Open AccessMolecular Characterisation of Plant Endoplasmic Reticulum
Author(s) -
Coughlan Sean J.,
Hastings Craig,
Winfrey Ron J.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00215.x
Subject(s) - endoplasmic reticulum , protein disulfide isomerase , biology , complementary dna , biochemistry , microbiology and biotechnology , chaperone (clinical) , molecular cloning , northern blot , gene , medicine , pathology
Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two‐step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components. A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity‐purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide‐isomerase. Northern and Southern blot analysis showed that the mRNA from a single‐copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide‐isomerase catalytic activity, thus confirming identity of this protein.