Cloning of the cDNA for glutaredoxin, an abundant sieve-tube exudate protein from Ricinus communis L. and characterisation of the glutathione-dependent thiol-reduction system in sieve tubes.
Sieve-tube exudate protein (STEP) from Ricinus communis L. seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum was raised. This antiserum cross-reacted with dominant protein species (molecular weights 10-30 kDa) present in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species. For further elucidation of the nature of individuals STEPs in the sieve tubes the anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA. Two clones that differed in the 3' untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences. Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate. Michaelis Menten constants (Km) for reduced glutathione and cysteine were 2 mM and 50 microns, respectively. Besides L-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate. Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations (up to 3 mM) with a ratio of total to oxidised glutathione of 3:1. It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins.
Szederkényi
J
J
Universität Bayreuth, Germany.
Komor
E
E
Schobert
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002367:Ricinus communis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant; D015870:Gene Expression; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010088:Oxidoreductases; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010947:Plants, Toxic; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism
D018076:DNA, Complementary; D018744:DNA, Plant; D054477:Glutaredoxins; D010940:Plant Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D010088:Oxidoreductases; D005978:Glutathione