Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238768
Title: The proteolytic processing of organellar proteins
Author: Bassham, Diane C.
ISNI:       0000 0001 3450 4563
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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Abstract:
Biogenesis of the chloroplast involves the activities of both the nuclear and chloroplast genomes. Nuclear-encoded chloroplast proteins are synthesised on free ribosomes in the cytosol and imported into the chloroplast post-translationally. Protein uptake is directed by an N-terminal presequence which is removed after import by specific processing peptidases. Thylakoid lumen proteins must cross three membranes to reach their site of function and have a composite presequence to enable their correct localisation, consisting of an envelope transit domain (ETD) and a thylakoid transfer domain (TTD). The ETD is removed after translocation across the envelope by a stromal processing peptidase (SPP) to produce an intermediate-sized protein and the TTD then allows transport into the thylakoid lumen where it is removed by a thylakoidal processing peptidase (TPP). SPP is a soluble, stromal- located metallopeptidase which also removes the presequences from stromal proteins. Despite its apparently high specificity, there is very little sequence similarity around SPP cleavage sites in stromal and thylakoidal proteins, and little is known about the structural features which SPP recognises. SPP was partially purified from pea chloroplasts and the SPP cleavage site within the presequences of three thylakoid lumen proteins was determined by radiosequencing of the cleavage products with the two-fold aim of identifying the residues around the cleavage site which SPP may recognise and of delineating the ETD and TTD regions of the presequences, allowing their comparison with signal sequences. This information was then used to create four mutant thylakoid lumen precursor proteins by site-directed mutagenesis with altered SPP processing sites. The four mutant proteins all showed a reduced rate and efficiency of processing in an organelle-free time course assay, although all were imported into chloroplasts, correctly localised and processed to the mature size in an in vitro assay. Although SPP has usually been considered to be highly specific for impoited chloroplast precursor proteins, it has recently been suggested that it may also process mitochondrial precursor proteins, which are cleaved in vivo and in vitro to the mature size by the mitochondrial processing peptidase (MPP). These two peptidases were therefore compared in terms of substrate specificity and mechanism by assaying the activity of the two enzymes against chloroplast and mitochondrial precursor proteins. MPP did not process any of the chloroplast precursor proteins available, suggesting that this enzyme is indeed highly specific for mitochondrial precursor proteins. A partially-purified SPP preparation cleaved the mitochondrial precursor proteins tested to a smaller size; however, the site of cleavage in at least some of these proteins was different to the authentic MPP cleavage site, with the stromal preparation cleaving N- terminal to MPP. This stromal processing activity was not due to mitochondrial contamination and evidence from inhibitor sensitivities and column chromatography suggests that SPP cleaves both chloroplast and mitochondrial precursor proteins. Hie SPP processing site within the presequences of two mitochondrial proteins was determined by radiosequencing and compared with the chloroplast cleavage sites, and this data should enable further analysis to determine the features which constitute a site which can be recognised by SPP. An SPP activity was also partially-purified from Chlamydomonas reinhardtii and shown to be located in the stroma. This activity is able to process chloroplast precursor proteins from C. reinhardtii and pea to an intermediate or mature size, emphasising the similarity of the specificity of SPP from these two species.
Supervisor: Not available Sponsor: Science and Engineering Research Council ; Schering Agrochemicals
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.238768  DOI: Not available
Keywords: QK Botany
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