The PA-TM-RING protein RING finger protein 13 is an endosomal integral membrane E3 ubiquitin ligase whose RING finger domain is released to the cytoplasm by proteolysis Jeffrey P. Bocock1, Stephanie Carmicle1, Saba Chhotani1, Michael R. Ruffolo1, Haitao Chu2 and Ann H. Erickson1
1 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA 2 Department of Biostatistics, University of North Carolina, Chapel Hill, NC, USA
Keywords E3 ubiquitin ligase; neurite outgrowth; protease-associated domain; proteolysis; RNF13
Correspondence A. Erickson, Department of Biochemistry and Biophysics, CB 7260 GM, University of North Carolina, Chapel Hill, NC 27599, USA Fax: +1 929 966 2852 Tel: +1 919 966 4694 E-mail: ann_erickson@med.unc.edu
(Received 1 November 2008, revised 23 December 2008, accepted 20 January 2009)
doi:10.1111/j.1742-4658.2009.06913.x
PA-TM-RING proteins have an N-terminal protease-associated domain, a structure found in numerous proteases and implicated in protein binding, and C-terminal RING finger and PEST domains. Homologous proteins include GRAIL (gene related to anergy in leukocytes), which controls T-cell anergy, and AtRMR1 (receptor homology region-transmembrane domain-RING-H2 motif protein), a plant protein storage vacuole sorting receptor. Another family member, chicken RING zinc finger (C-RZF), was identified as being upregulated in embryonic chicken brain cells grown in the presence of tenascin-C. Despite algorithm predictions that the cDNA encodes a signal peptide and transmembrane domain, the protein was found in the nucleus. We showed that RING finger protein 13 (RNF13), the murine homolog of C-RZF, is a type I integral membrane protein localized in the endosomal ⁄ lysosomal system. By quantitative real-time RT-PCR analysis, we demonstrated that expression of RNF13 is increased in adult relative to embryonic mouse tissues and is upregulated in B35 neu- roblastoma cells stimulated to undergo neurite outgrowth. We found that RNF13 is very labile, being subject to extensive proteolysis that releases both the protein-associated domain and the RING domain from the mem- brane. By analyzing microsomes, we showed that the ectodomain is shed into the lumen of vesicles, whereas the C-terminal half, which possesses the RING finger, is released to the cytoplasm. This C-terminal fragment of RNF13 has the ability to mediate ubiquitination. Proteolytic release of RNF13 from a membrane anchor thus provides unique spatial and tempo- ral regulation that has not been previously described for an endosomal E3 ubiquitin ligase.
Abbreviations APP, Alzheimer’s precursor protein; AtRMR1, Arabidopsis thaliana receptor homology region-transmembrane domain-RING-H2 motif protein; CHO, Chinese hamster ovary; C-RZF, chicken RING zinc finger; CTF, cytoplasmic C-terminal fragment; EEA1, early endosomal antigen 1; ER, endoplasmic reticulum; GRAIL, gene related to anergy in leukocytes; HA, hemagglutinin; HAF, hemagglutinin and 3· FLAG epitopes; HRP, horseradish peroxidase; ICD, intracellular domain; LAMP2, lysosomal-associated membrane protein 2; MPR, mannose 6-phosphate receptor; MVB, multivesicular body; NLS, nuclear localization signal; PA, protease-associated; PDI, protein disulfide isomerase; PNGase F, peptide: N-glycosidase F; RNF13, RING finger protein 13; TM, transmembrane.
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Proteins of the PA-TM-RING family have a protease- associated (PA) domain and a RING finger domain separated by a transmembrane (TM) domain. PA domains are 120–210 amino acid sequences located in the noncatalytic regions of diverse proteases [1,2]. They are found in multiple members of MEROPS peptidase
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papilla when chickens were exposed to acoustic trauma [19]. A truncated splice variant that lacks a complete RING-H2 domain was additionally identified in mice [19] but was not characterized. On the basis of immu- nofluorescence microscopy and nuclear fractionation experiments, Tranque et al. [18] reported that RNF13 is a nuclear protein, even though the tmpred algo- rithm [20] predicts that it has a TM domain. A recent study established that RNF13 is an E3 ubiquitin ligase whose expression is increased in pancreatic ductal ade- nocarcinoma tissues, suggesting that the protein may participate in pancreatic cancer development [21].
families [3], including the transferrin receptor, a cata- lytically inactive protease, prostate-specific membrane antigen [4], the human Golgi ⁄ endosomal signal pepti- dase peptidase-like proteins SPPL2a and SPPL2b [5], and streptococcal C5a peptidase [6]. PA domains have been proposed to serve as substrate or ligand recogni- tion domains [1] or as protease regulatory regions [2], yet they have been functionally characterized only in plant proteins. The BP-80 receptor, which targets pro- teases to the plant lytic vacuole through recognition of the NPIR sorting determinant, contains a PA domain. Binding of vacuolar proteases requires the PA domain as well as other regions of the BP-80 luminal domain [7].
