Limits...
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.

Show MeSH

Related in: MedlinePlus

Thylakoid-associated GFPΔTPP intermediate is partially protected from proteolysis and buried within the membrane. (A) Tobacco protoplasts transfected with a plasmid encoding pre-GFPΔTPP were pulsed for 3 h with 35S-Met and 35S-Cys. Chloroplasts were isolated and fractionated, and purified thylakoids were split into eight identical aliquots and incubated either in the absence or in the presence of different proteases and detergent (Triton X-100), as indicated. TL, thermolysin; PK, proteinase K; TRP, trypsin. (B) Protoplasts expressing pre-GFP were pulsed with 35S-Met and 35S-Cys as in A, after which the stromal fraction was prepared and analyzed directly after immunoprecipitation with GFP antibodies (−TL) or after incubation with thermolysin as in A (+TL). Proteins were immunoprecipitated using anti-GFP antisera and analyzed by SDS-PAGE and fluorography.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171186&req=5

fig8: Thylakoid-associated GFPΔTPP intermediate is partially protected from proteolysis and buried within the membrane. (A) Tobacco protoplasts transfected with a plasmid encoding pre-GFPΔTPP were pulsed for 3 h with 35S-Met and 35S-Cys. Chloroplasts were isolated and fractionated, and purified thylakoids were split into eight identical aliquots and incubated either in the absence or in the presence of different proteases and detergent (Triton X-100), as indicated. TL, thermolysin; PK, proteinase K; TRP, trypsin. (B) Protoplasts expressing pre-GFP were pulsed with 35S-Met and 35S-Cys as in A, after which the stromal fraction was prepared and analyzed directly after immunoprecipitation with GFP antibodies (−TL) or after incubation with thermolysin as in A (+TL). Proteins were immunoprecipitated using anti-GFP antisera and analyzed by SDS-PAGE and fluorography.

Mentions: The data shown in Figs. 4 and 5 point to interesting differences in the fate of pre-GFP and the pre-GFPΔTPP mutant. The latter associates to a significant extent with the thylakoid membrane, and this is dependent on a functional RR signal peptide (the corresponding KK mutant does not associate at all). This membrane association was investigated further by a more detailed analysis of the protease sensitivity of the bound intermediate as shown in Fig. 8. Thylakoids were isolated after the pulse period and incubated in buffer (Fig. 8, lanes denoted by minus sign) or with thermolysin, proteinase K, or trypsin. Parallel protease incubations were performed in the presence of the detergent Triton X-100 to allow full access of the protease to the lumenal contents. The data show that a significant proportion of the GFPΔTPP is resistant to digestion by thermolysin or proteinase K, with a further population of molecules digested to a DP that is marginally smaller than mature GFP. The presence of Triton X-100 results in complete digestion of the protein, and our conclusion is that the GFPΔTPP is associated with the membrane in a location that protects the protein, either wholly or substantially, from digestion. Trypsin, on the other hand, digests all of the GFPΔTPP to the DP form and/or smaller products that are not detectable.


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

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

Thylakoid-associated GFPΔTPP intermediate is partially protected from proteolysis and buried within the membrane. (A) Tobacco protoplasts transfected with a plasmid encoding pre-GFPΔTPP were pulsed for 3 h with 35S-Met and 35S-Cys. Chloroplasts were isolated and fractionated, and purified thylakoids were split into eight identical aliquots and incubated either in the absence or in the presence of different proteases and detergent (Triton X-100), as indicated. TL, thermolysin; PK, proteinase K; TRP, trypsin. (B) Protoplasts expressing pre-GFP were pulsed with 35S-Met and 35S-Cys as in A, after which the stromal fraction was prepared and analyzed directly after immunoprecipitation with GFP antibodies (−TL) or after incubation with thermolysin as in A (+TL). Proteins were immunoprecipitated using anti-GFP antisera and analyzed by SDS-PAGE and fluorography.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Thylakoid-associated GFPΔTPP intermediate is partially protected from proteolysis and buried within the membrane. (A) Tobacco protoplasts transfected with a plasmid encoding pre-GFPΔTPP were pulsed for 3 h with 35S-Met and 35S-Cys. Chloroplasts were isolated and fractionated, and purified thylakoids were split into eight identical aliquots and incubated either in the absence or in the presence of different proteases and detergent (Triton X-100), as indicated. TL, thermolysin; PK, proteinase K; TRP, trypsin. (B) Protoplasts expressing pre-GFP were pulsed with 35S-Met and 35S-Cys as in A, after which the stromal fraction was prepared and analyzed directly after immunoprecipitation with GFP antibodies (−TL) or after incubation with thermolysin as in A (+TL). Proteins were immunoprecipitated using anti-GFP antisera and analyzed by SDS-PAGE and fluorography.
Mentions: The data shown in Figs. 4 and 5 point to interesting differences in the fate of pre-GFP and the pre-GFPΔTPP mutant. The latter associates to a significant extent with the thylakoid membrane, and this is dependent on a functional RR signal peptide (the corresponding KK mutant does not associate at all). This membrane association was investigated further by a more detailed analysis of the protease sensitivity of the bound intermediate as shown in Fig. 8. Thylakoids were isolated after the pulse period and incubated in buffer (Fig. 8, lanes denoted by minus sign) or with thermolysin, proteinase K, or trypsin. Parallel protease incubations were performed in the presence of the detergent Triton X-100 to allow full access of the protease to the lumenal contents. The data show that a significant proportion of the GFPΔTPP is resistant to digestion by thermolysin or proteinase K, with a further population of molecules digested to a DP that is marginally smaller than mature GFP. The presence of Triton X-100 results in complete digestion of the protein, and our conclusion is that the GFPΔTPP is associated with the membrane in a location that protects the protein, either wholly or substantially, from digestion. Trypsin, on the other hand, digests all of the GFPΔTPP to the DP form and/or smaller products that are not detectable.

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