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Title: The uptake of proteins by the chloroplast and their subsequent routing to the thylakoid
Author: Shackleton, Jamie B.
ISNI:       0000 0001 3395 0772
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1991
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Proteins located in the chloroplast are encoded by both the chloroplast and the nuclear genomes. Those encoded by the nuclear genome are translated on cytosolic ribosomes as precursors with N-terminal pre-sequences. These pre-sequences target the proteins to their correct location within the chloroplast. Proteins destined for the thylakoid lumen contain a bipartite pre-sequence and are imported by a presumed two step process. The envelope transfer domain (ETD) targets the protein across the chloroplast envelope into the stroma where the precursor is cleaved to an intermediate by the stromal processing peptidase (SPP). This stromal intermediate is targeted by the thylakoid transfer domain (TTD) across the thylakoid membrane into the lumen where it is cleaved to the mature size by the thylakoidal processing peptidase (TPP). A cDNA clone encoding the precursor of the 33kDa protein (pre-33K) of the photosynthesis oxygen evolving complex (OEC), a nuclear encoded thylakoid lumen protein, was isolated and the section encoding the N-terminal pre-sequence was sequenced. This cDNA clone was used to confirm the two step model proposed for the targeting of thylakoid proteins. The question of whether import into the thylakoid lumen occurs via a stromal intermediate has been addressed by attempting to block the SPP recognition site using iodoacetate (IAA). By blocking the recognition of the SPP cleavage site of the precursor of the 23kDa protein (pre-23K) of the OEC, it was shown that the import of precursor protein into isolated chloroplasts and thylakoids can occur without the generation of the stromal intermediate. Import of this blocked precursor into the thylakoid lumen is assumed to occur by a one step process and represents the first demonstration of one step import. The TTD in this one step import model acts as an internal targeting signal, clearly indicating that the thylakoid targeting signal does require an N-terminal position to target a protein across the thylakoid membrane. When the pre-33K was treated with iodoacetate a stromal intermediate was generated but this failed to target across the thylakoid membrane (possibly due to a failure of the TTD to recognise the thylakoid receptor). Sequence analysis of thylakoid transfer domains has revealed a Ala X Ala conserved motif immediately before the TPP cleavage site which appears to be required for TPP cleavage. Site directed mutagenesis of the 33K precursor was used to define the reaction specificity of the TPP. A number of mutant proteins have been identified which are blocked for TPP cleavage when processed in vitro. These mutant proteins when imported into isolated chloroplasts are located in the thylakoid lumen and either processed to a 36kDa intermediate or to 33K and the 36kDa intermediate. These mutant proteins were expressed in Escherichia and were exported to the periplasmic space where they were either processed to 33K or to an intermediate slightly larger than 33K. When the reaction specificities of bacterial leader peptidase, eukaryotic signal peptidase and TPP (Fikes et al., 1990 and Folz et al., 1988 have carried out a similar site directed mutagenesis study to that carried out for TPP above) were compared, it was found that although all three peptidases possessed similar reaction specificities they were not identical.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QK Botany