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The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells

journal contribution
posted on 2023-05-17, 07:44 authored by Richard WilsonRichard Wilson, Allen, AJ, Oliver, J, Brookman, JL, High, S, Bulleid, NJ
We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results show that the intact ER within these cells can assemble proteins much more efficiently than vesicularized ER. Furthermore, the semi-permeabilized cells carried out the redox-dependent degradation of tissue-type plasminogen activator. This system has all the advantages of conventional cell-free systems, including speed and, importantly, the ability to manipulate the components of the assay, while retaining intracellular organelles and, therefore, allowing cellular processes to occur as they would in the intact cell.

History

Publication title

Biochemical Journal

Volume

307

Pagination

679-687

ISSN

0264-6021

Publisher

Portland Press

Place of publication

59 Portland Place, London, England, W1N 3Aj

Repository Status

  • Restricted

Socio-economic Objectives

Expanding knowledge in the health sciences

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