THE D1 C-TERMINAL PROCESSING PROTEASE OF PHOTOSYSTEM-II FROM SCENEDESMUS-OBLIQUUS - PROTEIN-PURIFICATION AND GENE CHARACTERIZATION IN WILD-TYPE AND PROCESSING MUTANTS

Polypeptide D1 of the photosystem II reaction center of oxygenic photo synthesis is expressed in precursor form (pre-D1), and it must be prot eolytically processed at its C terminus to enable assembly of the mang anese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtite r plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus . Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is pre sented. A comparison of the gene product with homologous proteases poi nts to a region of conserved residues that likely corresponds to the a ctive site of a new class of serine protease, The LF-1 mutant strain o f Scenedesmus (isolated by Dr, Norman Bishop) is incapable of processi ng pre-D1. We show here that the C-terminal processing protease gene i n this strain contains a single base deletion that causes a frame shif t and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophi c and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations prov ide the first definitive proof that the enzyme isolated is responsible for in vivo proteolytic processing of pre-D1 and that no other protea se can compensate for its loss.