The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the Rpn4 transcription factor upregulates the proteasome genes and is rapidly degraded by the proteasome. Previous work has identified Ubr2 and Rad6 as the cognate E3 and E2 enzymes for Rpn4 ubiquitylation. However, our recent attempts to ubiquitylate Rpn4 using purified Ubr2 and Rad6 proteins in a reconstitution system have been unsuccessful, suggesting that an additional factor is required for Rpn4 ubiquitylation. Here, we screened the entire collection of the single-gene-deletion yeast mutants generated by the Saccharomyces Genome Deletion Project and identified the mub1Δ mutant defective in ubiquitin-dependent degradation of Rpn4. An in vitro reconstitution ubiquitylation assay confirms that Mub1 is the missing factor for Rpn4 ubiquitylation. We further show that Mub1 directly interacts with Ubr2 and Rpn4. The MYND domain of Mub1 may play an important role in Rpn4 ubiquitylation. Interestingly, Mub1 itself is a short-lived protein and its degradation is dependent on the Ubr2/Rad6 ubiquitin ligase. Together, these data suggest that Mub1 and Ubr2 cooperate to transfer ubiquitin to Rpn4 from Rad6 and that Mub1 may switch from a partner to a substrate of the Ubr2/Rad6 ubiquitin ligase.
Abstract Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a multifunctional protein that intracellularly catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate in both glycolytic and gluconeogenesis pathways and extracellularly acts as a cytokine associated with aggressive tumor phenotype. Secreted PGI/AMF promotes cancer cell invasion and metastasis by stimulating cell motility via an autocrine manner following binding to its seven-transmembrane glycoprotein receptor gp78/AMFR. Gp78/AMFR also demonstrates an additional function in the endoplasmic reticulum (ER) as ubiquitin ligase (E3). Thus, gp78/AMFR represents a potential link between ubiquitination, ER-associated degradation (ERAD) and metastasis. We questioned if gp78/AMFR is involved in the ubiquitination of PGI/AMF, and whether the ubiquitinated PGI/AMF can be related to cancer progression and metastasis. In this study, we found that indeed PGI/AMF can be ubiquitinated and gp78/AMFR is involved in this process. PGI/AMF was predominantly ubiquitinated via K29-linked ubiquitin chains. Monoubiquitinated PGI/AMF increases the secretion of PGI/AMF and this is the first report to show that monoubiquitinated PGI/AMF is secreted out of the cells. As secretion of PGI/AMF plays a significant role in tumor cell migration and invasion during metastasis, these studies assist in understanding the regulation of PGI/AMF and the mechanisms of its secretion, which may provide a new therapeutic target for tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 212. doi:1538-7445.AM2012-212
gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.
Endoplasmic-reticulum-associated protein degradation
Background The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. The integrity of the Rpn4-proteasome feedback loop is critical for cell viability under stressed conditions. We have demonstrated that inhibition of Rpn4 degradation sensitizes cells to DNA damage, particularly in response to high doses of DNA damaging agents. The underlying mechanism, however, remains unclear. Methodology/Principal Findings Using yeast genetics and biochemical approach we show that inhibition of Rpn4 degradation displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. In addition, inhibition of Rpn4 degradation leads to a defect in repair of double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ). The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced by inhibition of Rpn4 degradation. We find that Rpn4 and the proteasome are recruited to a DSB, suggesting their direct participation in NHEJ. Inhibition of Rpn4 degradation may result in a concomitant delay of release of Rpn4 and the proteasome from a DSB. Conclusion/Significance This study provides the first evidence for the role of proteasomal degradation of Rpn4 in NHEJ.
Cotranslational protein degradation plays an important role in protein quality control and proteostasis. Although ubiquitylation has been suggested to signal cotranslational degradation of nascent polypeptides, cotranslational ubiquitylation occurs at a low level, suggesting the existence of an alternative route for delivery of nascent polypeptides to the proteasome. Here we report that the nuclear import factor Srp1 (also known as importin α or karyopherin α) is required for ubiquitin-independent cotranslational degradation of the transcription factor Rpn4. We further demonstrate that cotranslational protein degradation is generally impaired in the srp1-49 mutant. Srp1 binds nascent polypeptides emerging from the ribosome. The association of proteasomes with polysomes is weakened in srp1-49. The interaction between Srp1 and the proteasome is mediated by Sts1, a multicopy suppressor of srp1-49. The srp1-49 and sts1-2 mutants are hypersensitive to stressors that promote protein misfolding, underscoring the physiological function of Srp1 and Sts1 in degradation of misfolded nascent polypeptides. This study unveils a previously unknown role for Srp1 and Sts1 in cotranslational protein degradation and suggests a novel model whereby Srp1 and Sts1 cooperate to couple proteasomes to ribosome-bound nascent polypeptides.
Introduction: SM22 (aka transgelin) is known to be an actin-binding protein that is rapidly downregulated in response to vascular injury. Despite prevalent view regarding SM22 as a maker of VSMC dedifferentiation, recently a series of studies including our own demonstrate that SM22 downregulation promotes SMC dedifferentiate into a variety of SMC subtypes involving in proliferation, migration, inflammation, osteochondrogendesis and apoptosis. Hypothesis: In the present study, we assess the hypothesis that SM22 regulates the expression of col1a2, a fibrotic marker, in SMCs. Methods: We use molecular and cellular approaches to analyze the regulation of col1a2 in smooth muscle cells upon artery injury using Sm22 knockout mice. Results: We found that carotid injury induces high expression of Col1a2 in the medial layer of the vessel wall and the nuclear translocation of serum response factor (SRF), a key transcription factor that regulates muscle-specific genes as well as growth factor response genes. To determine whether col1a2, an extracellular matrix protein, is a target of SRF, we performed bioinformatics analysis: we identified an evolutionarily conserved SRF binding sequence, CC(A/T)6GG (known as the CArG box), in the col1a2 promoter. Luciferase transfection assay showed that Vp16-SRF significantly transactivated the col1a2 promoter; mutation of the CArG box abolished most of this stimulating effect. Gel shift and chromatin precipitation assays confirmed that SRF bound to the CArG box of the col1a2 promoter. To investigate whether loss of SM22 in SMCs contributes to increased nuclear SRF expression, we knocked down SM22 in PAC1 cells, a VSMC cell line, using SM22 siRNA. Consistent with the in vivo observation, knockdown SM22 also enhanced nuclear SRF expression. However, cola2 transcription was not upregulated, suggesting that additional signals may be involved in this regulation in vivo . Conclusions: SM22 deficiency increases the fibrosis in the arterial media, and the induction of nuclear SRF may contribute at least in part to col1a2 transcription in response to artery injury.
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