Results
RING finger proteins constitute a subfamily of the proteins that possess a pattern of cysteine and histidine residues that chelate zinc ions. The RING subfamily is thought to function exclusively in protein–protein interactions rather than protein–nucleic acid interac- tions [8]. Many RING finger proteins are E3 ubiquitin ligases [9]. The ubiquitination system functions in a variety of cellular processes, including protein degra- dation and protein trafficking. PA-TM-RING proteins We show that RNF13 is synthesized as an endoso- mal integral membrane protein rather than a soluble nuclear protein, consistent with other members of the PA-TM-RING family. We demonstrate that RNF13 mRNA is upregulated following initiation of neurite outgrowth, thus expanding on an array study that found RNF13 expression to be sufficient to induce neurite outgrowth [22]. We show that RNF13 is sub- ject to unexpected proteolysis that releases both the PA domain and the RING domain from the mem- brane, providing a biochemical basis for understanding the regulation of this family of multimodular endo- somal membrane E3 ubiquitin ligases.
Domain structure of RNF13
translocated across
that combine these two domains have been identified in plants, Xenopus, Drosophila and mammals, but not in yeast. The Arabi- dopsis thaliana PA-TM-RING receptor homology region–transmembrane domain–RING-H2 motif pro- tein (AtRMR1) was found to colocalize with a protein storage vacuole membrane marker and was predicted to be a receptor mediating targeting to the plant storage vacuole [10]. This organelle is a multivesicular body (MVB) containing segregated compartments of lytic and storage activity [11,12]. AtRMR1 was subsequently determined to be responsible for sorting the bean stor- age protein phaseolin to the protein storage vacuole [13] and was shown to bind to C-terminal vacuolar sorting determinants on tobacco chitinase and barley lectin [14], establishing that in plants the PA domain can serve as a ligand-binding domain.
to that
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The best-characterized mammalian PA-TM-RING family member is RNF128 ⁄ gene related to anergy in lymphocytes (GRAIL). GRAIL was first identified in a screen for genes upregulated in anergic CD4+ T-cells, which are unresponsive to antigen rechallenge [15]. It was further characterized as an E3 ubiquitin ligase that localizes to recycling endosomes, and was later con- firmed to be necessary for induction of T-cell anergy [16,17]. RING finger protein 13 (RNF13) was first designated chicken RING zinc finger (C-RZF), a pro- tein upregulated when chicken embryo brain cells were treated with the extracellular matrix component tenas- cin-C [18]. The protein was also upregulated in basilar RNF13 contains a number of protein domains likely to regulate its localization and function (Fig. 1). The first 34 amino acid residues at the N-terminus are pre- dicted by the algorithm signalp v.3.0 [23] to function as a transient signal peptide, suggesting that the newly synthesized polypeptide is the endoplasmic reticulum (ER) membrane cotranslation- ally. Residues 56–162 (numbering based on alignment in Fig. S1) have a high degree of sequence identity to PA domains. netnglyc 1.0 [24] predicts that this domain contains two N-linked glycosylation sites, resi- dues 43 and 88. Consistent with synthesis on the ER, the program tmpred [20] predicts that residues 182– 203 comprise a 22-residue integral membrane sequence, indicating that RNF13 might be a type I membrane protein. predictnls [25] predicts that RNF13 has a nuclear localization signal (NLS) (RRNRLRKD) at residues 214–221, in the cytoplasmic half of the pro- tein, near the membrane. psort [26] also predicts that RNF13 has an NLS (PVHKFKK) but at residues identified by 227–233, a site C-terminal predictnls. Residues 240–292 form a RING-H2 domain. Contiguous with the RING-H2 domain is a
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Fig. 1. RNF13 is a PA-TM-RING protein composed of several domains that might regulate other proteins. RNF13 is predicted by TMPRED [20] to be a TM protein, with the hydrophobic TM domain falling in the middle of the amino acid sequence (residues 182–203). Additional major domains include a predicted signal peptide (residues 1–34), a luminal PA domain (residues 56–162), and a cytoplasmic RING-H2 domain (resi- dues 240–292). The protein is also predicted to have an NLS (residues 214–221 or 227–233), a PEST sequence (residues 284–307), and a serine-rich region predicted to be phosphorylated (residues 309–381). We prepared expression constructs containing one or more of the following epitope tags: an HA tag at position 38, a FLAG tag at position 377, or a 3· FLAG tag at position 381.
24-residue sequence (residues 284–307) predicted, with a high probability score of 14.33 (significant if > 5), by the algorithm pestfind [27] to be a PEST domain. PEST domains, defined as hydrophilic stretches of at least 12 amino acids having a high concentration of proline, glutamic acid, serine, and threonine, are pro- tein domains that direct rapid degradation and thus are usually found in proteins with a short half-life [28]. The remainder of the C-terminal region is rich in ser- ine residues, similar to transcription factor activation domains. Multiple phosphorylation sites are predicted in the cytoplasmic half of the protein both by netph- osk1.0 [29] and by group-based phosphorylation scoring (GPS) 1.1 [30,31].
