The C-terminal extension of the beta 7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. beta 7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the alpha ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in beta subunit maturation. The effect of blm10 Delta on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4Cterminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.info:eu-repo/semantics/publishedVersio

PACemakers of proteasome core particle assembly

The 26S proteasome mediates ubiquitin-dependent proteolysis in eukaryotic cells. A number of studies including very recent ones have revealed that assembly of its 20S catalytic core particle is an ordered process that involves several conserved proteasome assembly chaperones (PACs). Two heterodimeric chaperones, PAC1-PAC2 and PAC3-PAC4, promote the assembly of rings composed of seven alpha subunits. Subsequently, P subunits join to form half-proteasome precursor complexes containing all but one of the 14 subunits. These complexes lack the beta 7 subunit but contain UMP1, another assembly chaperone, and in yeast, at least to some degree, the activator protein Blm10. Dimerization of two such complexes is triggered by incorporation of beta 7, whose C-terminal extension reaches out into the other half to stabilize the newly formed 20S particle. The process is completed by the maturation of active sites and subsequent degradation of UMP1 and PAC1-PAC2.Fundacao para a Ciencia e Tecnologia; Deutsche Forschungsgemeinschaf

Individual Risk Attitudes: New Evidence from a Large, Representative, Experimentally-Validated Survey

This paper presents new evidence on the distribution of risk attitudes in the population, using a novel set of survey questions and a representative sample of roughly 22,000 individuals living in Germany. Using a question that asks about willingness to take risks in general, on an 11-point scale, we find evidence of heterogeneity across individuals, and show that willingness to take risks is negatively related to age and being female, and positively related to height and parental education. We test the behavioral relevance of this survey measure by conducting a complementary field experiment, based on a representative sample of 450 subjects, and find that the general risk question is a good predictor of actual risk-taking behavior. We then use a more standard lottery question to measure risk preferences in our sample of 22,000, and find similar results regarding heterogeneity and determinants of risk preferences, compared to the general risk question. The lottery question also makes it possible to estimate the coefficient of relative risk aversion for each individual in the sample. Using five questions about willingness to take risks in specific domains - car driving, financial matters, sports and leisure, career, and health - the paper also studies the impact of context on risk attitudes, finding a strong but imperfect correlation across contexts. Using data on a collection of risky behaviors from different contexts, including traffic offenses, portfolio choice, smoking, occupational choice, participation in sports, and migration, the paper compares the predictive power of all of the risk measures. Strikingly, the general risk question predicts all behaviors whereas the standard lottery measure does not. The best predictor for any specific behavior is typically the corresponding context-specific measure.Risk Preferences, Experimental Validation, Field Experiment, SOEP, Gender Differences, Context, Age, Height, Subjective Well-Being, Migration, Occupational Choice, Health

SUMO-targeted ubiquitin ligases

Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved

Using a smartphone application for customer-centric banking

The banking industry is under pressure. In order to compete, banks should adapt to concentrating on the specific customer needs, following an outside-in perspective. This paper presents the design of a business model for banks that considers this development by providing flexible and comprehensive support for retail banking clients. It is demonstrated that the identification of customer processes and the consequent alignment of banking services to those processes implies great potential to increase customer retention in banking. It will be shown that information technology – especially smartphones – can serve as an interface between customer and suppliers to enable an alignment of offerings to customer processes. This approach enables the integration of banks into their customers’ lifestyle, creating emotional value added, improving the personal relationship and the customers’ affiliation with the bank. The paper presents the design of such a customer-process-centric smartphone application and derives success factors for implementation

Interaction with the Assembly Chaperone Ump1 Promotes Incorporation of the beta 7 Subunit into Half-Proteasome Precursor Complexes Driving Their Dimerization

