Mould, Ruth M. (1992) Protein targeting to the thylakoid lumen. PhD thesis, University of Warwick.

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Abstract

Chloroplast biogenesis involves the activities of two genetic systems, the nuclear and chloroplast genomes. The majority of chloroplast proteins are synthesised on cytosolic ribosomes as precursors with N-terminal extensions known as presequences. Presequences are involved in the posttranslational targeting of such proteins to the appropriate location within the chloroplast. The targeting of thylakoid lumen (TL) proteins is particularly complex since it involves transport across the chloroplast envelope membranes, through the stroma and across the thylakoid membrane. The presequences of nuclear-encoded TL proteins have a bipartite nature, the first (most N-terminal) domain directs transport across the chloroplast envelope and is (probably) removed by stromal processing peptidase (SPP) in the stroma to yield an intermediate size protein. The second domain directs transport across the thylakoid membrane and is removed by thylakoidal processing peptidase (TPP) in the lumen yielding the mature size protein.

When this Ph.D. project commenced, very little was known about the mechanism by which proteins are transported across the thylakoid membrane. Import assays using isolated chloroplasts had led to advances in the study of protein transport across the chloroplast envelope but were of limited use for the study of the import of nuclear-encoded TL proteins since transport across the thylakoid membrane could not be studied in isolation. Kirwin et al. (1989) achieved import of a TL protein into isolate? thylakoids, thus enabling study of transport across the thylakoid membrane in isolation from that across the envelope membranes. During the course of this project, the assay system of Kirwin et al. (1989) has been improved, in terms of the efficiency of protein import by isolated thylakoids achieved, and the import of another TL protein demonstrated. This improved assay system has since been used to study the import of several other thylakoid proteins by other workers. Work presented in this thesis describes the use of this assay and the intact chloroplast assay to study the energy requirements for the import of two TL proteins, 33kDa and 23kDa components of the oxygen evolving complex. The efficient transport of both proteins across the thylakoid membrane requires a transthylakoidal proton gradient and that of 33K may also require ATP. The mechanism by which a proton gradient drives transport has yet to be determined. The ability of the thylakoid transport machinery to import the precursor form of 23K (work presented in this thesis and by J. Shackleton, University of Warwick) and artificial intermediate forms of 23K and 33K has been demonstrated. The import of 23K into isolated thylakoids can occur without the addition of any stromal factors whereas the import of 33K requires at least one stromal factor other than SPP. The requirements for the transport of several other TL and thylakoid membrane proteins have recently been studied by other workers; these studies in conjunction with the results presented in this thesis, reveal that the requirements for transport into or across the thylakoid membrane varies depending on the protein in question. Whether these proteins have a common, extremely flexible, import pathway or whether more than one import pathway for thylakoid proteins operates within the chloroplast, has yet to be determined.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QK Botany
Library of Congress Subject Headings (LCSH):	Chloroplasts -- Formation, Thylakoids, Proteins -- Physiological transport, Proteins -- Secretion, Biological transport, Escherichia coli
Official Date:	February 1992
Dates:	Date Event February 1992 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Robinson, Colin, 1958-
Sponsors:	Science and Engineering Research Council (Great Britain)
Format of File:	pdf
Extent:	xviii, 197 leaves : illustrations
Language:	eng

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