Sequence alignment of RNF13 with other PA-TM-RING proteins
activity of the RING-H2 domain, as does mutation of the same conserved cysteine in the RING domain of the E3 c-Cbl [34]. The expressed proteins, which con- tained N-terminal 6· His epitope tags, were purified on Ni2+–nitrilotriacetic acid affinity columns, eluted, and resolved by SDS ⁄ PAGE. An antibody against 6· His recognized two proteins in a western blot of each eluate (Fig. S2A, lanes 1 and 2), establishing that both bands contained the N-terminal epitope tag. The lower band could result from early termination, but the two discrete bands were reproducibly equally intense. Thus, it is more likely that C-terminal cleavage of the protein by a bacterial enzyme produces the lower band. The size difference of 2 kDa indicates that only approxi- mately 18 residues are missing from the C-terminus. As the RING domain of RNF13 is composed of resi- dues 240–292 out of 381, both protein bands should contain an intact RING-H2 domain. The truncated RNF13 proteins eluted from the affinity columns were resolved on polyacrylamide gels that were stained with Coomassie Blue R250 to assess purity (Fig. S2B). Eluted protein was assayed for ubiquitin ligase activity without further purification.
Three PA-TM-RING proteins, plant AtRMR1, mouse little overall GRAIL and mouse RNF13, exhibit sequence identity, as shown in the alignment in Fig. S1. Only approximately 12% of the amino acids are identical between the three proteins, as determined by tcoffee alignment [32]. Most of the conserved residues (gray boxes) lie within either the PA domain or the RING-H2 domain.
RNF13 is an E3 ubiquitin ligase
cytosolic domain The
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RING finger sequences frequently mediate ubiquitin ligase activity [9]; however, at least three distinct roles have been described for RING domains [33]. We there- fore investigated whether the RING domain in the cytoplasmic half of RNF13 was capable of catalyzing polyubiquitination. of RNF13D1–205 comprising residues 206–381, and thus the entire RING-H2 domain, was expressed in bacteria with or without the point mutation C266A. This muta- tion was designed to inactivate E3 ubiquitin ligase When RNF13D1–205 was added to an in vitro ubiq- uitination reaction mixture including ubiquitin, puri- fied commercial E1 enzyme, and a commercial E2 enzyme, either UbcH5a, UbcH5c, or UbcH6, it was able to catalyze the formation of polyubiquitin chains, as shown by the appearance of a high molecular mass ladder of protein bands (Fig. S2C, lanes 1–3). As there were only four proteins present in this in vitro assay, and one of them was ubiquitin itself, these data sug- gest that, like many E3 ubiquitin ligases, RNF13 can ubiquitinate itself. All three E2s assayed interacted with RNF13, but UbcH6 appeared to produce more polyubiquitination (Fig. S2C, lane 3). As expected by analogy with c-Cbl, purified RNF13D1–205 C266A was unable to catalyze polyubiquitination when added to a similar assay (Fig. S2C, lanes 4–6), as seen by the
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RNF13 is an endosomal protein
subcellular the RNF13 is predicted to have a TM domain and signal peptide, suggesting that it is an integral membrane protein in the secretory pathway. C-RZF was localized to the nucleus in chicken embryo heart cells [18], but RNF13 was recently reported to be present in the ER and Golgi when expressed transiently in MiaPaca-2 pancreatic cancer cells [21]. As no other PA-TM- RING protein has been found in the nucleus or the ER, we performed immunofluorescence experiments to determine localization of mouse RNF13 (Figs 2 and 3). failure to produce the characteristic polyubiquitin ladder. This indicates that catalysis of polyubiquitin chains is specific to the RING-H2 domain of purified RNF13, as a single point mutant of a conserved cyste- ine can abrogate E3 ligase activity. Failure to catalyze polyubiquitination was also seen when assay mixtures were prepared that lacked any E2 enzyme (Fig. S2C, lanes 7 and 8). These data show that the RING finger of RNF13 requires active E2 enzyme to function as an E3 ubiquitin ligase. As expected, when any of the other essential components of the reaction, including the E1 or E3 enzyme, ATP, or ubiquitin, was not included in the reaction, polyubiquitination did not occur (data not shown).
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Fig. 2. Endogenous, transiently expressed and stably expressed RNF13 all show punctate staining consistent with localization to endoso- mal–lysosomal vesicles. (A, B) Primary cortical neurons prepared from embryonic day 14.5 mouse embryos were treated with MG132 for 12 h. Endogenous RNF13 was detected with antibodies directed against the 14 amino acid C-terminal peptide of mouse RNF13. Staining was observed with the use of secondary donkey anti-rabbit Alexa Fluor 488 serum. The size bar in (B) represents 10 lm. (C) PC12 cells sta- bly expressing RNF13 were treated with MG132 for 12 h. RNF13 expression was detected with mouse anti-FLAG serum and, as secondary antibody, donkey anti-mouse Alexa Fluor 568 serum. (D–F) COS cells were transiently transfected with the RNF13 expression plasmid pSG5X-RNF13 FLAG377. RNF13 (D) was detected with rabbit anti-FLAG serum and, as secondary antibody, anti-rabbit Texas Red serum. Cells were counterstained with mouse antibodies raised against PDI (E) and donkey anti-rabbit Alexa Fluor 488 serum. These panels are merged in (F). The size bar in (D–F) represents 20 lm. (G–I) HeLa cells stably expressing RNF13 were treated with MG132 for 12 h. RNF13 (G) was stained with mouse anti-FLAG and donkey anti-mouse Alexa Fluor 488 sera. Calnexin staining (H) was observed with rabbit anti- calnexin and goat anti-rabbit Alexa Fluor 568 sera. These panels were merged in (I). The size bar in (G–I) represents 5 lm. RNF13 did not colocalize with either of the two ER markers.
