Nucleotide sequence of a cDNA clone encoding the precursor of the 33 kDa protein of the oxygen-evolving complex from wheat

Abstract

The light-driven oxidation of water in the thylakoid membrane is carried out by photosystem II, a multisubunit complex containing both integral and extrinsic components. The wateroxidation reaction takes place on the lumenal side of the membrane and is catalysed by a group of proteins, often termed the oxygen-evolving complex, of which three proteins of 33, 23 and 16 kDa are particularly prominent [2,5]. The 33 kDa protein is believed to stabilise an essential manganese centre, whereas the 23 kDa and 16kDa proteins probably assume regulatory functions [ 1,6]. All three proteins are synthesised in the cytosol as larger precursors containing aminoterminal pre-sequences [11] and import into the thylakoid lumen is believed to take place by a two-step mechanism. Precursor proteins are initially transported into the stroma and cleaved to intermediate forms by a stromal processing peptidase, after which these intermediates are transferred across the thylakoid membrane and cleaved to the mature sizes by a second, thylakoid proces sing peptidase [ 3 ].We have determined the N-terminal amino acid sequence of the wheat 33 kDa protein, and have used specific antisera to isolate a full-length cDNA encoding the precursor protein from a wheat leaf library in 2gtll. The nucleotide and deduced amino acid sequences of this cDNA, p33K-2, are shown in Fig. 1. Amino acid residues 80-102 precisely match the sequence determined by N-terminal Edman degradation of the purified protein; the precursor protein therefore consists of a mature protein (246 residues) preceded by a pre-sequence of 79 amino acids. The mature wheat protein shows a high degree of conservation with the corresponding pea [9, 10] and spinach [7] proteins, with 82~o and 81~o of the residues being identical, respectively.The pre-sequence of the wheat 33 kDa protein is shorter than those of the corresponding spinach and pea proteins, by 5 and 2 residues, respectively. The intermediate cleavage site has yet to be determined, but it is believed that the envelope transfer and thylakoid transfer domains are of approximately equal length [3]. A comparison of the wheat and spinach pre-sequences is shown in Fig. 2; overall, these sequences exhibit considerably less homology (54 ~o residue identity) than do the mature sequences, but it is notable that the second, thylakoid transfer domains are more highly conserved than the envelope transfer domains. Only the amino-terminal sections of the envelope transfer domains are highly conserved, although it may be significant that the positions of basic residues are also conserved. The thylakoidThe nucleotide sequence data reported will appear in the EMBL GenBank and DDBJ Nucleotide Sequence Databases under the accession number X57408.

Characterization of Proteins Responsive to Gibberellin in the Leaf-Sheath of Rice (Oryza sativa L.) Seedling Using Proteome Analysis.

“…28) LS237 was identical to the oxygen-evolving enhancer protein-1 precursor from Triticum aestivum and stabilized the manganese cluster. 29) Proteins LS030 and LS034 were matched with heat-shock family proteins, namely endoplasmic reticulum (ER) and lumenal binding proteins (BiP), which act as molecular chaperones. 30) LS079 and LS083 were identical to calreticulin, which is a Ca 2ϩ -binding protein of the ER, and also functions as a molecular chaperone.…”

Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

“…p23K2A was made by digesting p23K2A-GUS with EcoRI and ApaI and cloning the fragment encoding 23K2A into pGEM11Zf1. A clone for the precursor of the 33-kD polypeptide (Meadows et al, 1991) was used as template to produce p23K2A-33K using primers D (introducing an ApaI site at the 59 of the transit peptide) and E (incorporating the tag sequence, a stop codon, and an NsiI site) in PCR. The amplified product was cut (ApaI/NsiI) and used to replace GUS in p23K2A-GUS.…”

A chloroplast processing enzyme functions as the general stromal processing peptidase

“…Amino acid sequences of identified CPE cleavage sites. The processing sites of preRBCA (Spinacia oleracea) (10), preLHCPI (Lycopersicon esculentum) (43), preLHCPII (Triticum aestivum) (15), preOEE1 (T. aestivum) (44), preOEE2 (T. aestivum) (45) and prePC (Silene pratensis) (19) are shown. The amino acids found by N-terminal sequencing of the cleavage product of preRBCA generated by immobilized CPE-B are underlined.…”