52 research outputs found

    Hsp70 and Hsp110 chaperones promote early steps of proteasome assembly

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    Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete alpha-ring that functions as a template for beta subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of alpha subunits in S. cerevisiae. Instead, we found that the CP subunits alpha 1, alpha 2, and alpha 4 each form independent small complexes. Purification of such complexes containing alpha 4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing alpha 1, alpha 2, and alpha 4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (alpha 3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with alpha subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual alpha subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.info:eu-repo/semantics/publishedVersio

    Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone

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    The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we elucidate the structural role of these chaperones in late steps of core particle biogenesis using biochemical, electron microscopy, cross-linking and mass spectrometry analyses. In 15S precursor complexes, Ump1 is largely unstructured, lining the inner cavity of the complex along the interface between alpha and beta subunits. The alpha and beta subunits form loosely packed rings with a wider alpha ring opening than in the 20S core particle, allowing for the Pba1-Pba2 heterodimer to be partially embedded in the central alpha ring cavity. During biogenesis, the heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening. In this way, the Pba1-Pba2 chaperone is recycled for a new round of proteasome assembly

    Regulatory mechanisms controlling biogenesis of ubiquitin and the proteasome

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    Analysis of several Saccharomyces cerevisiae ump mutants with defects in ubiquitin (Ub)-mediated proteolysis yielded insights into the regulation of the polyubiquitin gene UB14 and of proteasome genes. High-molecular weight Ub-protein conjugates accumulated in ump mutants with impaired proteasome function with a concomitant decrease in the amount of free Ub. In these mutants, transcriptional induction of UB14 was depending in part on the transcription factor Rpn4. Deletion of UB14 partially suppressed the growth defects of ump1 mutants, indicating that accumulation of polyubiquitylated proteins is deleterious to cell growth. Transcription of proteasome subunit genes was induced in ump mutants affecting the proteasome, as well as under conditions that mediate DNA damage or the formation of abnormal proteins. This induction required the transcriptional activator Rpn4. Elevated Rpn4 levels in proteasome-deficient mutants or as a response to abnormal proteins were due to increased metabolic stability. Up-regulation of proteasome genes in response to DNA damage, in contrast, is shown to operate via induction of RPN4 transcription. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.info:eu-repo/semantics/publishedVersio

    Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1

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    SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions

    Measuring Trust: Experiments and Surveys in Contrast and Combination

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    Trust is a concept that has attracted significant attention in economic theory and research within the last two decades: it has been applied in a number of contexts and has been investigated both as an explanatory and as a dependent variable. In this paper, we explore the questions of what exactly is measured by the diverse survey-derived scales and experiments claiming to measure trust, and how these different measures are related. Using nationally representative data, we test a commonly used experimental measure of trust for robustness to a number of interferences, finding it to be mostly unsusceptible to stake size, the extent of strategy space, the use of the strategy method, and the characteristics of the experimenters. Inspired by criticism of the widespread trust question used in many surveys, we created a new, improved survey trust scale consisting of three short statements. We show that the dimension of this scale is distinct from trust in institutions and trust in known others. Our new scale is a valid and reliable measure of trust in strangers. The scale is valid in the sense that it correlates with trusting behaviour in the experiment. Both survey and experimental measure correlate with related factors such as risk aversion, being an entrepreneur or a shareholder. Furthermore, we demonstrate that the survey measure's test-retest reliability (six weeks) is high. The experimental measure of trust is, on the other hand, not significantly correlated with trust in institutions nor with trust in known others. We conclude that the experimental measure of trust refers not to trust in a general sense, but specifically to trust in strangers
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