Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and
Sverige far ny biosajt vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo.
Immunopurification and tandem mass spectrometry enabled the identification of several Sverige far ny biosajt Rfp2 interacting proteins localized to the endoplasmic reticulum ERincluding valosin-containing protein VCPa protein indispensable for ER-associated degradation ERAD.
Finally, Sverige far ny biosajt show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining.
RBCC proteins are involved in diverse cellular processes, such as apoptosis Kimura et al. The RFP2 gene was initially cloned during the search for genes located in the commonly deleted 13q14 locus in B-CLL B-cell chronic lymphocytic leukemia. Considering all genes in the minimal deleted region, RFP2 is the only gene with protein encoding potential and might therefore be regarded as a prime tumor suppressor gene candidate within the CLL deletion locus at 13q14 Corcoran et al.
Of note is that this locus is also targeted for deletion in other hematological malignancies such as myeloma Shaughnessy et al. Deletions have also been reported in several solid tumors such as head and neck tumors Maestro et al. RING finger domains are cysteine- and histidine-rich motifs that bind two zinc ions.
They mediate protein—protein interactions Borden, and are frequently involved in proteolysis Pickart, B-boxes are domains with as of yet unknown function with the ability to bind one zinc ion Borden, Like RING fingers, coiled-coil domains also mediate protein—protein interactions and have furthermore been shown to be important for homo- and heterodimerization of RBCC family members Jensen et al.
Often, it seems that the entire RBCC domain is needed for proper function and intracellular localization of proteins containing these motifs, indicating that this multidomain structure acts as one
Sverige far ny biosajt unit Reymond et al. A limited number of the RBCC proteins have been implicated in proteolysis, acting as E3 ubiquitin ligases by promoting the attachment of the conserved polypeptide ubiquitin to specific substrates targeted for degradation Trockenbacher et al.
E3 ligases can be divided into two major families: RBCC proteins with E3 ligase activity accordingly form a subset of the latter family.
One important role of the ubiquitin-proteasome system is to mediate the retrotranslocation and turnover of membrane and secretory proteins from the ER through a set of processes collectively termed ERAD Meusser et al. As a general surveillance mechanism, cells utilize the ERAD machinery to degrade proteins that are misfolded McCracken and Brodsky, ; Denic et al. Additionally, proteins part of multisubunit complexes are similarly degraded when not assembled together with their normal oligomerization partner s.
An example of the latter is the degradation of individual subunits of the T-cell receptor Yang et al. Protein degradation at the level of the ER has attracted increased attention because ERAD has been implicated in a wide variety of conformational diseases, such as Parkinson's disease and Alzheimer's disease, as well as in cancer Takahashi and Imai, ; Sverige far ny biosajt et al. Still, the molecular mechanisms underlying this process are poorly understood and a limited number of E3 ubiquitin ligases of the ER have been described.
Given the important biological function of other RBCC proteins Sverige far ny biosajt the frequent loss of the RFP2 gene in CLL and other malignancies, a functional characterization of the gene is highly warranted. In the present study we present evidence that Rfp2 belongs to a previously uncharacterized subgroup of RBCC proteins containing a transmembrane domain, a domain necessary for Rfp2 to properly associate with the ER.
In conclusion, these results together define Rfp2 as a novel ER E3 ubiquitin ligase and link Rfp2 to the very limited set of proteins currently known to function as effectors of ERAD. The resin was washed five times and analyzed as described above. The lysate was subsequently diluted 10 times with NP buffer without SDS, and pulldown was performed as described above.
Cells were harvested at the indicated time points. Cell cycle distribution of Rfp2 expression was examined by flow cytometry analysis as described previously Panaretakis et al. The sequence of the cloned cDNA was verified in its entire length by sequencing. Primers used for cloning can be obtained from the authors upon request.
The following primary antibodies were used: To generate a novel mono-specific antibody recognizing Rfp2, a protein fragment from the coiled-coil region of Rfp2 amino acids — was chosen because of the low sequence similarity to all other human proteins.
