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Query: UNIPROT:P06889 (Mol)
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The split-Ubiquitin (split-Ub) technique was used to map the molecular environment of a membrane protein in vivo. Cub, the C-terminal half of Ub, was attached to Sec63p, and Nub, the N-terminal half of Ub, was attached to a selection of differently localized proteins of the yeast Saccharomyces cerevisiae. The efficiency of the Nub and Cub reassembly to the quasi-native Ub reflects the proximity between Sec63-Cub and the Nub-labeled proteins. By using a modified Ura3p as the reporter that is released from Cub, the local concentration between Sec63-Cub-RUra3p and the different Nub-constructs could be translated into the growth rate of yeast cells on media lacking uracil. We show that Sec63p interacts with Sec62p and Sec61p in vivo. Ssh1p is more distant to Sec63p than its close sequence homologue Sec61p. Employing Nub- and Cub-labeled versions of Ste14p, an enzyme of the protein isoprenylation pathway, we conclude that Ste14p is a membrane protein of the ER. Using Sec63p as a reference, a gradient of local concentrations of different t- and v-SNARES could be visualized in the living cell. The RUra3p reporter should further allow the selection of new binding partners of Sec63p and the selection of molecules or cellular conditions that interfere with the binding between Sec63p and one of its known partners.
Mol Biol Cell 1999 Aug
PMID:Probing the molecular environment of membrane proteins in vivo. 1043 9

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.
Mol Biol Cell 1999 Aug
PMID:The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast. 1043 14

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.
Mol Cell Biol 2000 Sep
PMID:Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures. 1093 31

A fully automatic procedure for predicting the amino acid sequences compatible with a given target structure is described. It is based on the CHARMM package, and uses an all atom force-field and rotamer libraries to describe and evaluate side-chain types and conformations. Sequences are ranked by a quantity akin to the free energy of folding, which incorporates hydration effects. Exact (Branch and Bound) and heuristic optimisation procedures are used to identifying highly scoring sequences from an astronomical number of possibilities. These sequences include the minimum free energy sequence, as well as all amino acid sequences whose free energy lies within a specified window from the minimum. Several applications of our procedure are illustrated. Prediction of side-chain conformations for a set of ten proteins yields results comparable to those of established side-chain placement programs. Applications to sequence optimisation comprise the re-design of the protein cores of c-Crk SH3 domain, the B1 domain of protein G and Ubiquitin, and of surface residues of the SH3 domain. In all calculations, no restrictions are imposed on the amino acid composition and identical parameter settings are used for core and surface residues. The best scoring sequences for the protein cores are virtually identical to wild-type. They feature no more than one to three mutations in a total of 11-16 variable positions. Tests suggest that this is due to the balance between various contributions in the force-field rather than to overwhelming influence from packing constraints. The effectiveness of our force-field is further supported by the sequence predictions for surface residues of the SH3 domain. More mutations are predicted than in the core, seemingly in order to optimise the network of complementary interactions between polar and charged groups. This appears to be an important energetic requirement in absence of the partner molecules with which the SH3 domain interacts, which were not included in the calculations. Finally, a detailed comparison between the sequences generated by the heuristic and exact optimisation algorithms, commends a note of caution concerning the efficiency of heuristic procedures in exploring sequence space.
J Mol Biol 2000 Aug 18
PMID:Automatic protein design with all atom force-fields by exact and heuristic optimization. 1096 79

