Divergent N-terminal sequences of a deubiquitinating enzyme modulate substrate specificity

Ubiquitin-specific processing proteases (UBPs) are characterized by a conserved core domain with surrounding divergent sequences, particularly at the N-terminal end. We previously cloned two isoforms of a testis UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini that target the two isoforms to different subcellular locations (Lin, H., Keriel, A., Morales, C. R., Bedard, N., Zhao, Q., Hingamp, P., Lefrancois, S., Combaret, L., and Wing, S. S. (2000) Mol. Cell. Biol. 20, 6568-6578). To determine whether the N termini also influence the biochemical functions of the UBP, we expressed UBP-t1, UBP-t2, and the common core domain, UBP core, in Escherichia coli. The three isoforms cleaved branched triubiquitin at >20-fold faster rates than linear diubiquitin, suggesting that UBP-testis functions as an isopeptidase. Both N-terminal extensions inhibited the ability of UBP-core to generate free ubiquitin when linked in a peptide bond with itself, another peptide, or to small adducts. The N-terminal extension of UBP-t2 increased the ability of UBP-core to cleave branched triubiquitin. UBP-core removed ubiquitin from testis ubiquitinated proteins more rapidly than UBP-t2 and UBP-t1. Thus, UBP enzymes appear to contain a catalytic core domain, the activities and specificities of which can be modulated by N-terminal extensions. These divergent N termini can alter localization and confer multiple functions to the various members of the large UBP family.     

Compartmentalization of Mammalian Pantothenate Kinases

The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.     

Alternative splicing of Staufen2 creates the nuclear export signal for CRM1 (Exportin 1)

Mammalian Staufen2 (Stau2), a brain-specific double-stranded RNA-binding protein, is involved in the localization of mRNA in neurons. To gain insights into the function of Stau2, the subcellular localization of Stau2 isoforms fused to the green fluorescence protein was examined. Fluorescence microscopic analysis showed that Stau2 functions as a nucleocytoplasmic shuttle protein. The nuclear export of the 62-kDa isoform of Stau2 (Stau2(62)) is mediated by the double-stranded RNA-binding domain 3 (RBD3) because a mutation to RBD3 led to nuclear accumulation. On the other hand, the shorter isoform of Stau2, Stau2(59), is exported from the nucleus by two distinct pathways, one of which is RBD3-mediated and the other of which is CRM1 (exportin 1)-dependent. The nuclear export signal recognized by CRM1 was found to be located in the N-terminal region of Stau2(59). These results suggest that Stau2 may carry a variety of RNAs out of the nucleus, using the two export pathways. The present study addresses the issue of why plural Stau2 isoforms are expressed in neurons.     

RBEL1 is a novel gene that encodes a nucleocytoplasmic Ras superfamily GTP-binding protein and is overexpressed in breast cancer

Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.     

Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T

The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.     

The 14-3-3s

Multiple members of the 14-3-3 protein family have been found in all eukaryotes so far investigated, yet they are apparently absent from prokaryotes. The major native forms of 14-3-3s are homo- and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors. There are currently 133 full-length sequences available in GenBank for this highly conserved protein family. A phylogenetic tree based on the conserved middle core region of the protein sequences shows that, in plants, the 14-3-3 family can be divided into two clearly defined groups. The core region encodes an amphipathic groove that binds the multitude of client proteins that have conserved 14-3-3-recognition sequences. The amino and carboxyl termini of 14-3-3 proteins are much more divergent than the core region and may interact with isoform-specific client proteins and/or confer specialized subcellular and tissue localization.     

KRP3A and KRP3B: candidate motors in spermatid maturation in the seminiferous epithelium

We have identified KRP3, a novel kinesin-related protein expressed in the mammalian testis, and have examined the tissue distribution and subcellular localization of isoforms of this protein. Isolation of KRP3 clones, using the head domain identified in a previous PCR screen as probe, identified at least two KRP3 isoforms in the rat. We have isolated coding sequences of two highly related cDNAs from the rat testis that we have termed KRP3A and KRP3B (kinesin-related protein 3, A and B). Both cDNAs code for predicted polypeptides with the three-domain structure typical of kinesin superfamily members; namely a conserved motor domain, a region capable of forming a limited coiled-coil secondary structure, and a globular tail domain. Although almost identical in their head and stalk domains, these motors diverge in their tail domains. This group of motors is found in many tissues and cell types. The KRP3B motor contains DNA-binding motifs and an RCC1 (regulator of chromosome condensation 1) consensus sequence in its tail domain. Despite this similarity, KRP3B is not associated with the same structures as RCC1. Instead, KRP3 isoforms localize with the nuclei of developing spermatids, and their immunolocalization in the testis overlaps with that of the small GTPase Ran. Like Ran, KRP3 motors are associated in a polarized fashion with the nucleus of maturing spermatids at various stages of elongation. Our findings suggest a possible role for KRP3 motor isoforms in spermatid maturation mediated by possible interaction with the Ran GTPase.     

Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain.

Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta. We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains. Several C-terminal deletion mutants of mouse topoisomerase IIalpha were constructed and expressed in yeast top2 cells. We found that the CTD of topoisomerase IIalphais dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo. Interestingly, the CTD of topoisomerase IIbetawas also able to function as a signal for nuclear targeting. We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo . The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells. As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIalphaor IIbeta. Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIalpha-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIbeta-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus. Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly.     

Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A(2) catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.     

Spatiotemporal changes of levels of a moonlighting protein,phospholipid hydroperoxide glutathione peroxidase,in subcellular compartments during spermatogenesis in the rat testis

We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1). Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2). mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15-17; 3). nuclear labeling density suddenly increased in steps 12-14 to a maximum; 4). in cytoplasmic matrix, the density remained low in all steps; and 5). the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.     

Stage-specific expression of rat transition protein 2 mRNA and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a nonradioactive riboprobe.

The present study has used methoxyacetic acid (MAA)-induced depletion of specific germ cell types in the rat and in situ hybridization with nonradioactive riboprobes to determine the stages of the spermatogenic cycle at which there is expression of the mRNA for the basic chromosomal protein transition protein 2 (TP2). On Northern blots, an abundant mRNA was detectable in samples from control adult rats, but the amount of message was markedly reduced when RNA was extracted from the testes of rats treated 14 and 21 days previously with methoxyacetic acid. These testes were depleted specifically of step 7-12 spermatids, suggesting that these cells contain TP2 mRNA. When tissue sections were subjected to in situ hybridization, the TP2 mRNA was localized at the cellular and subcellular levels. Messenger RNA for TP2 was first detectable in spermatids at step 7. In these spermatids, at high magnification, in addition to some positive reaction in the cytoplasm, intense staining was located to a perinuclear structure consistent with localization of mRNA within the chromatoid body. The amount of TP2 mRNA in the cytoplasm increased as remodelling of the early spermatid nucleus progressed and was highest in step 10 and 11 spermatids at stages X and XI. Thereafter, the mRNA decreased until it was undetectable in step 14 spermatids at stage XIV. The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long-lived mRNAs utilized later in spermiogenesis.     

Structural and functional studies of USP20 ZnF‐UBP domain by NMR

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia‐inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N‐terminal zinc‐finger ubiquitin binding domain (ZnF‐UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF‐UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF‐UBP domain and investigated its binding properties with mono‐ubiquitin and poly‐ubiquitin (K48‐linked di‐ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF‐UBP domain forms a spherically shaped fold consisting of a central β‐sheet with either one α‐helix or two α‐helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF‐UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF‐UBP's ubiquitin binding capacity was revealed by NMR data‐driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF‐UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di‐glycine motif and the conserved and non‐conserved residues of USP20 ZnF‐UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF‐UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.     

Dissecting the sequence specific functions of alternative N-terminal isoforms of mouse bullous pemphigoid antigen 1

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins that is involved in cross-linking the cytoskeletal elements and attaching them to cell junctions. BPAG1 null mice develop severe degeneration of sensory neurons that was attributed in part due to the absence of a splice variant called BPAG1a that harbors an actin-binding domain at the N-terminus. Additional alternative splicing also results in BPAG1a isoforms with different first exons, leading to three additional types of BPAG1a called isoforms 1, 2 and 3 (or BPAG1a1, BPAG1a2, and BPAG1a3). These unique N-terminal extensions of the BPAG1a isoforms are of variable length. In this study, we characterized these N-terminal isoforms and evaluated the influence of these unique N-terminal sequences to the actin-binding properties. The unique N-terminal region of isoform 1 is very short and was not expected to affect the property of the ABD that followed it. In contrast, transfection studies and mutagenesis analyses signified that the N-terminal sequences of isoform 2 had the ability to bundle actin filaments and the N-terminal region that contained isoform 3 showed cortical localization. Isoforms 1, 2 and 3 also displayed differential tissue expression profiles. Taken together, these data suggested that the unique N-terminal regions of these isoforms have different roles that may be tailored to meet tissue specific functions.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.     

Subcellular translocation signals regulate Geminin activity during embryonic development

BACKGROUND INFORMATION: Geminin (Gem) is a protein with roles in regulating both the fidelity of DNA replication and cell fate during embryonic development. The distribution of Gem is predominantly nuclear in cells undergoing the cell cycle. Previous studies have demonstrated that Gem performs multiple activities in the nucleus and that regulation of Gem activation requires nuclear import in at least one context. In the present study, we defined structural and mechanistic features underlying subcellular localization of Gem and tested whether regulation of the subcellular localization of Gem has an impact on its activity in cell fate specification during embryonic development. RESULTS: We determined that nuclear localization of Gem is dependent on a bipartite NLS (nuclear localization signal) in the N-terminus of Xenopus Gem protein. This bipartite motif mapped to a Gem N-terminal region previously shown to regulate neural cell fate acquisition. Microinjection into Xenopus embryos demonstrated that import-deficient Gem was incapable of modulating ectodermal cell fate, but that this activity was rescued by fusion to a heterologous NLS. Cross-species comparison of Gem protein sequences revealed that the Xenopus bipartite signal is conserved in many non-mammalian vertebrates, but not in mammalian species assessed. Instead, we found that human Gem employs an alternative N-terminal motif to regulate the protein's nuclear localization. Finally, we found that additional mechanisms contributed to regulating the subcellular localization of Gem. These included a link to Crm1-dependent nuclear export and the observation that Cdt1, a protein in the pre-replication complex, could also mediate nuclear import of Gem. CONCLUSIONS: We have defined new structural and regulatory features of Gem, and showed that the activity of Gem in regulating cell fate, in addition to its cell-cycle-regulatory activity, requires control of its subcellular localization. Our data suggest that rather than being constitutively nuclear, Gem may undergo nucleocytoplasmic shuttling through several mechanisms involving distinct protein motifs. The use of multiple mechanisms for modulating Gem subcellular localization is congruent with observations that Gem levels and activity must be stringently controlled during cell-cycle progression and embryonic development.     

A unique spacer domain of synaptotagmin IV is essential for Golgi localization

Synaptotagmin (Syt) family members consist of six separate domains: a short amino terminus, a single transmembrane domain, a spacer domain, a C2A domain, a C2B domain and a short carboxyl (C) terminus. Despite sharing the same domain structures, several synaptotagmin isoforms show distinct subcellular localization. Syt IV is mainly localized at the Golgi, while Syt I, a possible Ca(2+)-sensor for secretory vesicles, is localized at dense-core vesicles and synaptic-like microvesicles in PC12 cells. In this study, we sought to identify the region responsible for the Golgi localization of Syt IV by immunocytochemical and biochemical analyses as a means of defining the distinct subcellular localization of the synaptotagmin family. We found that the unique C-terminus of the spacer domain (amino acid residues 73-144) between the transmembrane domain and the C2A domain is essential for the Golgi localization of Syt IV. In addition, the short C-terminus is probably involved in proper folding of the protein, especially the C2B domain. Without the C-terminus, Syt IVdeltaC proteins are not targeted to the Golgi and seem to colocalize with an endoplasmic reticulum (ER) marker (i.e. induce crystalloid ER-like structures). On the basis of these results, we propose that the divergent spacer domain among synaptotagmin isoforms may contain certain signals that determine the final destination of each isoform.     

Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization

Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.     

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.