The wealth of USPs found in the human proteome likely reflects substrate specificity, but potentially also complementation in terms of expression profiles and subcellular distribution. We therefore sought to analyze the intracellular distribution pattern of a subset of our crossreactive DUBs, using confocal microscopy. The analysis of a genome-wide set of C-terminal GFP fusion proteins for yeast had shown remarkably few with altered function or subcellular distribution (,5%), validating the choice of such Cterminal modifications [32] . Using anti-G/YFP antibodies, we also utilized this tag to assay for activity of DUBs in cell lysate. We cloned and transiently expressed five USP-EYFP constructs in 293T cells: USP5, USP13, USP14, USP3, and USP36 ( Figure 6A ). Lysate of USP14 EYFP transfected cells was incubated with the ubiquitin and the ISG15 probe, and assayed by anti-YFP immunoblot analysis ( Figure 6B ). Whereas the USP14 EYFP construct reacted with both probes, the respective C114S mutant Mutation of the catalytic cysteine residue to serine (C114S) in USP14 abolishes its reactivity toward UbVME and ISG15VS. When stored for longer periods at room temperature, the probes polymerize covalently, presumably by formation of secondary amine bonds between internal lysine residues and the reactive Michael acceptor at the C-terminus, thus resembling isopeptide-linked polyubiquitin. Such polymeric probes of UbVME likely caused the additional high-molecular mass adducts observed for USP14. Note that the smallest version of these adducts (a UbVME dimer) has a maximum electrophoretic mobility similar to that of the diubiquitin-like ISG15VS when complexed to USP14. The absence of any adducts in the C114S mutant of USP14 excludes the possibility of multiple binding sites for the probes. (C) Purified recombinant USP14 labels with UbVME and ISG15VS and results in the same abnormal mobility shift for ISG15VS-USP14 as seen in the IVT labeling experiments. (D) MALDI-TOF mass spectroscopic analysis of USP14 after incubation with ISG15VS. As described above for UbVME, ISG15VS also engages in internal polymerization. Molecular masses consistent with tri-and tetrameric ISG15VS are marked in this spectrogram by the numbers in superscript. Monovalently modified USP14 results in an adduct of predicted size, indicated with a red arrow. This complex is unique to the mixture containing both USP14 and ISG15VS, and is absent in the mass spectra of either component alone (data not shown). doi:10.1371/journal.pone.0000679.g003 did not, in agreement with the results of our IVT screen. With respect to subcellular distribution, we were particularly interested in the expression pattern of USP5 and USP13, given that these two isoforms displayed different specificity in UbVME and ISG15VS labeling experiments. USP5 EYFP was found throughout the cell ( Figure 6C, upper left panel) , similar to USP18 [33] . In contrast, its close relative USP13 EYFP was expressed mainly in the nucleus in a speckled pattern ( Figure 6C, upper right panel) . Consistent with the in vivo interaction between USP14 and the proteasome [5] , we observed USP14 EYFP predominantly in the cytoplasm, though we also noticed fluorescence in the nucleus ( Figure 6C, lower left panel) . To demonstrate that the presence of the C-terminal YFP fusion does not interfere with the endogenous distribution of these enzymes, we analyzed USP3 EYFP ( Figure 6C , lower right panel) and USP36 EYFP ( Figure 6C , lower right panel). USP3 is predicted to be a nuclear protein [34] and USP36 was . Reactivity of USP14 toward ISG15VS is augmented by proteasomal association. USP14 and USP5 were generated by IVT. Their activity toward ISG15VS and UbVME was analyzed in the presence of increasing concentrations of purified human 26S proteasomes. (A) Activity of USP14 toward UbVME and ISG15VS increases as a function of the concentration of added purified 26S proteasomes (in mg/ml). (B) The activity of USP5 remains unaffected. (C) Quantification of the radioactive signal of covalently modified USP5 and USP14. Binding affinity is depicted on the y-axis as percent in labeling intensity, determined by the ratio of labeled versus unlabeled USP5 or USP14. The ratio in the absence of exogenous proteasomes is defined as 100%. doi:10.1371/journal.pone.0000679.g004 Figure 5 . Proteasome-associated USP14 has ISG15-specific isopeptidase activity. (A) Scheme depicting the UbL-peptide conjugate used to assay isopeptidase activity. The biotinylated peptide heptamer is attached to either ISG15 or SUMO1 in isopeptide-linkage. Upon hydrolysis of the isopeptide bond by a specific DUB, the heptamer is released and the biotin signal lost. (B) Incubation of proteasomeenriched fraction (''5 hr pellet'') with UbL-peptide conjugate. After overnight incubation, the ISG15-peptide conjugate is completely cleaved, resulting in loss of the biotin-signal (significant proteolysis occurs already after one hour, data not shown). This activity is sensitive to NEM. Hydrolysis is not observed for the SUMO1-peptide conjugate. (C) Anti-HA immunoblot of HA-ISG15VS treated subcellular fractions. Based on previous identification [28] and on electrophoretic mobility, USP5 is the dominant ISG15-reactive DUB in the five-hour supernatant, which is enriched for uncomplexed proteins of light and moderate size (red asterisk). The five-hour pellet represents heavy cytosolic complexes, in particular the 26S proteasome, and contains USP14 as the only ISG15reactive DUB (blue asterisk). doi:10.1371/journal.pone.0000679.g005