ATUBP3 AND ATUBP4 ARE 2 CLOSELY-RELATED ARABIDOPSIS-THALIANA UBIQUITIN-SPECIFIC PROTEASES PRESENT IN THE NUCLEUS

The ubiquitin-specific proteases (UBPs) are a class of enzymes vital t o the ubiquitin pathway. These enzymes cleave ubiquitin at its C-termi nus from two types of substrates containing (i) ubiquitin in an alpha- amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiqui tin in an epsilon-amino linkage, as found in multiubiquitin chains eit her unattached or conjugated to cellular proteins. We have isolated cD NAs for two Arabidopsis thaliana genes, At UBP3 and AtUBP4, which enco de UBPs that are 93% identical. These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to da te in any organism. Using in vivo assays in Escherichia coli that allo w the coexpression of a UBP with a putative substrate, we have shown t hat AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial s ubstrate Ub-X-beta-gal but cannot act upon the natural alpha-amino-lin ked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquit in. Affinity-purified antibody prepared against AtUBP3 expressed in E. coli recognizes both AtUBP3 and AtUBP4. AtUBP3 and/or AtUBP4 are pres ent in all Arabidopsis organs examined and at multiple developmental s tages. Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts. Possible physiological roles for the se UBPs are discussed.