Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating via their C-propeptides (C-terminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen ‘mini-gene’ which lacks part of the triple-helical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the C-propeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed suggested that inter-chain disulphide bonds had formed within the C-telopeptide (C-terminal telopeptide). However, when hydroxylation of prolyl and lysyl residues was inhibited no inter-chain disulphide bonds were formed in the C-telopeptide, indicating that hydroxylation is required for the initial nucleation of the triple-helical domain. Mutant chains which lacked the ability to form inter-chain disulphide bonds within the C-propeptide or the C-telopeptide could still assemble to form trimeric triple-helical molecules linked by inter-chain disulphide bonds within the N-propeptide (N-terminal propeptide). These results indicate that inter-chain disulphide bond formation within the C-propeptide or the C-telopeptide is not required for chain association and triple-helix formation.
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Research Article|
July 01 1996
Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation
Neil J. BULLEID;
Neil J. BULLEID
1School of Biological Sciences, University of Manchester, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, U.K.
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Richard WILSON;
Richard WILSON
1School of Biological Sciences, University of Manchester, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, U.K.
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Janice F. LEES
Janice F. LEES
1School of Biological Sciences, University of Manchester, 2.205, Stopford Building, Oxford Road, Manchester M13 9PT, U.K.
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Publisher: Portland Press Ltd
Received:
January 26 1996
Revision Received:
February 26 1996
Accepted:
March 01 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 195–202.
Article history
Received:
January 26 1996
Revision Received:
February 26 1996
Accepted:
March 01 1996
Citation
Neil J. BULLEID, Richard WILSON, Janice F. LEES; Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation. Biochem J 1 July 1996; 317 (1): 195–202. doi: https://doi.org/10.1042/bj3170195
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