The emerging regulatory potential of SCFMet30 -mediated polyubiquitination and proteolysis of the Met4 transcriptional activator

The yeast SCFMet30 ubiquitin ligase plays a critical role in cell division by regulating the Met4 transcriptional activator of genes that control the uptake and assimilation of sulfur into methionine and S-adenosyl-methionine. The initial view on how SCFMet30 performs its function has been driven by the assumption that SCFMet30 acts exclusively as Met4 inhibitor when high levels of methionine drive an accumulation of cysteine. We revisit this model in light of the growing evidence that SCFMet30 can also activate Met4. The notion that Met4 can be inhibited or activated depending on the sulfur metabolite context is not new, but for the first time both aspects have been linked to SCFMet30, creating an interesting regulatory paradigm in which polyubiquitination and proteolysis of a single transcriptional activator can play different roles depending on context. We discuss the emerging molecular basis and the implications of this new regulatory phenomenon

Systematic Analysis of Essential Genes Reveals Important Regulators of G Protein Signaling

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly-represented. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways

The emerging regulatory potential of SCF-mediated polyubiquitination and proteolysis of the Met4 transcriptional activator-0

Assembly between Met4 and cofactors (A), and that the dissociation of Met4 homodimers by SCF(B, ) is necessary to stabilize and/or rearrange the protein-protein interactions within the Met4/Met28/Cbf1 complex, triggering its proper assembly (B). The low abundance of SCFsuggests that proteolysis of the remaining Met4 molecule occupying SCF(B, ) is necessary for SCFrecycling (B, ). As a result, 'two stepping' with SCFcould be necessary for each round of Met4 activity at a promoter (all steps involving SCFare marked in blue). The stabilizing effect of cofactors on the SCF-Met4 interaction ('tight complex', note exposure of the UIM domain in Met4) allows Met4 inhibition by polyubiquitination only (C), unless methionine (D) or cysteine (D) is available to destabilize the tight interaction between Met4 and SCF, allowing Met4 proteolysis. Disassembly of the Met4 complex by the proteasome could link activation of methionine biosynthesis to cell division by releasing the DNA binding cofactors Cbf1 and Met31/32 from promoters (D), which, at least in the case of Cbf1, would make it available for its cell division role. Dissociation of Met4 prior to its proteolysis could protect the cofactors and SCFfrom effects of the proteasome-mediated disassembly (D), preventing Cbf1 and/or Met31/32 release. See text for details.<p><b>Copyright information:</b></p><p>Taken from "The emerging regulatory potential of SCF-mediated polyubiquitination and proteolysis of the Met4 transcriptional activator"</p><p>http://www.celldiv.com/content/3/1/11</p><p>Cell Division 2008;3():11-11.</p><p>Published online 25 Jul 2008</p><p>PMCID:PMC2526995.</p><p></p

Release of Ubiquitin-Charged Cdc34-S‫ف‬Ub from the RING Domain Is Essential for Ubiquitination of the SCF Cdc4 -Bound Substrate Sic1

case of HECT (homology to E6-AP C terminus)-typ

PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

<div><p>Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.</p></div

Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28) regulator during early antiviral response mediated by IFNβ in mouse pancreatic β-cells.

Autoimmune destruction of insulin producing pancreatic β-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic β-cells. Of special interest to immunoproteasome activation in β-cells are the effects of IFNβ, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic β1(i), β2(i) and β5(i), and 11S mRNAs upon exposure to IFNβ or IFNγ. Higher concentrations of IFNβ than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNβ-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic β-cells exposed to IFNβ, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function