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Fig. 3. RNF13 is localized in MVBs and endosomes. COS cells (A–L) or HeLa cells (M–O) were transiently transfected with RNF13-FLAG377, which was detected using rabbit anti-FLAG sera (B, E, H, K, N). Cells were costained with mouse anti-human Golgin 97 (A) serum, mouse anti-human LAMP2 serum (D), mouse anti-human CD63 serum (G), mouse anti-human MPR serum (J) or mouse anti-human EEA1 serum (M). Primary antibodies were visualized with the secondary antibodies donkey anti-mouse AlexaFluor 488 serum (A, D, G, J), goat anti-rabbit Texas Red serum (B, E, H, K), donkey anti-rabbit AlexaFluor 488 serum (N) and goat anti-mouse AlexaFluor 568 serum (M). RNF13 colocalized with LAMP2 (F), CD63 (I), and MPR, (L), but not with Gol- gin 97 (C) or EEA1 (O). Images were obtained with a Zeiss LSM 210 confocal microscope. The size bars represent 10 lm (A–C, J–L) and 20 lm (D–I).
the rat adrenal medulla and are frequently used as a model for neuronal differentiation (Fig. 2C). The same pattern was observed when epitope-tagged RNF13 was expressed either transiently in COS cells (Fig. 2D–F) or stably in HeLa cells (Fig. 2G–I). Thus, ectopic expression from the vectors utilized in this study does not appear to alter the localization of RNF13 relative to the endogenous protein.
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RNF13 observed in embryonic mouse cortical neu- rons using an antiserum specific for the C-terminal 14 amino acids of RNF13 showed punctate, non-nuclear staining characteristic of endosomes and lysosomes (Fig. 2A,B). To facilitate detection of RNF13 by immunofluorescence and to enable us to determine the origin of the biosynthetic forms detected by western blotting, we constructed vectors to express RNF13 with an N-terminal hemagglutinin (HA) epitope and a C-terminal FLAG tag. Stably expressed, epitope- tagged RNF13 exhibited punctate staining in PC12 cells, which are derived from a pheochyromocytoma of RNF13 was recently reported to be localized in the ER, on the basis of transient expression in pancreatic tumor cells [21]. In contrast, we found that the protein is not present in the ER, as it failed to colocalize with
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stably or
No accumulation of RNF13 in the nucleus could be detected at steady-state by immunofluorescence stain- ing of primary neurons or of cells expressing the pro- tein either transiently (Figs 2 and 3). Similarly, nuclear RNF13 was not observed in pancre- atic cancer cells transiently expressing RNF13 [21].
RNF13 undergoes extensive post-translational proteolysis two different ER chaperone proteins. RNF13 did not colocalize with endogenous protein disulfide isomerase (PDI) when expressed transiently in COS cells (Fig. 2D–F). Similarly, RNF13 expressed stably in HeLa cells did not colocalize with calnexin (Fig. 2G– I). Consistent with this, RNF13 did not accumulate with the trans-Golgi network protein golgin 97 (Fig. 3A–C), indicating that our ectopically expressed, epitope-tagged RNF13 is able to traverse the secretory pathway efficiently. Our immunofluorescence constructed viral expression vectors
also partially
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Anti-FLAG
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confocal microscopy studies indicated that RNF13 is localized in the endo- system (Fig. 3). RNF13 showed somal–lysosomal significant colocalization with lysosomal-associated membrane protein 2 (LAMP2), which localizes to the membranes of endosomes and lysosomes (Fig. 3D–F). colocalized with CD63 RNF13 (Fig. 3G–I), a tetraspanin that localizes to multivesic- ular endosomes [35], and with mannose 6-phosphate receptors (MPRs) (Fig. 3J–L), which are enriched in late endosomes. RNF13 failed to colocalize with the tether early endosomal antigen 1 early endosomal (EEA1) (Fig. 3M–O) at several planes of depth in the cell. Consistent with this, RNF13 did not colocalize with fluorescently labeled transferrin internalized for either 7.5 or 30 min by receptor-mediated endocytosis (data not shown). To characterize the biosynthetic processing of RNF13, we encoding mouse RNF13 with an HA epitope at position 38 and a 3· FLAG epitope at position 381 (RNF13-HAF) that we used to infect Chinese hamster ovary (CHO) cells to produce the CHO-RNF13-HAF cell line, which stably expresses RNF13. FLAG-positive RNF13-spe- cific bands were not detected by western blot analysis of cells expressing empty vector (Fig. 4A, lane 1). Sur- prisingly, RNF13-specific FLAG-positive bands were barely detectable in cell stably expressing RNF13 were treated with dimethylsulfoxide vehicle for 8 h (Fig. 4A, lane 2). When these cells were incubated with the protease inhibitor MG132 in dimethylsulfoxide for 8 h prior to harvest, however, a specific RNF13 banding pattern indicative of extensive reproducibly post-translational modification was
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Fig. 4. RNF13 undergoes extensive post-translational proteolysis. (A) CHO cells (lane 1) or CHO cells stably expressing RNF13-HAF (lanes 2 and 3) were treated, as indicated, with dimethylsulfoxide (DMSO) or MG132 for 8 h. Equal quantities of cellular protein were resolved on a 12% polyacrylamide gel. Biosynthetic forms of RNF13 were visualized on a western blot with mouse anti-FLAG serum. Prestained molecular mass markers are indicated on the left. (B) CHO cells (lane 1) or CHO cells stably expressing RNF13-HAF (lanes 2–5) were treated, as indi- cated, with dimethylsulfoxide, MG132 or epoxomicin for 10 h. RNF13 was visualized with anti-HA serum. (C) CHO cells transiently express- ing pSG5X-RNF13-HAF were pulse-labeled with [35S]methionine for 45 min. RNF13 was immunoprecipitated with anti-FLAG serum and resolved on a 12% polyacrylamide gel (lane 1). To detect nonspecific protein bands, normal whole serum (NS) was substituted for specific affinity-purified anti-FLAG serum (lane 2).
