Degradation of the 32-kilodalton thylakoid-membrane polypeptide of Chlamydomonas reinhardi Y-1.

Isolated thylakoid membranes of Chlamydomonas reinhardi Y-1 with the 32-kDa polypeptide either radioactively labelled or unlabelled were incubated in vitro under various conditions in order to gain information about the degradation of the 32-kDa polypeptide. The degradation was higher at pH 6 compared with pH 7 and pH 8 and exhibited a temperature maximum between 20° C and 25° C (pH 6, pH 8). A light-dependent part of the total degradation was linearly dependent on white light of energy fluence rate between 1 and 20 mW·cm(-2) at 25° C and leveled out at higher fluence rates. The degradation in light was only slightly stimulated by ATP but was reduced by 3-(3'-4'-dichlorophenyl)-1,1-dimethylurea. Adenosine-5'-diphosphate and heparin (2.7 mM and 200 μg per 100 μl, respectively) known to inhibit kinases, caused a 50% decrease in degradation indicating that a phosphorylation step is involved in degradating the 32-kDa polypeptide. Out of various inhibitors specific for different types of proteases, only those for thiol- and endoproteases showed intense effects. These results point to a proteolytic degradation of the 32-kDa polypetide by a thylakoid-membrane-bound thiol-endoprotease. Its activity yields soluble breakdown products with relative molecular masses (Mrs) of 23, 16.5, 11.3 and 10.7 kDa, and these are accumulated in the in-vitro system. Partial proteolytic digestion of thylakoids with Staphylococcus aureus V8 protease results in at least two labelled breakdown products (Mrs 23, and 16.5 kDa). It is assumed that cleaving at identical amino-acid residues of the 32-kDa polypeptide by the thylakoid-membrane-bound thiolendoprotease and the V8 protease results in these two breakdown products. They are derived from subsequent cleavage at amino-acid residues 60-242 and 60-189 according to the deduced protein sequence (Erickson et al. 1984, EMBO J. 3, 2753-2762).