Large-scale translocation reversal within the thylakoid Tat system in vivo.
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.
Di Cola
Alessandra
A
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England, UK.
Robinson
Colin
C
D020816:Amino Acid Motifs; D002736:Chloroplasts / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010441:Peptide Chain Elongation, Translational / Q000235:genetics / Q000502:physiology*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011523:Protoplasts / Q000378:metabolism; D020524:Thylakoids / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D014026:Nicotiana / Q000378:metabolism
D026901:Membrane Transport Proteins; D010940:Plant Proteins; D049452:Green Fluorescent Proteins