Identification, characterization, and DNA sequence of a functional "double" groES-like chaperonin from chloroplasts of higher plants.

Abstract

Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpnlO; also known as groES). In pea (Pisum satvum), chloroplast cpnlO was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (i) form a stable complex with bacterial cpn6O in the presence ofMg'ATP.The subunit size of the pea protein is -24 kDa-about twice the size of bacterial cpnlO. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpnlO was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpnlO molecules in tandem. Moreover, both halves of this "double" cpnl'0 are highly conserved at a number of residues that are present in all cpnlOs that have been examined. Upon import into chloroplasts the spinach cpnl0 precursor is processed to its mature form of -24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpnlO are identical at 13 of 21 residues.Molecular chaperones play a vital role in protein folding and protein translocation in eukaryotic and prokaryotic cells (for a recent review see ref.

Molecular chaperones are present in the thylakoid lumen of pea chloroplasts

“…3a, lane 1). This form is also detected after import and processing of the precursor form of stromal cpn 10 into chloroplasts [9]. This indicates that the lower molecular weight form of stromal cpnl0 could represent a degradation product.…”

“…OEE33 and SSU were synthesized by overexpression in E. coli using the pET-vector system (Novagen, Madison/ USA) and purified as insoluble inclusion bodies [20]. An antiserum was raised in a rabbit against a 21 amino acids long peptide of stromal cpnl0 (DEEGNRIPLPVCSGNTVLYSC) coupled to rabbit serum albumin [9]. The antiserum to hsp70 from tomato is described in [21] (gift from L. Nover, Frankfurt).…”

“…The cpn60/cpnl0 system operating in the chloroplast stroma [9][10][11][12] is unique in comparison to other organelles or prokaryotes [13][14][15]. Stromal cpn60 exists in two isoforms ~ andfl in the chloroplasts [11,12] and its co-chaperonin 10 is comprised of two distinct cpnl0 like domains, fused in tandem to give a binary co-chaperonin structure [9,16].…”

“…Stromal cpn60 exists in two isoforms ~ andfl in the chloroplasts [11,12] and its co-chaperonin 10 is comprised of two distinct cpnl0 like domains, fused in tandem to give a binary co-chaperonin structure [9,16]. The reasons for the presence of the special composition of the stromal cpn60/cpnl0 is unknown in the moment, because it has been demonstrated that *Corresponding author.…”

Comparative Biochemical Characterization of Two GroEL Homologs from the CyanobacteriumSynechococcus elongatusPCC 7942

“…5) The presence of multiple copies of groEL is not limited to bacterial species, for chloroplasts of higher plants also possess two kinds of cpns (ch-cpn60). 6) The functions of multiple GroELs are not fully understood, except the few examples explored. 7,8) The R. leguminosarum three GroEL homologs show distinct in vitro properties, and only one of the three groEL genes is required for the growth of this organism.…”