Analysis of Pea Chloroplast Inner and Outer Envelope Membrane Proteins by Two-Dimensional Gel Electrophoresis and Their Comparison with Stromal Proteins

Abstract

Analysis of inner and outer pea chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the 2 membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the 2 membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by 2-dimensional gel electrophoresis. An 86 kilodalton band found in both membranes represents at least 2 different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by 2-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.