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were not recognized with anti-HA serum, suggesting that the N-terminal portion of the protein containing the HA epitope was lost from these proteins by pro- teolysis.
detected (Fig. 4A, lane 3). The pattern included a het- erogeneous collection of proteins of approximately 80 kDa, a second series of proteins that occasionally resolved into four discrete bands at approximately 65 kDa (e.g. Fig. 5A, lane 2), three protein bands of approximately 45 kDa, and one protein band of approximately 36 kDa. As all these proteins were visu- alized with antiserum that recognizes the 3· FLAG epitope at residue 381, they all contain the C-terminus of RNF13. An identical protein pattern was obtained when RNF13-HAF was expressed stably in B35 rat neurons (data not shown). To determine which of the RNF13 bands is the ini- tial biosynthetic product, we pulsed transiently trans- fected CHO cells expressing RNF13-HAF with [35S]methionine and immunoprecipitated RNF13 using antibodies specific for the FLAG epitope (Fig. 4C). The major protein detected after a 45 min pulse migrated at 65 kDa (Fig. 4C, lane 1). This protein band was absent upon immunoprecipitation with nor- mal serum as a negative control (Fig. 4C, lane 2).
RNF13 acquires carbohydrate modification
possesses that RNF13
1 2 3 4
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We next utilized antiserum specific for the N-termi- nal HA epitope tag (residue 38) to determine which of the RNF13 proteins in the banding pattern contain the N-terminus. Cells were treated as indicated (Fig. 4B). The specific proteasome inhibitor epoxomicin stabi- lized RNF13 (Fig. 4B, lane 5), as did MG132 (Fig. 4B, lane 4). Both the heterogeneous bands at (cid:2) 80 kDa and the group of bands at (cid:2) 65 kDa were recognized by the anti-HA serum (Fig. 4B, lanes 4 and 5). As these proteins are also recognized by the anti-FLAG serum, they must possess both residues 38 and 381 and therefore be close to full-length RNF13. The lower molecular mass bands around 45 kDa and at 36 kDa
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Fig. 5. RNF13 is modified with N-linked sugars and chondroitin sulfate. (A) pSG5X-RNF13-FLAG377 was expressed transiently in CHO cells. Immunoprecipitated RNF13 was treated with PNGase F from two different manufacturers (lanes 3 and 4). Transfected cells were incubated overnight with tunicamycin to block high-mannose sugar addition (lane 5). Cellular proteins were resolved on a 12% gel, and RNF13 was identified by western blotting using an antise- rum specific for the FLAG epitope. (B) Mouse RNF13-HAF was expressed transiently in CHO cells, and immunoprecipitated with antiserum specific for the FLAG epitope. The immunoprecipitate was split into two equal parts, which were incubated overnight in the absence (lane 1) or presence (lane 2) of chondroitinase ABC prior to resolution on a 12% polyacrylamide gel.
As we observed forms of RNF13 that migrated more slowly on polyacrylamide gels than the 43 kDa form predicted by the primary sequence alone, we assayed the protein for sugar modification. The netnglyc 1.0 algorithm predicts two sequences in the N-terminal domain that could acquire N-linked carbohydrate. Transiently expressed RNF13 was immunoprecipitated and treated with peptide: N-glycosidase F (PNGase F), which removes both asparagine-linked high-mannose and complex oligosac- charides [36]. The 65 kDa region resolved, on this gel, into four distinct proteins in the absence of endogly- cosidase treatment (Fig. 5A, lane 2). After endoglycosi- bands two dase disappeared, with a concomitant increase of the lowest band. Identical results were obtained with drug prepa- rations from two different suppliers (Fig. 5A, lanes 3 and 4). To confirm this result, CHO cells transiently expressing FLAG-tagged RNF13 were cultured in the presence of tunicamycin, an antibiotic that inhibits transfer of N-acetylglucosamine 1-phosphate to doli- cholmonophosphate [37], thus blocking the synthesis of asparagine-linked oligosaccharide chains on glyco- proteins. Tunicamycin treatment reproducibly reduced the amount of the upper band and resulted in loss of the middle band. These results confirm that two N-linked sugar chains can be removed from RNF13, supporting the predictions made by netnglyc 1.0.
