Plant Molecular Biology

, Volume 30, Issue 1, pp 39–50 | Cite as

Carboxyl-terminal processing protease for the D1 precursor protein: cloning and sequencing of the spinach cDNA

  • Noritoshi Inagaki
  • Yumiko Yamamoto
  • Hitoshi Mori
  • Kimiyuki Satoh
Research Article
Received:
Accepted:

Abstract

A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D1 precursor protein (pD1) of the photosystem II reaction center is a monomeric protein of about 45 kDa. Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library. In order to determine the 5′ end of the transcript, the rapid amplification of cDNA end (5′-RACE) technique was applied. By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues. By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence. This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids. The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e. (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp. PCC 6803, based on genetic experiments and (2) proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis.

Key words

cloning and sequencing C-terminal processing D1 protein photosystem II protease spinach (Spinacia oleracea) 

Abbreviations

DFP

diisopropyl fluorophosphate

kDa

kilodalton

NEM

N-ethylmaleimide

ORF

open reading frame

p-ABSF

4-(2-aminoethyl)benzenesulfonyl fluoride

PAGE

polyacrylamide gel electrophoresis

PCR

polymerase chain reaction

pD1

D1 precursor protein

PSII

photosystem II

PVDF

polyvinylidene difluoride

RACE

rapid amplification of the cDNA end

RT-PCR

reverse transcription-polymerase chain reaction

SDS

sodium dodecyl sulfate

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Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Noritoshi Inagaki
    • 1
  • Yumiko Yamamoto
    • 2
  • Hitoshi Mori
    • 3
  • Kimiyuki Satoh
    • 1
    • 2
  1. 1.Division of Biological Regulation and PhotobiologyNational Institute for Basic BiologyOkazakiJapan
  2. 2.Department of BiologyOkayama UniversityOkayamaJapan
  3. 3.School of Agricultural ScienceNagoya UniversityNagoyaJapan

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