Biology Reference
In-Depth Information
1
Semipermeabilized Cells
to Study Procollagen Assembly
Richard R. Wilson and Neil J. Bulleid
1. Introduction
This chapter will describe the preparation and use of a semipermeabilized
(SP) cell system that reconstitutes the initial stages in the assembly and modi-
fication of proteins entering the secretory pathway
(1)
. The procedure involves
treating cells grown in culture with the detergent digitonin and isolating the
cells free from their cytosolic component
(2)
. The expression of proteins in an
SP cell system allows protein assembly to be studied in an environment that
more closely resembles that of the intact cell. As this is an in vitro system, the
individual components can be manipulated easily, providing a means by which
cellular processes can be studied under a variety of conditions. In addition,
membrane-permeable chemical crosslinking reagents can be added in order to
facilitate the study of interaction between proteins within the endoplasmic
reticulum (ER) lumen
(1
,
3)
. Furthermore, as the ER remains morphologically
intact, the spatial localization of folding and transport processes within the
reticular network may also be examined.
The basic protocol involves translation of an mRNA transcript encoding the
protein of interest in a rabbit reticulocyte lysate supplemented with the SP
HT1080 cells prepared as outlined below (
Subheadings 3.1.
to
3.3.
). This
particular cell line was initially chosen because it can carry out the complex
co- and post-translational modifications required for the assembly of
procollagen molecules into thermally stable triple helices
(4)
. Other cell lines
have been used to study the initial stages in the biosynthesis of a wide range of
proteins demonstrating the flexibility of this approach. The mRNA transcript
coding for the protein of interest is translated in the presence of a radio-labeled
amino acid (
35
S-methionine) such that the protein synthesized can be visual-
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