The increase in the p53 activity in response to DNA damage is thought to be one of the important mechanisms by which p53 contributes to transcriptional activation of p21(wafl), mdm2, and other downstream regulatory genes. To investigate the p53 response to ultraviolet (UV) type of DNA damage, p53 protein level, its transcriptional activity and in vivo ubiquitination were compared in repair-proficient normal human fibroblasts (NHFs) and repair-deficient xeroderma pigmentosum (XP) group A and group C (XP-C) fibroblasts subsequent to irradiation with UV light. Accumulation of p53 protein level was observed with increasing UV doses in all the cell lines; however, discordance between p53 and p21(waf1) and mdm2 levels was observed in NHF and XP-A cells. Induction of p21(waf1) and mdm2 was inhibited by UV irradiation, requiring higher doses in NHF and lower doses in XP-A cells. However, inhibition of p21(waf1) and mdm2 induction was not observed in XP-C cells. Ubiquitin-p53 conjugates could be detected in irradiated or unirradiated NHF and XP-A cells but not in XP-C cells irradiated with 30 and 50 J/m(2) UV light. Using a p53 reporter assay, p53 transcriptional activities were found to be induced by 10 J/m(2) UV exposure and dramatically inhibited with increasing UV doses in NHF cells. Compared with repair-proficient NHF cells, UV inhibition of p53 transcriptional activity was relatively more sensitive in XP-A cells but resistant in XP-C cells. These results indicate that DNA damage by UV, in addition to inducing p53, acts as a trigger for inhibition of p53 transcriptional activity. Overall, recognition of DNA damage links both p53 induction and p53 degradation to DNA repair mechanisms.
Mol Carcinog 2000 Aug
PMID:Modulation of transcriptional activity of p53 by ultraviolet radiation: linkage between p53 pathway and DNA repair through damage recognition. 1097 91

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.
Mol Cell Biol 2000 Oct
PMID:Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis. 1100 57

Ubiquitin-mediated degradation plays a crucial role in many fundamental biological pathways, including the mediation of cellular responses to changes in environmental conditions. A family of ubiquitin ligase complexes, called SCF complexes, found throughout eukaryotes, is involved in a variety of biological pathways. In Saccharomyces cerevisiae, an SCF complex contains a common set of components, namely, Cdc53p, Skp1p, and Hrt1p. Substrate specificity is defined by a variable component called an F-box protein. The F- box is a approximately 40-amino-acid motif that allows the F-box protein to bind Skp1p. Each SCF complex recognizes different substrates according to which F-box protein is associated with the complex. In yeasts, three SCF complexes have been demonstrated to associate with the ubiquitin-conjugating enzyme Cdc34p and have ubiquitin ligase activity. F-box proteins are not abundant and are unstable. As part of the SCF(Met30p) complex, the F-box protein Met30p represses methionine biosynthetic gene expression when availability of L-methionine is high. Here we demonstrate that in vivo SCF(Met30p) complex activity can be regulated by the abundance of Met30p. Furthermore, we provide evidence that Met30p abundance is regulated by the availability of L-methionine. We propose that the cellular responses mediated by an SCF complex are directly regulated by environmental conditions through the control of F-box protein stability.
Mol Cell Biol 2000 Nov
PMID:The abundance of Met30p limits SCF(Met30p) complex activity and is regulated by methionine availability. 1102 56

Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex. Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells. Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-gamma-S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap. To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry. Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex). Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction. Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies.
Mol Biol Cell 2000 Oct
PMID:Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. 1102 46

Selective protein degradation by the ubiquitin-proteosome pathway has recently emerged as a powerful regulatory mechanism in a wide variety of cellular processes. Ubiquitin conjugation requires the sequential activity of three enzymes or protein complexes called the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin-protein ligase (E3). In most eukaryotes, there are a small number of similar E1 isoforms without apparent functional specificity. The specific selection of target proteins is accomplished by the E2 and E3 proteins. One of the best-characterized families of E3s are the SCF complexes. The SCF is composed of a cullin (Cdc53), SKP1, RBX1 and one member of a large family of proteins called F-box proteins. The function of the F-box protein is to interact with target proteins. In some cases, the stability of the F-box protein may regulate activity of the SCF complex. In addition, post-translational modification of the cullin subunit by the ubiquitin-like protein RUB/NEDD8 appears to regulate SCF function. In plants, the SCF has so far been implicated in floral development, circadian clock, and response to the plant growth regulators auxin and jasmonic acid.
Plant Mol Biol 2000 Sep
PMID:F-box proteins and protein degradation: an emerging theme in cellular regulation. 1111 56

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.
Mol Cell 2000 Dec
PMID:Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. 1116 10


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