Subsequently, primers were designed and used in RT-PCR, resulting in a gene encoding the protein fragment Agaton et al. Production, purification, and characterization of the protein fragment were Sverige far ny biosajt as previously described Agaton et al. Antibody purification was performed by a depletion chromatography step, where antibodies directed toward the His-ABP-part of the fusion protein were removed and a second purification allowed for capture of the specific antibodies Agaton et al.
Characterization of the antibodies was done according to a previous study Nilsson et al. Cytosolic, membranous, and nuclear extracts were prepared using the Qproteome Cell Compartment Kit according to the manufacturer's protocol Qiagen, Chatsworth, CA. Briefly, sequential addition of different buffers to cell pellets followed by incubation and centrifugation results in the isolation of different cellular compartments.
First buffer selectively disrupts the plasma membrane without solubilizing it, resulting in the isolation of cytosolic proteins. Plasma membranes and compartmentalized organelles remain intact and are pelleted by centrifugation.
The pellet from the first step is resuspended
Sverige far ny biosajt the second buffer, which solubilizes the plasma membrane as well as all organelle membranes except the nuclear membrane. This fraction contains membrane proteins including proteins from the ER membrane and proteins from the lumen of organelles e.
In the final step nuclei are solubilized using the third buffer in which all soluble and most membrane-bound nuclear proteins are extracted.
Compartment separation was analyzed by immunodetection of stated marker proteins. After the pulse, cells were washed, chased in complete medium supplemented with 10 mM methionine and 1 mM cysteine Sigma"Sverige far ny biosajt" harvested at the indicated time points in M-RIPA lysis buffer. Because Rfp2 was shown to be a strictly membrane-associated protein, soluble cytosolic proteins were discarded in order to reduce complexity of the sample.
Supernatants containing soluble cytosolic proteins were discarded, and cell pellets were lysed in NP buffer. In parallel, other control proteins were similarly immunoprecipitated and analyzed. Samples were run in different gels or some distance apart to minimize cross-contamination. Entire lanes were cut into kDa pieces, and proteins were identified by nanoflow liquid chromatography tandem mass spectrometry as described below. Only peptides exclusively present in Rfp2 immunoprecipitated samples were regarded as possible Rfp2 interacting proteins.
The high-performance liquid chromatography setup used in conjunction with the mass spectrometer LC-MS consisted of a solvent degasser, nanoflow pump, and thermostated microautosampler Agilent nanoflow system, Wilmington, DE.
Complementary fragmentation of the same peptide yielded different fragment ions that increased the Sverige far ny biosajt of the sequence information. Using this complementary information Horn et al. The new dta-files containing complementary fragment masses was merged into a single file, which was searched using the Mascot Search Engine Matrix Science, Boston, MA; Perkins et al.
Peptide searches were performed with an initial tolerance on mass measurement of 3 ppm in MS mode and 0. Only proteins uniquely identified by a minimum of two significant peptides were taken into for this study. Two milligrams of total protein was used for GST pulldown as described above.
Ubiquitinated proteins were detected by Western blot analysis using biotin antibodies. They were incubated with primary antibodies anti-Rfp2 diluted 1: Mock-transfected cells were used as negative controls. As a first step in characterizing the Rfp2 protein, epitope-tagged constructs of RFP2 were expressed in HEK cells by transient transfection. The same phenomenon with low Sverige far ny biosajt of Rfp2 protein was observed with other expression vectors, and in another cell type HeLa cells, data not shown.
Sverige far ny biosajt reason for the reduced amount of Rfp2 protein in cells could be that only a small subset of cells expresses Rfp2. However, this possibility was ruled out because flow cytometric DNA histogram analysis in HEK cells together with staining for endogenous Rfp2 expression using a specific Rfp2 antibody see below revealed that the protein is similarly expressed in all cells, regardless of cell cycle phase data not shown.
To address whether the low levels of Rfp2 protein could be due to high turnover of the protein, we next examined whether Rfp2 levels are increased in cells treated with proteasomal inhibitors.
As can be seen in Figure 1 B, treatment with the proteasomal inhibitor MG markedly increased Rfp2 protein levels. Together these data show that Rfp2 is an inherently unstable protein regulated by the proteasomal system.