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As a percentage of certain integral membrane pro- teins, such as the Alzheimer’s precursor protein (APP) [38] and the immunoglobulin invariant chain [39,40], acquire chondroitin sulfate glycosaminoglycan chains, we also assayed RNF13 for this modification. RNF13 possesses one potential Ser-Gly dipeptide acceptor sequence [41] in its luminal domain. When immuno- precipitated RNF13 was treated with chondroitin- ase ABC, the intensity of the diffusely staining bands
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RNF13 is a type I integral membrane protein
least a proportion of indicates that at at 80 kDa dramatically decreased, whereas the 65– 70 kDa bands increased in intensity (Fig. 5B). This result the RNF13 protein is modified with chondroitin sulfate.
Proteolysis releases N-terminal and C-terminal fragments of RNF13 from the membrane
that like the C-terminal domain,
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To further characterize the 36 kDa FLAG-positive RNF13 band observed in cell lysates, CHO cells were transfected transiently with a construct that encodes only the C-terminal half of RNF13. This variant (RNF13D1–204) is initiated a few residues beyond the putative TM sequence and retains the FLAG epitope. It was found to comigrate with the 36 kDa protein in cell lysates (Fig. 6A, lane 2 versus lane 4), suggesting the 36 kDa band is derived from full-length that RNF13 by proteolysis at or near the TM sequence. An HA-positive protein of approximately the same size was reproducibly detected when blots probed with anti-HA serum were overexposed (Fig. 6B, lane 5). This protein band always appeared fuzzy, consistent with the presence of carbohydrate. Detection of this the N-terminal domain of protein suggests RNF13, is released by proteolysis from the TM anchor localized approxi- mately in the middle of the protein.
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+MG132
By isolating microsomes and stripping them of periph- eral proteins, we confirmed the prediction of Mahon & Bateman [1] that RNF13 is synthesized as an integral membrane, not a nuclear, protein. We prepared micro- somal membranes, by Dounce homogenization in the presence of sucrose to maintain microsome integrity, supernatant of MG132-treated from a postnuclear CHO-RNF13-HAF cells. Proteins in both the postnu- clear supernatant, which contains soluble cytoplasmic proteins, and in the pelleted microsomes were resolved on a polyacrylamide gel (Fig. 7A). A western blot was probed for both the FLAG and HA epitopes. The 36 kDa protein, which comigrated with the expressed soluble C-terminal cytoplasmic half of the protein (Fig. 6A), was detected in the postnuclear superna- tant ⁄ cytoplasmic fraction (Fig. 7A, lane 1), establish- ing that the FLAG-tagged C-terminal half of RNF13 is released from the membrane by proteolysis and thus resembles the intracellular domain (ICD) of other inte- gral membrane proteins such as APP and Notch. Recovery of the FLAG-tagged C-terminal fragment in the cytoplasmic fraction also indicates that RNF13 is a type I membrane protein that has its PA domain either in the lumen of vesicles or on the cell surface and its C-terminal half in the cell cytoplasm. All other biosynthetic forms of RNF13, including the N-termi- nal HA-tagged domain (Fig. 7B, lane 3), were present indicating they are either in the microsome fraction, embedded in the microsomal membrane or present inside vesicles.
Fig. 6. RNF13 undergoes proteolysis on both sides of its trans- membrane domain. (A) B35-Con cells (lane 1), B35-RNF13-HAF cells (lanes 3 and 4) or CHO cells transiently expressing RNF13D1– 204, the ICD (lane 2), were treated, as indicated, with dimethylsulf- oxide (DMSO) or with MG132 for 8 h. Equal quantities of protein (600 lg) were loaded in lanes 1, 3 and 4, and 100 lg of protein was loaded in lane 2. Biosynthetic forms of RNF13 were visualized on a western blot of a 10% polyacrylamide gel with anti-FLAG-HRP serum. (B) CHO-RNF13-HAF cells were treated with MG132 for 9 h, and RNF13 was visualized on a blot of a 12% gel with anti-HA serum.
To confirm that RNF13 is an integral, not a periph- eral membrane protein, we isolated microsomes from CHO-RNF13-HAF cells and lysed them in high-pH carbonate buffer (Fig. 7B). Freezing and thawing microsomes in pH 11.5 buffer lyses vesicles and solu- bilizes peripheral membrane proteins not embedded in the membrane bilayer [42,43]. The luminal lysosomal protease cathepsin L, detected as a control, was present in the soluble fraction, confirming that soluble content proteins are released by carbonate treatment (data not shown).
