Characterization of envelope membrane polypeptides from spinach chloroplasts.

Chloroplast envelope polypeptides from spinach were separated into several fractions on the basis of their solubility in a mixture of chloroform/methanol (2:1, v/v) or in 0.1 N NaOH. Using a simple two dimension procedure for gel electrophoresis, we resolved up to 70 polypeptides. The envelope is characterized by the preponderance of high molecular weight polypeptides (Mr > 54,000), many of which are not extracted by NaOH, indicating that they are integral membrane proteins. The polypeptide pattern of envelope membranes is very different from that of the stroma or of the thylakoids: very few polypeptides from the three fractions have identical mobility in the different electrophoretic systems used. Most of the major envelope polypeptides are peripheral proteins. Two of these, E14 and E54, were identified by crossed immunoelectrophoresis as the small and the large subunit of ribulose bisphosphate carboxylase, respectively, However, polypeptide E30 (involved in phosphate transport across the envelope) was the only major polypeptide to be xtracted by chloroform/methanol. This polypeptide is also an integral membrane polypeptide since it is not extracted by NaOH. Using double immunodiffusion of envelope, stroma, and thylakoids against antibodies to two integral (E30 and E10) and two peripheral (E37 and E24) envelope polypeptides, we demonstrated that these four polypeptides are not found in the thylakoid membranes or in the stroma. Furthermore, we also have shown that there is no cross-reaction between the envelope membranes and antibodies prepared against the major thylakoid membrane polypeptides (a, p, y, and S subunits of coupling factor CF1 and polypeptides 5, 6, and 11). These results do not support a direct biogenetic relation between the two chloroplast membrane systems (envelope and thylakoids). If indeed thylakoid membranes are derived from the inner envelope membranes, considerable changes must occur in the composition of the vesicles following the initial steps of membrane invagination.