Glycerol Gradient Centrifugation Research Articles

Analysis of mammalian circadian clock protein complexes over a circadian cycle

Circadian rhythmicity is maintained by a set of core clock proteins including the transcriptional activators CLOCK and BMAL1, and the repressors PER (PER1, PER2, and PER3), CRY (CRY1 and CRY2), and CK1δ. In mice, peak expression of the repressors in the early morning reduces CLOCK- and BMAL1-mediated transcription/translation of the repressors themselves. By late afternoon the repressors are largely depleted by degradation, and thereby their expression is reactivated in a cycle repeated every 24h. Studies have characterized a variety of possible protein interactions and complexes associated with the function of this transcription-translation feedback loop. Our prior investigation suggested there were two circadian complexes responsible for rhythmicity, one containing CLOCK-BMAL and the other containing PER2, CRY1, and CK1δ. In this investigation, we acquired data from glycerol gradient centrifugation and gel filtration chromatography of mouse liver extracts obtained at different circadian times to further characterize circadian complexes. In addition, anti-PER2 and anti-CRY1 immunoprecipitates obtained from the same extracts were analyzed by liquid chromatography-tandem mass spectrometry to identify components of circadian complexes. Our results confirm the presence of discrete CLOCK-BMAL1 and PER-CRY-CK1δ complexes at the different circadian time points, provide masses of 255 and 707kDa, respectively, for these complexes, and indicate that these complexes are composed principally of the core circadian proteins.

DDB1 and CUL4 Associated Factor 7 (DCAF7) Is Essential for Hematopoiesis

Mutations in DDB1 And CUL4 Associated Factor 7 (DCAF7) have been detected in a subset of patients with Myeloid Leukemia in Down syndrome. DCAF7 is a highly conserved WD40 domain protein with 100% homology among vertebrates and is a prominent binding partner of DYRK1A, a kinase located on chromosome 21. Analysis of the DepMap database revealed that hematopoietic cell lines are highly dependent on DCAF7 compared to cell lines from other tumors. However, the function of DCAF7 during normal hematopoiesis is currently unknown. Thus, we aimed to study its function in hematopoietic cell lines and in a conditional knockout mouse model. We first deleted DCAF7 from a panel of hematopoietic cell lines by CRISPR/Cas9 editing and observed its absence led to cell cycle arrest and decreased proliferation. Rescue experiments in which a CRISPR resistant DCAF7 allele was expressed in the targeted clones restored proliferation and cell cycling. A comparison of RNA-seq data between parental HEL cells and those without DCAF7 showed decreased expression of genes involved in differentiation, TNF signaling and the IFN pathway, indicating that DCAF7 is important for cell differentiation. Next, we created a Dcaf7 conditionally targeted mouse strain (Dcaf7F/F Mx-1 Cre) and assessed its role in hematopoiesis in vivo. Following Cre induction by pIpC, we observed a decrease in white blood cells, hemoglobin and platelet counts in KO mice. Analysis of the bone marrow showed an accumulation of LT-HSCs and multipotent progenitors. In addition, we observed a defect in the generation of B-cells, reminiscent of the phenotype observed upon Dyr1ka deletion. We then tested the recovery of Dcaf7 KO hematopoietic cells after different types of stress, which included ex-vivo culture, transplantation, and myelosuppression. Dcaf7 deficient HSCs showed defective expansion in PVA-containing media and poor differentiation in single cell colony assays in methylcellulose. In non-competitive transplants, KO cells reconstituted hematopoiesis although the number of mature myeloid and lymphoid cells was dramatically decreased. In competitive transplants, Dcaf7 KO cells were outcompeted by wild-type cells. Lastly, upon myelosuppression with 5-FU, Dcaf7 deficient mice were not able to recover and died acutely. Overall, these results indicate that DCAF7 plays a critical role in HSCs during homeostasis and regeneration. To uncover the mechanisms by which DCAF7 contributes to hematopoiesis, we performed IP-MS using tagged DCAF7 as bait in a hematopoietic cell line. In addition to DYRK1A, we found that DCAF7 interacts with multiple components of the polycomb repressive complex1 (PRC1), specifically the non-canonical PRC1.5 complex, which has been previously implicated in gene activation and transcription. We confirmed that DCAF7 engages in the PRC1.5 complex by glycerol gradient centrifugation. Upon DCAF7 absence the size the PRC1 complex shifts towards a lower molecular weight, indicating that DCAF7 is a core subunit of the complex. We then performed RNASeq analysis in sorted LT-HSCs (Lin-cKit+Sca1+CD48-CD150+) and found that 70% of the differentially expressed genes were downregulated in KO cells. These downregulated genes were associated with diminished activation of GATA1, GATA2, RUNX1 and SP1 target genes, in agreement with the functional role of the PRC1.5 complex in gene activation and cell differentiation. Together our results reveal that DCAF7 regulates hematopoiesis by participating in transcriptional regulatory complexes, and we predict that DCAF7 mutations contribute to leukemia by cooperating with DYRK1A and altering the activity of the PRC1 complex.

Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

Utilization of Grafix for the Detection of Transient Interactors of <em>Saccharomyces cerevisiae</em> Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Truncated forms of the PA protein containing only the C-terminal domains are associated with the ribonucleoprotein complex within H1N1 influenza virus particles.

We have examined the expression profile of the influenza virus PA protein in pH1N1/2009 virus-infected cells. Immunoblotting analysis of virus-infected MDCK cells revealed the presence of full-length PA protein from 8 h post-infection, together with the simultaneous appearance of PA protein species of approximately 50, 35/39 and 20/25 kDa (collectively referred to as PA*). PA* was also detected in H1N1/WSN-virus-infected cells, indicating that its presence was not virus-specific, and it was also observed in virus-infected A549 and chick embryo fibroblast (CEF) cells, indicating that its presence was not cell-type-specific. PA* was detected in cells expressing the recombinant PA protein, indicating that the PA* formation occurred in the absence of virus infection. These data collectively indicated that PA* formation is an intrinsic property of PA gene expression. The association of PA* with purified influenza virus particles was demonstrated by immunoblotting, and a protease protection assay provided evidence that PA* was packaged into virus particles. The ribonucleoprotein (RNP) complex was isolated from purified influenza virus particles using glycerol gradient centrifugation, which demonstrated that PA* was associated with the RNP complex. To the best of our knowledge, this is the first report to demonstrate that PA protein species containing only segments of the C-terminal domain form during influenza virus infection. Furthermore, these truncated PA protein species are subsequently packaged into virus particles as part of the functional RNP complex.

Protein Phosphatase-1 Regulates Expression of Neuregulin-1.

Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner.

CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.

Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component.

A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis.

trans-Acting small interfering RNAs (tasiRNAs) participate in the regulation of organ morphogenesis and determination of developmental timing in plants by down-regulating target genes through mRNA cleavage. The production of tasiRNAs is triggered by microRNA173 (miR173) and other specific microRNA-mediated cleavage of 5'-capped and 3'-polyadenylated primary TAS transcripts (pri-TASs). Although pri-TASs are not thought to encode functional proteins, they contain multiple short open reading frames (ORFs). For example, the primary TAS2 transcript (pri-TAS2) contains 11 short ORFs, and the third ORF from the 5' terminus (ORF3) encompasses the miR173 target site. Here, we show that nonsense mutations in ORF3 of pri-TAS2 upstream of the miR173 recognition site suppress tasiRNA accumulation and that ORF3 is translated in vitro. Glycerol gradient centrifugation analysis of Arabidopsis (Arabidopsis thaliana) plant extracts revealed that pri-TAS2 and its miR173-cleaved 5' and 3' fragments are fractionated together in the polysome fractions. These and previous results suggest that the 3' fragment of pri-TAS2, which is a source of tasiRNAs, forms a huge complex containing SGS3, miR173-programmed AGO1 RNA-induced silencing complex, the 5' fragment, and ribosomes. This complex overaccumulated, moderately accumulated, and did not accumulate in rdr6, sde5, and sgs3 mutants, respectively. The sgs3 sde5 and rdr6 sde5 double mutants showed phenotypes similar to those of sgs3 and sde5 single mutants, respectively, with regard to the TAS2-related RNA accumulation, suggesting that the complex is formed in an SGS3-dependent manner, somehow modified and stabilized by SDE5, and becomes competent for RDR6 action. Ribosomes in this complex likely play an important role in this process.

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM

We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.

Study on mitotic spindle and midbody extraction

Objective Mitotic spindle and midbody are both microtubule-based temporary structures during cell growth and play essential roles in mitosis.The purpose of this study was to establish a mature and efficient method to extract mitotic spindle and midbody.Methods Through the cell cycle synchronization method,mitotic spindle or midbody was made appear inside cells.Low permeability swelling and glycerol gradient centrifugation principles were then used to extract spindle and midbody.Results By Western Blot and immunofluorescence staining,the extracts were identified as mitotic spindle and midbody.Conclusions The successful extraction of mitotic spindle and midbody from synchronized Hela cells will provide foundation for identifying the proteins located in cell during mitosis,and be of great significance to the study of molecular regulation mechanisms of mitosis and tumorigenesis. Key words: Mitotic spindle; Midbody; Cell cycle synchronization; Mitosis

The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris)

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S-13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.