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All forms of RNF13 present in microsomes and recognized by the anti-FLAG serum were present in the membrane fraction and were not solubilized when vesi- cles were lysed at high pH, indicating they are integral, not peripheral, membrane proteins (Fig. 7B, lane 2). The HA-tagged 36 kDa fragment of RNF13 was also detectable within microsomes (Fig. 7B, lane 3), sug- gesting that the luminal domain is shed within endo- somes. With this protocol, an additional HA-positive protein in the RNF13 pattern was reproducibly detect-
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Proteolytic regulation of RNF13
Model for RNF13 Biosynthetic Forms
Cell fractionation
A
B
C
Lysed microsomes stripped of peripheral proteins
HA
HA
HA
HA
Anti-HA
Anti-FLAG
Anti-FLAG
Anti-HA
3
1 2 1 2
1 2
1 2
97-
95
-
97
-
72
-
-
55
54-
-
54
F L A G
F L A G
F L A G
F L A G
F L A G
F L A G
37-
37
-
80
36
39-46
~kDa:
36
70
63
-
36
Sol Mb Sol Mb Microsomes
Full-length
CTF ICD
Cyto Mb Cyto Mb
Fig. 7. Proteolysis releases a C-terminal fragment of RNF13 into the cytoplasm. Microsomes were prepared by Dounce homogenization of CHO cells stably expressing RNF13-HAF treated with MG132. (A) RNF13 in the soluble cytoplasmic fraction (lane 1, Cyto) and in the pelleted microsome fraction (lane 2, Mb) was visualized by probing a blot with antibodies specific for the C-terminal FLAG or N-terminal HA epitope, as indicated. (B) Pelleted microsomes were lysed and stripped of peripheral membrane proteins by resuspension and incubation in pH 11.5 carbonate buffer. RNF13 was visualized in the soluble (Sol) and membrane (Mb) fractions by probing a blot of a minigel with antibodies specific for the C-terminal FLAG or N-terminal HA epitope, as indicated. The arrows mark an HA-tagged form that markedly decreases in intensity when microsomes are lysed (B). This protein is present, but more difficult to resolve, on commercial minigels [(B), all lanes except lane 3]. For the minigel, 100 lg of protein was resolved in each lane. (C) A model of the epitope-tagged protein bands detected is presented. The three short horizontal lines on the full-length protein represent chrondroitin sulfate modification.
in the membrane
lysosomal cathepsins
fraction below 65 kDa able (Fig. 7A,B, arrow). This protein, which lacks a FLAG-tag and thus presumably has lost its C-terminal sequences, was readily detectable in intact microsomes (Fig. 7A), but was less apparent once microsomes were lysed (Fig. 7B). It can often be visualized in whole cell extracts after long exposure of the western blot to film, suggesting that it corresponds to an authentic biosyn- thetic form of RNF13 that is stable in intact micro- somes (data not shown).
that raise the pH of vesicles in an attempt to inhibit lysosomal proteolysis of RNF13. Bafilomycin A1 inhibits the vacuolar ATPase [44,45], and ammonium chloride raises the pH of lysosomes and blocks the light–heavy chain cleavage of lysosomal cathepsin L [46]. Although MG132 is commonly employed as a it has also been reported to proteasome inhibitor, inhibit [47], [47,48], calpains and BACE1 [49]. Stably expressed RNF13 was barely detectable in cell extracts unless the cells were pretreated with MG132 (Fig. 8A, lane 1 versus lane 2). Inhibition of the vacuolar ATPase with bafilomycin A1 or by treating cells with ammonium chloride (Fig. 8A, lanes 3 and 4, respectively) stabilized biosynthetic forms of RNF13, but not as efficiently as did MG132 treatment of cells. The data suggest that other proteases primar- ily mediate the turnover of RNF13 in vesicles distinct from mature lysosomes.
A model summarizing the major biosynthetic forms of RNF13 and their relationship to membranes, based on the observed molecular mass and presence or absence of the epitope tags, is presented in Fig. 7C. The three cytoplasmic C-terminal fragments (CTFs) or ‘stubs’ remaining after loss of the PA domain could be generated by multiple proteases or could result from multiple cleavages by one enzyme. Other biosynthetic intermediates may be present but not detectable by our gel system. Additionally, the ratio of the forms may vary with the cell type and the metabolic condition of the cells expressing RNF13. Ectopically expressed RNF13 ICD does not localize in the nucleus when expressed transiently from a plasmid
Inhibiting the vacuolar ATPase only partially stabilizes RNF13
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Since RNF13 localizes to the endosomal–lysosomal membrane system, we treated cells with two inhibitors RNF13 is predicted to have one or two NLSs (Fig. 1), but we detected only RNF13 in punctate structures by confocal microscopy (Figs 2 and 3). Similarly, we were unable to detect the FLAG-tagged 36 kDa ICD in preparations of purified nuclei (data not shown). We
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Proteolytic regulation of RNF13
A
2
C l
3
O
1
S
4
G
a fil o
M
B
ci n H N
M
D
+
m y +
+
+
97
Anti- FLAG
Despite the presence of two sequences predicted to be NLSs, RNF13D1–204 3· FLAG381 localized to the cytoplasm (Fig. 9C). This is in agreement with our cell fractionation data, which also indicated that it is local- ized to the cytosol (data not shown).
