude the possibility that this species is in fact generated by general stromal proteases before engaging the Tat machinery. This is unlikely because pre-GFPΔTPP is not cleaved to the mature size and this construct contains only two mutations (deletion of the terminal Ala and the NH2-terminal residue of the mature protein). Nevertheless, we substituted the RR motifs in both pre-GFP and pre-GFPΔTPP to completely block any interaction with, or translocation by, the Tat system (Fig. 1). These RR motifs are far removed from the site of cleavage and should not be involved in recognition by a hypothetical nonspecific protease that cleaves at the presequence-mature protein junction. Fig. 5 shows chloroplast fractionations from protoplasts expressing these KK mutants, denoted pre–KK-GFP and pre–KK-GFPΔTPP. Pre-GFP and pre-GFPΔTPP were analyzed simultaneously to provide markers for the intermediate and mature sizes; these behave as in Fig. 4, with the majority of imported pre-GFP found as mature GFP in the stroma and the pre-GFPΔTPP found as a mixture of the stromal- and thylakoid-bound intermediate forms. Completely different results are obtained with the KK mutants: both proteins are found exclusively as intermediate proteins in the stroma. We also analyzed a pre-23K mutant in which the RR motif was substituted by KK, and the data are shown in Fig. 5 B. As with the GFP mutant depicted in Fig. 5 A, the presence of the KK motif leads to a complete block in maturation and the intermediate protein accumulates in the stroma.


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

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

Complete maturation of pre-GFP and pre-23K is totally dependent on an intact RR motif. Pre-GFP, pre-GFPΔTPP, pre–KK-GFP, pre–KK-GFPΔTPP, and pre–KK-23K were expressed in protoplasts, followed by pulse and chase treatments as described in Fig. 4. The chloroplasts were isolated and samples prepared of chloroplasts (C), protease-treated chloroplasts (C+), stroma (S), thylakoids (T), and thermolysin-treated thylakoids (T+) as for chloroplast import experiments in Fig. 2. Samples were immunoprecipitated using antibodies to GFP (A) or 23K (B).
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Related In: Results  -  Collection

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

fig5: Complete maturation of pre-GFP and pre-23K is totally dependent on an intact RR motif. Pre-GFP, pre-GFPΔTPP, pre–KK-GFP, pre–KK-GFPΔTPP, and pre–KK-23K were expressed in protoplasts, followed by pulse and chase treatments as described in Fig. 4. The chloroplasts were isolated and samples prepared of chloroplasts (C), protease-treated chloroplasts (C+), stroma (S), thylakoids (T), and thermolysin-treated thylakoids (T+) as for chloroplast import experiments in Fig. 2. Samples were immunoprecipitated using antibodies to GFP (A) or 23K (B).
Mentions: The GFP observed in Fig. 4 is mature, which strongly suggests that it is generated by TPP, but we cannot exclude the possibility that this species is in fact generated by general stromal proteases before engaging the Tat machinery. This is unlikely because pre-GFPΔTPP is not cleaved to the mature size and this construct contains only two mutations (deletion of the terminal Ala and the NH2-terminal residue of the mature protein). Nevertheless, we substituted the RR motifs in both pre-GFP and pre-GFPΔTPP to completely block any interaction with, or translocation by, the Tat system (Fig. 1). These RR motifs are far removed from the site of cleavage and should not be involved in recognition by a hypothetical nonspecific protease that cleaves at the presequence-mature protein junction. Fig. 5 shows chloroplast fractionations from protoplasts expressing these KK mutants, denoted pre–KK-GFP and pre–KK-GFPΔTPP. Pre-GFP and pre-GFPΔTPP were analyzed simultaneously to provide markers for the intermediate and mature sizes; these behave as in Fig. 4, with the majority of imported pre-GFP found as mature GFP in the stroma and the pre-GFPΔTPP found as a mixture of the stromal- and thylakoid-bound intermediate forms. Completely different results are obtained with the KK mutants: both proteins are found exclusively as intermediate proteins in the stroma. We also analyzed a pre-23K mutant in which the RR motif was substituted by KK, and the data are shown in Fig. 5 B. As with the GFP mutant depicted in Fig. 5 A, the presence of the KK motif leads to a complete block in maturation and the intermediate protein accumulates in the stroma.

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