LeSBT1, a subtilase from tomato plants. Overexpression in insect cells, purification, and characterization.
The cDNA of a tomato subtilase designated LeSBT1 was cloned from a tomato flower cDNA library. The deduced amino acid sequence indicated for LeSBT1 the structure of a prepro-protein targeted to the secretory pathway by virtue of an amino-terminal signal peptide. LeSBT1 was expressed in the baculovirus/insect cell system and a processed 73-kDa form of LeSBT1, lacking both signal peptide and prodomain, was purified to homogeneity from culture supernatants. This 73-kDa LeSBT1, however, lacked proteolytic activity. Zymogen activation to yield 68-kDa LeSBT1 required the additional processing of an amino-terminal autoinhibitory peptide in a strictly pH-dependent manner. Mature 68-kDa LeSBT1 showed highest activity at acidic pH consistent with its presumed localization in the apoplast of the plant cell. In comparison to other plant subtilases, LeSBT1 exhibited a narrower substrate specificity in that it cleaves only polypeptide substrates preferentially but not exclusively carboxyl-terminal of glutamine residues. The possible involvement of LeSBT1 in selective proprotein processing is discussed with reference to the related mammalian proprotein convertases.
Janzik
I
I
Institute of Plant Sciences, Federal Institute of Technology Zürich, Universitätstrasse 2, CH-8092 Zürich, Switzerland.
Macheroux
P
P
Amrhein
N
N
Schaller
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000254:growth & development; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D013381:Subtilisins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013381:Subtilisins; C119297:subtilase, plant