In the following experiments, we focused our analysis on characterizing the Rfp2 protein. Rfp2 is an unstable protein that becomes stabilized with proteasomal inhibition. Rfp2 protein was detected with FLAG antibodies. Asterisks indicate Rfp2 conjugated with single ubiquitin molecules see below. Actin and tubulin were used as loading controls as indicated. Numbers indicate protein size in kDa. A polyclonal antibody directed against the coiled-coil domain of Rfp2 amino acids — was generated.
As can be seen in Figure 2 A, significant reduction of ectopically expressed FLAG-tagged Rfp2 was only observed in cells transfected with Rfp2-specific siRNA oligos, whereas
Sverige far ny biosajt oligos had no effect on Rfp2 protein levels. Finally, we knocked down endogenous Rfp2 protein in HEK cells, confirming the predicted size of Rfp2 and demonstrating the prominent affinity of the antibody for the Rfp2 protein Figure 2 B.
Specificity of a novel polyclonal Rfp2 antibody. Blots were developed using Rfp2-specific polyclonal antibodies to detect exogenous Rfp2 protein. Detection of tubulin was used as loading control. Interestingly, MG treatment or long exposure of filters probed with Rfp2 antibodies revealed the existence of additional distinct bands, of higher molecular weight, extending upward from the Rfp2 band Figure 1 B. This, together with the fact that the Rfp2 protein is regulated by the proteasomal system, prompted us to investigate whether these additional bands represent ubiquitinated conjugates of Rfp2.
Bands of expected and higher molecular weight were observed exclusively in the lane containing transfected Rfp2 protein indicated by arrowheads. Although the band corresponding to the predicted size of GST-tagged full-length Rfp2 73 kDa, band 1 only contained tryptic peptides from Rfp2 and GST, both the slower migrating bands bands 2 and 3 were additionally found to contain peptides of ubiquitin see table in Figure 3 B.
From these mass spectrometric analyses, however, we were unable to detect any diagnostic branched ubiquitin peptides between Rfp2 and the C-terminus of ubiquitin and could therefore not determine the exact Rfp2 lysine residue s conjugated to ubiquitin.
Rfp2 is monoubiquitinated in cells. A Analysis of endogenous protein levels of Rfp2 in a panel of human cell lines. Nonubiquitinated Rfp2 is marked with a solid arrowhead, and asterisks indicate Rfp2 conjugated with single ubiquitin molecules. Tubulin was used as a loading control. B Identification of Rfp2 bands with higher molecular weight as ubiquitinated forms of the protein. Each Rfp2 band was excised, subjected to in-gel tryptic digestion, and analyzed by mass spectrometry. The bands analyzed are marked with numbers 1—3 and solid arrowheads.
High-molecular-mass Rfp2 bands are marked with asterisks monoubiquitinated Rfp2. Boxed text summarizes the mass spectrometric data and indicates the number of peptides identified and corresponding sequence coverage of the isolated nonubiquitinated band 1 and monoubiquiti-nated
Sverige far ny biosajt protein bands bands 2 and 3. This was followed by Western blot analysis with HA antibodies to detect ubiquitinated Rfp2 left and Rfp2 antibodies to detect both unconjugated and ubiquitinated Rfp2 panel.
Molecular size markers are shown on either side in kDa. To directly demonstrate the occurrence of mono-ubiquitinated Rfp2 "Sverige far ny biosajt," cells were cotransfected with GST-tagged Rfp2 and an HA-tagged expression plasmid. I menyn under "Analys " kan du se hur besökare och utställare igen", " Roligt med labbmässa i Sverige" och "Bra mässa, fick mycket nya ideer och.
11/27/ Streck sponsors free webinar on quality pillars in flow cytometry. 10/ 26/ Leading scientist highlights the potential of molecular kits for blood. microscope with Axiovision software (Thornwood, NY) and processed either as . Merged pictures (far right) show considerable overlap "Sverige far ny biosajt" Rfp2 and . [ PubMed]; Nuber U., Schwarz S. E., Scheffner M. The ubiquitin-protein ligase.
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