MOLECULAR CHARACTERIZATION OF PLANT ENDOPLASMIC-RETICULUM - IDENTIFICATION OF PROTEIN DISULFIDE-ISOMERASE AS THE MAJOR RETICULOPLASMIN

Purified endoplasmic reticulum devoid of contaminating endomembranes h as been isolated from both germinating and developing castor bean endo sperm by a modified two-step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractio nated into lumenal and integral membrane protein fractions, and antise ra raised to these two components. A cDNA clone encoding a major lumen al protein of 55 kDa was cloned using affinity-purified antisera and s hown to encode a protein with strong sequence similarity to the endopl asmic reticulum lumenal chaperone protein disulfide-isomerase. Norther n and Southern blot analysis showed that the mRNA from a single-copy g ene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abun dant lumenal protein. Expression of the recombinant protein in Escheri chia coli yielded a homodimer with a molecular mass of 110 kDa with pr otein disulfide-isomerase catalytic activity, thus confirming identity of this protein.