Vies, Saskia Maria van der (1989) Isolation and expression of a plastid alpha chaperonin cDNA sequence from "triticum aestivum". PhD thesis, University of Warwick.

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Abstract

This thesis describes the discovery of a new class of related proteins which has been named the chaperonins (Hemmingsen, Woolford, van der Vies, Tilly, Dennis, Georgopoulos, Hendrix & Ellis, Nature 333, 330-334, 1988). The proteins in this highly conserved class are structurally and immunologically related and ubiquitous in their occurrence in plastids, mitochondria and bacteria. The chaperonins comprise one class of the larger family of molecular chaperones since their function of assisting in the folding and assembly of other polypeptides without being components of the final structure meets the criteria suggested for molecular chaperones (Ellis, Nature 328, 378-379, 1987).

The chaperonin class of proteins was discovered during studies on the assembly of the hexadecameric enzyme ribulose-1.5-bisphosphate carboxylase-oxygenase (Rubisco); this enzyme is found in the chloroplasts of plants where it catalyzes the first step in the pathways of both photosynthesis and photorespiration. The assembly of Rubisco in vivo had been proposed to require the activity of another chloroplast protein, originally known as the Rubisco large subunit binding protein because it binds to Rubisco large subunits newly- synthesized in isolated chloroplasts of Pisum sativum (Barraclough & Ellis, Biochim. Biophys, Acta 608, 19-31, 1980). Newly-imported Rubisco small subunits have since been shown to bind to the same chloroplast protein, which has therefore been renamed the Rubisco subunit binding protein (abbreviated to binding protein), or the plastid chaperonin.

Antibodies raised against the plastid chaperonin purified from P.sativum recognize two subunit polypeptides with an apparent Mr of 61 500 and 60 500 (termed alpha and beta respectively) in extracts of Triticum aestivum leaves. With the aid of these antibodies a cDNA fragment has been isolated and sequenced from a lambda gtl 1 expression library of cDNA from leaves of Triticum aestivum. The cDNA fragment of 1834 bp encodes the entire mature plastid chaperonin alpha subunit plus two amino acids of the presequence. The amino acid sequence of the T.aestivum alpha chaperonin shows 46% identity to a protein from Escherichia coli known as the groEL protein; this protein had previously been shown to be essential for cell viability and is required for the assembly of bacteriophage capsids. An identity of 59% is found between the wheat alpha chaperonin and a groEL-like protein present in Mycobacterium leprae and M. tuberculosis. Immunologically related proteins were also detected in a variety of prokaryotes including cyanobacteria and Prochlorothrix hollandica, the eukaryote Chlamydomonas reinhardii and in mitochondrial fractions from P.sativum leaves and Solarum tuberosum tubers. All these related proteins comprise the new class of chaperonins.

Amino acid sequences of all the known chaperonins from plastids, mitochondria and bacteria were compared and show 41%-58% amino acid identity. The chloroplast alpha chaperonin is as closely related to the chloroplast beta chaperonin (amino acid identity is 50%), as it is to the bacterial and mitochondrial chaperonins. The most interesting finding to emerge from the analysis of the deduced amino acid sequences is the presence of a possible dinucleotide binding site. A highly conserved region of 36 amino acids shows 9 out of 11 matches reported for the dinucleotide binding sit fingerprint (Wierenga, Terpstra & Holl, J. Mol. Biol. 187, 101-107, 1986), but only when the chaperonin sequence is read from the carboxy to the aminoterminus and two additional amino acids are allowed. Other proteins such as the Ca2+-ATPase of the sarcoplasmic reticulum and the ecdysone-induced protein Eip 28129 of Drosophila melanogaster also contain this reversed dinucleotide binding site sequence. This finding raises the novel possibility that a given binding site can be constructed from a set of amino acids running in either direction along the polypeptide chain; this possibility should be tested for other consensus sequences.

When the T.aestivum chloroplast alpha chaperonin is synthesized in E.coli cells, it forms a hybrid oligomeric complex with the host chaperonin. The T.aestivum Rubisco large subunits that are synthesized in E.coli are found associated with either the E.coli chaperonin or with the hybrid chaperonin complex, whereas co-synthesized Rubisco small subunits bind neither to the large subunits nor to the chaperonin complexes. The T.aestivum Rubisco subunits fail to assemble into an enzymically active oligomer when synthesized in the presence of the T.aestivum chloroplast alpha chaperonin. This work is discussed in light of the conclusion emerging from studies in several laboratories that chaperonins function in many processes within the cell, the common feature of which is the requirement to prevent folding and assembly occurring between transiently exposed interactive protein surfaces.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QK Botany Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Proteins, Molecular chaperones, Photosynthesis
Official Date:	November 1989
Dates:	Date Event November 1989 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Ellis, R. J. (Reginald John), 1935-
Sponsors:	University of Warwick
Format of File:	pdf
Extent:	238, [5] leaves :illustrations
Language:	eng

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