Sequence of the cDNA Encoding the 17-Kilodalton Protein of the Photosynthetic Oxygen-Evolving Complex of Pea

Abstract

The OEC2 of PSII is the enzyme responsible for the photosynthetic cleavage of water and the evolution of molecular oxygen. The OEC consists of at least three nuclear-encoded proteins, OE33, OE23, and OE 17, with associated Mn-, Ca , and Cl ions. The OEC proteins are tightly bound to the lumenal face of the thylakoid membrane-embedded PSII core proteins. OE 17 is the 1 7-kD protein of the OEC in eukaryotic plants; it is homologous to the OEE3 gene in Chlamrydomonas reinhardtii. OE 17 has been shown to enhance rates of oxygen evolution, possibly by binding Ca>, and Cl ions (7). However, the individual contributions of the OEC proteins to the oxygen evolution process have yet to be completely determined (4). The biogenesis of OE 17 is a complex process. The protein is translated in the cytosol on unbound polyribosomes as a precursor containing a large N-terminal transit peptide. The precursor is posttranslationally translocated through the outer and inner chloroplast envelope membranes. In the stroma a portion of the transit peptide is thought to be removed by a stromal peptidase resulting in a protein of intermediate molecular mass. The OE17 intermediate is translocated across the thylakoid membrane and is concomitantly processed to its mature molecular weight by a thylakoid peptidase (10). OE 17 accumulates as a pool of unassembled subunits free in the thylakoid lumen before eventual assembly into an active OEC (2). Translocation of OE 17 into isolated intact chloroplasts and across isolated thylakoid membranes has been studied using precursor proteins synthesized by sequential in vitro transcription-translation systems (1, 8). Translocation of the protein across the chloroplast envelope membranes is an ATP dependent reaction (10). Transit of the OE17 stromal intermediate across the thylakoid membrane into the lumen is solely dependent on a trans-thylakoid proton gradient (1). The transport of both OE 17 and OE23 across the thylakoid membrane is noteworthy in that these processes represent the first energy-dependent protein translocation events that do not require ATP hydrolysis. The cDNA for pea OE 17 (Table I, Fig. 1) was isolated from a pea leaf X gtO cDNA library (3) using a maize OE 1 7 cDNA as a heterologous probe (R. Sayre). The cDNA was subcloned into the EcoRl site of the plasmid pGEM 7Zf(+). The nucleotide sequence of both strands of the cDNA were determined by dideoxy sequencing. The cDNA is 888 bp long and includes a 51 bp untranslated 5' region, a 651 bp open reading frame, and a 187 bp untranslated 3' end bearing a 76 bp poly(A) tail. The 651 bp open reading frame encodes a 217 amino acid precursor protein with a molecular mass of 23.1 kD and a pl of 8.4. The first eight residues of the open reading frame, MAQAMASM..., are identical with the first eight residues of the OE17 precursor from spinach (5) indicating