Biogenesis of the eukaryotic 20S proteasome core particle (PC) is a complex process assisted by specific chaperones absent from the active complex. The first identified chaperone, Ump1, was found in a precursor complex (PC) called 15S PC. Yeast cells lacking Ump1 display strong defects in the autocatalytic processing of beta subunits, and consequently have lower proteolytic activity. Here, we dissect an important interaction of Ump1 with the beta 7 subunit that is critical for proteasome biogenesis. Functional domains of Ump1 and the interacting proteasome subunit beta 7 were mapped, and the functional consequences of their deletion or mutation were analyzed. Cells in which the first sixteen Ump1 residues were deleted display growth phenotypes similar to ump1 increment , but massively accumulate 15S PC and distinct proteasome intermediate complexes containing the truncated protein. The viability of these cells depends on the transcription factor Rpn4. Remarkably, beta 7 subunit overexpression re-established viability in the absence of Rpn4. We show that an N-terminal domain of Ump1 and the propeptide of beta 7 promote direct interaction of the two polypeptides in vitro. This interaction is of critical importance for the recruitment of beta 7 precursor during proteasome assembly, a step that drives dimerization of 15S PCs and the formation of 20S CPs

Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA

Polyamines are essential organic polycations with multiple cellular functions relevant for cell division, cancer and ageing(1-3). Regulation of polyamine synthesis is mainly achieved by controlling the activity of ornithine decarboxylase (ODC) through an unusual mechanism involving ODC antizyme(1,4), the binding of which disrupts homodimeric ODC and targets it for ubiquitin-independent degradation by the 26S proteasome(5). Whereas mammals express several antizyme genes(6), we have identified a single orthologue, termed OAZ1, in Saccharomyces cerevisiae(7). Similar to its mammalian counterparts, OAZ1 synthesis is induced with rising intracellular polyamine concentrations, which also inhibit ubiquitin-dependent degradation of the OAZ1 protein(7). Together, these mechanisms contribute to a homeostatic feedback regulation of polyamines(1,7,8). Antizyme synthesis involves a conserved +1 ribosomal frameshifting (RFS) event at an internal STOP codon during decoding of its messenger RNA(6-10). Here we used S. cerevisiae OAZ1 to dissect the enigmatic mechanism underlying polyamine regulation of RFS. In contrast with previous assumptions, we report here that the nascent antizyme polypeptide is the relevant polyamine sensor that operates in cis to negatively regulate upstream RFS on the polysomes, where its own mRNA is being translated. At low polyamine levels, the emerging antizyme polypeptide inhibits completion of its synthesis causing a ribosome pile-up on antizyme mRNA, whereas polyamine binding to nascent antizyme promotes completion of its synthesis. Thus, our study reveals a novel autoregulatory mechanism, in which binding of a small metabolite to a nascent sensor protein stimulates the latter's synthesis co-translationally

Proteasomal Proteomics: Identification of Nucleotide-sensitive Proteasome-interacting Proteins by Mass Spectrometric Analysis of Affinity-purified Proteasomes

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-γ-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

Proteomics analyses of microvesicles released by Drosophila Kc167 and S2 cells

Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions depending on their origin. One subtype designated circulating microvesicles (MVs) provides a novel form of intercellular communication and recent work suggested the release and uptake of morphogens in vesicles by Drosophila cells. In this study, we have examined cells of the hemocyte-like cell lines Kc167 and S2 and identified secreted vesicles in the culture supernatant. The vesicles were isolated and found to have characteristics comparable to exosomes and plasma membrane MVs released by mammalian cells. In wingless-transfected cells, the full-length protein was detected in the vesicle isolates. Proteomics analyses of the vesicles identified 269 proteins that include various orthologs of marker proteins and proteins with putative functions in vesicle formation and release. Analogous to their mammalian counterparts, the subcellular origin of the vesicular constituents of both cell lines is dominated by membrane-associated and cytosolic proteins with functions that are consistent with their localization in MVs. The analyses revealed a significant overlap of the Kc167 and S2 vesicle proteomes and confirmed a close correlation with non-mammalian and mammalian exosomes