AGE-DEPENDENT APPEARANCE OF POLYPEPTIDES WITH IMMUNOREACTIVITY TO UBIQUITIN ANTIBODIES IN CHLOROPLAST MEMBRANES OF VICIA-FABA L

Thylakoid membrane proteins of differently developed chloroplasts from Vicia faba L. were separated by SDS-PAGE and immunoblotted with ubiqu itin antibodies. Samples from 9-day-old, non-expanded leaves contained an immunoreactive doublet (18/19 kD) and a 15 kD polypeptide when mem branes were isolated in the presence of the protease inhibitors p-chlo romercuribenzoic acid and leupeptin. Without these inhibitors an addit ional band of 42 kD was stained. These proteins were also found in eti oplasts during chloroplast biogenesis. Two-dimensional immunoblotting revealed a basic character of the polypeptides. Thylakoid membranes fr om 14- and 21-day-old primary leaves showed an immunoresponse at 30 kD . When no protease inhibitors were used during the preparations, some high molecular weight ubiquitin-conjugates were additionally found. Fr ee ubiquitin was not detected in any developmental stage. The results indicate that proteins bound to ubiquitin are present in the thylakoid membranes and the pattern of immunoresponding proteins is characteris tic for the developmental stage. An involvement of ubiquitin in chloro plast protein turnover is therefore assumed.