Ubiquitin-specific proteases from Arabidopsis thaliana: cloning of AtUBP5 and analysis of substrate specificity of AtUBP3, AtUBP4, and AtUBP5 using Escherichia coli in vivo and in vitro assays.
A cDNA for a new ubiquitin-specific protease (UBP), AtUBP5, was identified from Arabidopsis thaliana flower mRNA using an oligonucleotide made against the conserved UBP cysteine (Cys) box. The 924-amino-acid AtUBP5 contains the regions characteristic of all UBPs and has 35% identity and 53% similarity overall to a mammalian UBP (Unp), resulting from additional significant similarity outside these regions. AtUBP5 has 48% identity and 58% similarity overall to two uncharacterized Arabidopsis genomic sequences but is distinct outside the UBP conserved regions from two other previously published Arabidopsis UBPs, AtUBP3 and -4. Using in vivo Escherichia coli assays, which allow co-expression of GSTAtUBPs and substrates, we show that all three UBPs were active. AtUBP5 was active without 311 amino acids N-terminal to the active site cysteine, or without 233 nonconserved amino acids between the Cys and His boxes, or without both, indicating the core region was sufficient. In in vivo and in vitro assays, GSTAtUBP3, -4, and -5 exhibited preference for specific Ub-Ub linkages, suggesting accessibility and/or conformation is important and demonstrating that these enzymes cleave post-translationally. A chimeric UBP consisting of the AtUBP5 Cys box with AtUBP3 amino acids was active and exhibited AtUBP3 specificity, indicating that the modular nature of UBPs and specificity for cleavage sites is not determined by the Cys box.
Rao-Naik
C
C
Section of Molecular and Cellular Biology, University of California-Davis, One Shields Avenue, Davis, California 95616, USA.
Chandler
J S
JS
McArdle
B
B
Callis
J
J
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins; D015139:Blotting, Southern; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D064570:Ubiquitin-Specific Proteases; D014452:Ubiquitins / Q000031:analogs & derivatives / Q000378:metabolism*
D029681:Arabidopsis Proteins; D007527:Isoenzymes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C491066:AtUBP3 protein, Arabidopsis; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases