Interaction of the Doa4 deubiquitinating enzyme with the yeast 26S proteasome

e Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin-proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.     

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.     

Nuclear-cytoplasmic shuttling of the oncogenic mouse UNP/USP4 deubiquitylating enzyme

The oncogenic deubiquitylating enzyme (DUB) Unp/Usp4, which binds to the retinoblastoma family of tumor suppressor proteins, was originally described as a nuclear protein. However, more recent studies have shown it to be cytoplasmic. In addition, analysis of its subcellular localization has been complicated by the existence of the paralog Usp15. In this study, we resolved this controversy by investigating the localization of exogenously expressed Usp4 (using red fluorescent protein-Usp4) and of endogenous Usp4 (using highly specific antibodies that can distinguish Usp4 from Usp15). We found that by inhibiting nuclear export with leptomycin B, both exogenous and endogenous Usp4 accumulate in the nucleus. Further, using a Rev-green fluorescent protein-based export assay, we confirmed the existence of a nuclear export signal ((133)VEVYLLELKL(142)) in Usp4. In addition, a functional nuclear import signal ((766)QPQKKKK(772)) was also identified, which was specifically recognized by importin alpha/beta. Finally, we show that the equilibrium of Usp4 subcellular localization varies between different cell types. Usp4 is thus the first DUB reported to have nucleocytoplasmic shuttling properties. The implications of this shuttling for its function as a DUB are discussed.     

A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14

A C-terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site-directed probe that irreversibly modifies a subset of Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [(125)I]UbVS labeling with N-ethylmaleimide and Ub aldehyde, but also by genetic analysis. [(125)I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [(125)I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S-associated USP14 with [(125)I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.     

Multivalent endosome targeting by homodimeric EEA1.

Early endosome autoantigen localization to early endosomes is mediated by a C-terminal region, which includes a calmodulin binding motif, a Rab5 interaction site, and a FYVE domain that selectively binds phosphatidyl inositol 3-phosphate. The crystal structure of the C-terminal region bound to inositol 1,3-bisphosphate reveals an organized, quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal localization in which domain organization, dimerization, and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues. A unique mode of membrane engagement deduced from the quaternary structure of the C-terminal region provides insight into the structural basis of endosome tethering.     

Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme

Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell. A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit. Here, we report the X-ray crystal structure of an Ubp3-Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer interacts with two L-shaped beta-strand-turn-alpha-helix motifs of Ubp3. The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through antiparallel beta-sheet hydrogen bonds and van der Waals contacts. Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3-Bre5 association in vitro and Ubp3 function in vivo. Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3-Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.     

A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.     

The FYVE domain of early endosome antigen 1 is required for both phosphatidylinositol 3-phosphate and Rab5 binding. Critical role of this dual interaction for endosomal localization

Early endosome antigen 1 (EEA1) is 170-kDa polypeptide required for endosome fusion. EEA1 binds to both phosphtidylinositol 3-phosphate (PtdIns3P) and to Rab5-GTP in vitro, but the functional role of this dual interaction at the endosomal membrane is unclear. Here we have determined the structural features in EEA1 required for binding to these ligands. We have found that the FYVE domain is critical for both PtdIns3P and Rab5 binding. Whereas PtdIns3P binding only required the FYVE domain, Rab5 binding additionally required a 30-amino acid region directly adjacent to the FYVE domain. Microinjection of glutathione S-transferase fusion constructs into Cos cells revealed that the FYVE domain alone is insufficient for localization to cellular membranes; the upstream 30-amino acid region required for Rab5 binding must also be present for endosomal binding. The importance of Rab5 in membrane binding of EEA1 is underscored by the finding that the increased expression of wild-type Rab5 increases endosomal binding of EEA1 and decreases its dependence on PtdIns3P. Thus, the levels of Rab5 are rate-limiting for the recruitment of EEA1 to endosome membranes. PtdIns3P may play a role in modulating the Rab5 EEA1 interaction.     

The N-Terminal Domains of Vps3 and Vps8 Are Critical for Localization and Function of the CORVET Tethering Complex on Endosomes

Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.     

A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host-pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein--its binding partner within replication complexes--leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.     

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

Biochemical characterization of a multidomain deubiquitinating enzyme Ubp15 and the regulatory role of its terminal domains

Deubiquitinating enzymes (DUBs) have emerged as essential players in a myriad of cellular processes, yet the regulation of DUB function remains largely unknown. While some DUBs rely on the formation of complex for regulation of enzymatic activity, many DUBs utilize interdomain interactions to regulate catalysis. Here we report the biochemical characterization of a multidomain deubiquitinating enzyme, Ubp15, from Saccharomyces cerevisiae. Steady-state kinetic investigation showed that Ubp15 is a highly active DUB. We identified active-site residues that are required for catalysis. We have also identified key residues on Ubp15 required for ubiquitin binding and catalysis. We further demonstrated that Ubp15's enzymatic activity is regulated by the N- and C-terminal domains that flank the catalytic core domain. Moreover, we demonstrated that Ubp15 physically interacts with a WD40 repeat-containing protein, Cdh1, by copurification experiments. Interestingly, unlike other DUBs that specifically interact with WD40 repeat-containing proteins, Cdh1 does not function in stimulating Ubp15's activity. The possible cellular function of Ubp15 in cell cycle regulation is discussed in view of the specific interaction between Ubp15 and Cdh1, an activator of the anaphase-promoting complex/cyclosome (APC/C).     

A Global Census of Fission Yeast Deubiquitinating Enzyme Localization and Interaction Networks Reveals Distinct Compartmentalization Profiles and Overlapping Functions in Endocytosis and Polarity

Ubiquitination and deubiquitination are reciprocal processes that tune protein stability, function, and/or localization. The removal of ubiquitin and remodeling of ubiquitin chains is catalyzed by deubiquitinating enzymes (DUBs), which are cysteine proteases or metalloproteases. Although ubiquitination has been extensively studied for decades, the complexity of cellular roles for deubiquitinating enzymes has only recently been explored, and there are still several gaps in our understanding of when, where, and how these enzymes function to modulate the fate of polypeptides. To address these questions we performed a systematic analysis of the 20 Schizosaccharomyces pombe DUBs using confocal microscopy, proteomics, and enzymatic activity assays. Our results reveal that S. pombe DUBs are present in almost all cell compartments, and the majority are part of stable protein complexes essential for their function. Interestingly, DUB partners identified by our study include the homolog of a putative tumor suppressor gene not previously linked to the ubiquitin pathway, and two conserved tryptophan-aspartate (WD) repeat proteins that regulate Ubp9, a DUB that we show participates in endocytosis, actin dynamics, and cell polarity. In order to understand how DUB activity affects these processes we constructed multiple DUB mutants and find that a quintuple deletion of ubp4 ubp5 ubp9 ubp15 sst2/amsh displays severe growth, polarity, and endocytosis defects. This mutant allowed the identification of two common substrates for five cytoplasmic DUBs. Through these studies, a common regulatory theme emerged in which DUB localization and/or activity is modulated by interacting partners. Despite apparently distinct cytoplasmic localization patterns, several DUBs cooperate in regulating endocytosis and cell polarity. These studies provide a framework for dissecting DUB signaling pathways in S. pombe and may shed light on DUB functions in metazoans.     

The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor

Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.     

The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion.

Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.     

SNX-BAR-mediated endosome tubulation is co-ordinated with endosome maturation

Endosomal sorting is essential for cell homeostasis. Proteins targeted for degradation are retained in the maturing endosome vacuole while others are recycled to the cell surface or sorted to the biosynthetic pathway via tubular transport carriers. Sorting nexin (SNX) proteins containing a BAR (for Bin-Amphiphysin-Rvs) domain are key regulators of phosphoinositide-mediated, tubular-based endosomal sorting, but how such sorting is co-ordinated with endosomal maturation is not known. Here, using well-defined Rab GTPases as endosomal compartment markers, we have analyzed the localization of SNX1 [endosome-to-trans-Golgi network (TGN) transport as part of the SNX-BAR-retromer complex], SNX4 (cargo-recycling from endosomes to the plasma membrane) and SNX8 (endosomes-to-TGN trafficking in a retromer-independent manner). We show that these SNX-BARs are primarily localized to early endosomes, but display the highest frequency of tubule formation at the moment of early-to-late endosome transition: the Rab5-to-Rab7 switch. Perturbing this switch shifts SNX-BAR tubulation to early endosomes, resulting in SNX1-decorated tubules that lack retromer components VPS26 and VPS35, suggesting that both early and late endosomal characteristics of the endosome are important for SNX-BAR-retromer-tubule formation. We also establish that SNX4, but not SNX1 and SNX8, is associated with the Rab11-recycling endosomes and that a high frequency of SNX4-mediated tubule formation is observed as endosomes undergo Rab4-to-Rab11 transition. Our study therefore provides evidence for fine-tuning between the processes of endosomal maturation and the formation of endosomal tubules. As tubulation is required for SNX1-, SNX4- and SNX8-mediated sorting, these data reveal a previously unrecognized co-ordination between maturation and tubular-based sorting.     

Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting

Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3.     

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.     

A novel membrane targeting domain mediates the endosomal or Golgi localization specificity of small GTPases Rab22 and Rab31

Rab22 and Rab31 belong to the Rab5 subfamily of GTPases that regulates endocytic traffic and endosomal sorting. Rab22 and Rab31 (a.k.a. Rab22b) are closely related and share 87% amino acid sequence similarity, but they show distinct intracellular localization and function in the cell. Rab22 is localized to early endosomes and regulates early endosomal recycling, while Rab31 is mostly localized to the Golgi complex with only a small fraction in the endosomes at steady state. The specific determinants that affect this differential localization, however, are unclear. In this study, we identify a novel membrane targeting domain (MTD) consisting of the C-terminal hypervariable domain (HVD), interswitch loop (ISL), and N-terminal domain as a major determinant of endosomal localization for Rab22 and Rab31, as well as Rab5. Rab22 and Rab31 share the same N-terminal domain, but we find Rab22 chimeras with Rab31 HVD exhibit phenotypic Rab31 localization to the Golgi complex, while Rab31 chimeras with the Rab22 HVD localize to early endosomes, similar to wildtype Rab22. We also find that the Rab22 HVD favors interaction with the early endosomal effector protein Rabenosyn-5, which may stabilize the Rab localization to the endosomes. The importance of effector interaction in endosomal localization is further demonstrated by the disruption of Rab22 endosomal localization in Rabenosyn-5 knockout cells and by the shift of Rab31 to the endosomes in Rabenosyn-5-overexpressing cells. Taken together, we have identified a novel MTD that mediates localization of Rab5 subfamily members to early endosomes via interaction with an effector such as Rabenosyn-5.     

The Ubp6 family of deubiquitinating enzymes contains a ubiquitin-like domain: SUb.

A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases. An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving alpha-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.