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

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

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

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

A Conserved Protein with AN1 Zinc Finger and Ubiquitin-like Domains Modulates Cdc48 (p97) Function in the Ubiquitin-Proteasome Pathway

Regulated protein degradation mediated by the ubiquitin-proteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1-type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1-proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48(Npl4-Ufd1) complex leads to much greater impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes

Dual role of a GTPase conformational switch for membrane fusion by mitofusin ubiquitylation

Mitochondria are essential organelles whose function is upheld by their dynamic nature. This plasticity is mediated by large dynamin-related GTPases, called mitofusins in the case of fusion between two mitochondrial outer membranes. Fusion requires ubiquitylation, attached to K398 in the yeast mitofusin Fzo1, occurring in atypical and conserved forms. Here, modelling located ubiquitylation to alpha 4 of the GTPase domain, a critical helix in Ras-mediated events. Structure-driven analysis revealed a dual role of K398. First, it is required for GTP-dependent dynamic changes of alpha 4. Indeed, mutations designed to restore the conformational switch, in the absence of K398, rescued wild-type-like ubiquitylation on Fzo1 and allowed fusion. Second, K398 is needed for Fzo1 recognition by the pro-fusion factors Cdc48 and Ubp2. Finally, the atypical ubiquitylation pattern is stringently required bilaterally on both involved mitochondria. In contrast, exchange of the conserved pattern with conventional ubiquitin chains was not sufficient for fusion. In sum, alpha 4 lysines from both small and large GTPases could generally have an electrostatic function for membrane interaction, followed by posttranslational modifications, thus driving membrane fusion events

Phenotypes on demand via switchable target protein degradation in multicellular organisms

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation

Multivalent interactions of the SUMO-interaction motifs in RING finger protein 4 determine the specificity for chains of the SUMO

RNF4 (RING finger protein 4) is a STUbL [SUMO (small ubiquitin-related modifier)-targeted ubiquitin ligase] controlling PML (promyelocytic leukaemia) nuclear bodies, DNA double strand break repair and other nuclear functions. In the present paper, we describe that the sequence and spacing of the SIMs (SUMO-interaction motifs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length