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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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GFP and 23K constructs used in this study. In these constructs, 23K and GFP are preceded by bipartite presequences containing a chloroplast-import (“transit”) signal followed by an RR signal peptide. The figure shows the NH2-terminal sections of the signal peptides with the RR motifs in bold (mutated to KK in some mutants), together with the COOH-terminal regions of the signal peptides ending with the AXA motif specifying cleavage by TPP (TPP site). In the GFPΔTPP and 23KΔTPP mutants, the terminal Ala is deleted to block cleavage by TPP. The region underlined in the GFP constructs is the linker peptide; two Mets were introduced at the extreme COOH terminus of the 23K protein (bold) to aid in labeling the protein.
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fig1: GFP and 23K constructs used in this study. In these constructs, 23K and GFP are preceded by bipartite presequences containing a chloroplast-import (“transit”) signal followed by an RR signal peptide. The figure shows the NH2-terminal sections of the signal peptides with the RR motifs in bold (mutated to KK in some mutants), together with the COOH-terminal regions of the signal peptides ending with the AXA motif specifying cleavage by TPP (TPP site). In the GFPΔTPP and 23KΔTPP mutants, the terminal Ala is deleted to block cleavage by TPP. The region underlined in the GFP constructs is the linker peptide; two Mets were introduced at the extreme COOH terminus of the 23K protein (bold) to aid in labeling the protein.

Mentions: A variety of constructs were prepared for expression in tobacco protoplasts or in vitro import assays, as shown in Fig. 1. The coding sequence for the GFP variant mGFP5 (Siemering et al., 1996) was fused behind the presequence of pea 23K cDNA (Wales et al., 1989), with a linker of 13 amino acids from the cytochrome b6 coding sequence introduced between the 23K presequence and GFP. This construct, termed pre-GFP, was expressed in transfected tobacco protoplasts under the control of the cauliflower mosaic virus 35S promoter. A variant was generated in which the terminal residue of the presequence (Ala 73) was deleted. TPP cleaves after an Ala-Xaa-Ala consensus motif (Shackleton and Robinson, 1991), and the removal of the −1 Ala thus blocks processing; this construct is denoted by the term pre-GFPΔTPP. To assess the specificity of Tat-mediated translocation, we generated two mutants in which the essential RR motif in the signal peptide (Chaddock et al., 1995) was converted to twin-lysine (KK; pre–KK-GFP and pre–KK-GFPΔTPP). This mutation completely blocks translocation by the Tat pathway. Finally, we prepared a second set of constructs in which all the aforementioned mutations were introduced in the background of an authentic Tat substrate, pea pre-23K (Fig. 1). Two Met residues were incorporated at the extreme COOH terminus of the 23K protein (see Materials and methods) to facilitate labeling with [35S]-Met because wild-type pea 23K does not contain Met residues (indicated by “MM” in the pre-23K structure in Fig. 1). The extra Met residues do not affect the targeting characteristics of pre-23K in chloroplast import assays (unpublished data).


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

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

GFP and 23K constructs used in this study. In these constructs, 23K and GFP are preceded by bipartite presequences containing a chloroplast-import (“transit”) signal followed by an RR signal peptide. The figure shows the NH2-terminal sections of the signal peptides with the RR motifs in bold (mutated to KK in some mutants), together with the COOH-terminal regions of the signal peptides ending with the AXA motif specifying cleavage by TPP (TPP site). In the GFPΔTPP and 23KΔTPP mutants, the terminal Ala is deleted to block cleavage by TPP. The region underlined in the GFP constructs is the linker peptide; two Mets were introduced at the extreme COOH terminus of the 23K protein (bold) to aid in labeling the protein.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171186&req=5

fig1: GFP and 23K constructs used in this study. In these constructs, 23K and GFP are preceded by bipartite presequences containing a chloroplast-import (“transit”) signal followed by an RR signal peptide. The figure shows the NH2-terminal sections of the signal peptides with the RR motifs in bold (mutated to KK in some mutants), together with the COOH-terminal regions of the signal peptides ending with the AXA motif specifying cleavage by TPP (TPP site). In the GFPΔTPP and 23KΔTPP mutants, the terminal Ala is deleted to block cleavage by TPP. The region underlined in the GFP constructs is the linker peptide; two Mets were introduced at the extreme COOH terminus of the 23K protein (bold) to aid in labeling the protein.
Mentions: A variety of constructs were prepared for expression in tobacco protoplasts or in vitro import assays, as shown in Fig. 1. The coding sequence for the GFP variant mGFP5 (Siemering et al., 1996) was fused behind the presequence of pea 23K cDNA (Wales et al., 1989), with a linker of 13 amino acids from the cytochrome b6 coding sequence introduced between the 23K presequence and GFP. This construct, termed pre-GFP, was expressed in transfected tobacco protoplasts under the control of the cauliflower mosaic virus 35S promoter. A variant was generated in which the terminal residue of the presequence (Ala 73) was deleted. TPP cleaves after an Ala-Xaa-Ala consensus motif (Shackleton and Robinson, 1991), and the removal of the −1 Ala thus blocks processing; this construct is denoted by the term pre-GFPΔTPP. To assess the specificity of Tat-mediated translocation, we generated two mutants in which the essential RR motif in the signal peptide (Chaddock et al., 1995) was converted to twin-lysine (KK; pre–KK-GFP and pre–KK-GFPΔTPP). This mutation completely blocks translocation by the Tat pathway. Finally, we prepared a second set of constructs in which all the aforementioned mutations were introduced in the background of an authentic Tat substrate, pea pre-23K (Fig. 1). Two Met residues were incorporated at the extreme COOH terminus of the 23K protein (see Materials and methods) to facilitate labeling with [35S]-Met because wild-type pea 23K does not contain Met residues (indicated by “MM” in the pre-23K structure in Fig. 1). The extra Met residues do not affect the targeting characteristics of pre-23K in chloroplast import assays (unpublished data).

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