Adriamycin Enhances Proteasome-Mediated Generation of the Proapoptotic Processed Form of MAGE-A4 in Hepatoma Cells

Background: Melanoma antigen (MAGE)-A4 is processed to generate a C-terminal fragment with proapoptotic activity. Here we demonstrate that Adriamycin promotes generation of the processed MAGE-A4 by activating the proteasome. The proteasome is known to prevent accumulation of toxic proteins to maintain cellular homeostasis. Methods and Results: Treatment of hepatoma cells expressing MAGE-A4 with a sublethal dose of Adriamycin increased the MAGE-A4 processing and sensitized the cells to Adriamycin-induced apoptosis. The processing of MAGE-A4 was inhibited by the proteasome inhibitors MG115, MG132, lactacystin and epoxamicin. MAGE-A4 was coimmunoprecipitated with the S6 proteasomal ATPase, and present in the fractions containing the proteasome during glycerol gradient centrifugation. Consistent with the notion that the proteasome cleaves MAGE-A4, the 26S proteasome, ubiquitin, and cell lysates were necessary for efficient in vitrocleavage of MAGE-A4. Conclusions: The present study suggests that a low dose of Adriamycin increases the proteasome activity, which either maintains cellular homeostasis or leads to apoptosis depending, at least under the present conditions, on the expression of MAGE-A4.

Evidence for a Complex of Transcription Factor IIB (TFIIB) with Poly(A) Polymerase and Cleavage Factor 1 Subunits Required for Gene Looping

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

The Cdc45·Mcm2–7·GINS Protein Complex in Trypanosomes Regulates DNA Replication and Interacts with Two Orc1-like Proteins in the Origin Recognition Complex

Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.

Virion glycosylation governs integrity and infectivity of infectious pancreatic necrosis virus.

The possible importance of the O‐linked glycosylation in virion stability and infectivity of infectious pancreatic necrosis virus (IPNV) was analysed. Enzymatic treatment with O‐glycosidase of radiolabelled virions under different ionic conditions, to allow for possible alternative exposure of glycosidic enzyme cleavage sites, did not alter the specific infectivity of virions re‐isolated after rate‐zonal centrifugation in glycerol gradients. As an alternative method to assess the significance of carbohydrates in IPNV integrity, periodate oxidation in the presence of an aldehyde quencher was chosen. Following re‐isolation of viruses, a 3–5 10log‐unit reduction in specific infectivity was revealed and, at higher concentrations, a total disruption or virion aggregation was observed. The loss of infectivity of intact virions was not because of a lack of attachment to cells. Additionally, re‐evaluation of reading values from UV‐spectra of purified IPNV yielded a specific infectivity of 3 × 1011 TCID50‐units mg−1 of protein and a ratio of 40 virions per TCID50‐unit in the CHSE‐214 cell system.

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

DrosophilaSet1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.

Membrane-associated Ubiquitin Ligase Complex Containing gp78 Mediates Sterol-accelerated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway in the yeast Saccharomyces cerevisiae is mediated by two membrane-bound ubiquitin ligases, Doa10 and Hrd1. These enzymes are found in distinct multiprotein complexes that allow them to recognize and target a variety of substrates for proteasomal degradation. Although multiprotein complexes containing mammalian ERAD ubiquitin ligases likely exist, they have yet to be identified and characterized in detail. Here, we identify two ER membrane proteins, SPFH2 and TMUB1, as associated proteins of mammalian gp78, a membrane-bound ubiquitin ligase that bears significant sequence homology with mammalian Hrd1 and mediates sterol-accelerated ERAD of the cholesterol biosynthetic enzyme HMG-CoA reductase. Co-immunoprecipitation studies indicate that TMUB1 bridges SPFH2 to gp78 in ER membranes. The functional significance of these interactions is revealed by the observation that RNA interference (RNAi)-mediated knockdown of SPFH2 and TMUB1 blunts both the sterol-induced ubiquitination and degradation of endogenous reductase in HEK-293 cells. These studies mark the initial steps in the characterization of the mammalian gp78 ubiquitin ligase complex, the further elucidation of which may yield important insights into mechanisms underlying gp78-mediated ERAD.

Herp Regulates Hrd1-mediated Ubiquitylation in a Ubiquitin-like Domain-dependent Manner

Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.