54
RNF13 expression is higher in adult than in embryonic tissues
B Anti- α Tubulin
1 2 3 4
[18]. We
Fig. 8. Inhibitors stabilize RNF13 cleavage fragments. (A) CHO cells stably expressing RNF13-HAF were treated, as indicated, with dimethylsulfoxide (DMSO) (lane 1), MG132 (lane 2) or bafilo- mycin A1 (lane 3) for 8 h, or with NH4Cl for 24 h (lane 4). Biosyn- thetic forms of RNF13 were visualized on a western blot of a 12% polyacrylamide gel with mouse anti-FLAG serum. Migration of pre- stained molecular mass markers is indicated on the right. All lanes shown are derived from the same exposure of the same blot. (B) Equal quantities of total cellular protein were loaded in each lane, as verified by blotting for a-tubulin.
to that similar
transiently expressed RNF13D1–204
Genome sequencing suggests that RNF13 is ubiqui- tously expressed. This is supported by expression data from the Stanford Microarray Database, which show RNF13 to be widely expressed in many cell types, including throughout tissues of the human immune initial northern and nervous systems [50]. However, the chicken blot analysis of expression of C-RZF, the protein was homolog of RNF13, showed that expressed in embryonic heart and brain, but not in liver therefore analyzed mouse RNF13 expression by quantitative real-time RT-PCR, isolating mRNA from both embryonic and adult mouse tissues. The oligonucleotides used in this assay were specifi- cally designed to bind only the full-length RNF13 transcript. Expression of RNF13 in adult heart tissue was in spleen. We observed fold increases of 5.7, 2.6 and 1.9 for adult kidney, liver and brain, respectively, relative to spleen (Table 1; see Table S3 for statistical analysis). The PA-TM-RING family member GRAIL has been found to have similar expression in mouse tissues, using northern blots [15], but it has been primarily studied in T-cells. We also observed that RNF13 expression levels in adult tissues were higher than in the corresponding embryonic tissue. For example, there was a four-fold increase in adult brain as compared to embryonic brain after 14.5 or 16.5 days of development (Table 1). Our analysis of embryonic tissue showed similar expression of RNF13 therefore 3· FLAG381 (Fig. 9), containing only the C-terminal half of RNF13 including the putative NLS, to determine whether RNF13 could be observed in the nucleus when expression of the ICD was high. Figure 9A, showing cells treated with dimethylsulfoxide alone, the anti-FLAG serum. establishes the specificity of
A
B
C
RNF13 +DMSO
RNF13 +MG132
RNF13 ICD
Fig. 9. Expressed RNF13 ICD is not localized in the nucleus. (A, B) CHO cells stably expressing RNF13-HAF were plated on coverslips, incu- bated for 10 h with either dimethylsulfoxide (DMSO) vehicle (A) or MG132 (B), and stained with anti-FLAG serum, as indicated. (C) CHO cells were transiently transfected with a plasmid encoding RNF13D1–204 3· FLAG381, the soluble ICD of RNF13, and stained with anti-FLAG serum. RNF13 was visualized by confocal microscopy. The size bar in (C) represents 10 lm.
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Table 1. RNF13 expression in adult and embryonic tissues. Organs were harvested from adult mice and from embryonic mice at embryonic day (E) 14.5 and E16.5. RNF13 expression was normal- ized using 18S rRNA as an internal control. Each RNA sample was analyzed in duplicate, using quantitative real-time RT-PCR. Stati- stical analysis of the data is presented in Table S3.
Group
Organ
Fold expression
Mean DCT
Adult Adult Adult Adult Adult Adult E14.5 E16.5 Adult E14.5 E16.5
Brain Heart Kidney Liver Spleen Liver Liver Liver Brain Brain Brain
9.4 10.3 8.1 9.1 10.9 9.1 12.6 11.2 9.4 11.8 11.3
2.9 1.5 6.7 3.6 1.0 11.5 1.0 2.7 5.5 1.0 1.5
Fig. 10. RNF13 expression is increased following induction of neu- rite outgrowth. B35 cells were plated on dishes coated with fibro- nectin (5 mgÆmL)1) and incubated with dibutyryl-cAMP (100 lM) to induce neurite outgrowth. RNA was harvested using Trizol reagent, each plate being harvested separately as a single sample. Sample numbers of n = 9 were collected for control cells, and sample num- bers of n = 6 were collected for both time points of outgrowth. Quantitative real-time RT-PCR was performed in duplicate for each RNA sample, using ABI PRISM 7700 hardware and software. Data were analyzed using the 2DDCT method. The error bars represent the 95% confidence level. Statistical analysis is summarized in Table S3.
in both liver and brain, consistent with our findings in adult tissue.
RNF13 is upregulated during neurite outgrowth
endocytosis and in the formation of multivesicular endosomes is well established [52], but few endosomal E3s have been characterized, and none has been shown to be regulated by domain-specific proteolysis.
of cells
Discussion
Quantitative real-time RT-PCR data indicated that RNF13 is expressed in brain tissue (Table 1). A previ- ous array study identified the RNF13 gene as one of five genes that were able to promote neurite extension when expressed ectopically in PC12 neuronal precursor cells cultured on collagen [22]. The level of RNF13 expression was not assayed under these conditions. We therefore determined whether endogenous RNF13 gene expression increases during neurite outgrowth. B35 neuroblastoma cells were treated with dibutyryl-cAMP, a reagent that stimulates neurite extension by these cells [51], and RNF13 expression was analyzed using quantitative real-time RT-PCR (Fig. 10). We observed a two-fold increase in RNF13 mRNA after 72 h of outgrowth. A similar two-fold increase of RNF13 expression was observed after 5 days of outgrowth. Thus, RNF13 ubiquitin ligase activity may play a role in the regulation of nerve cell development.
PA-TM-RING family members are predicted by algorithms to be synthesized with transient signal peptides, placing the proteins in the membranes of the secretory pathway. RNF13 was recently reported to reside in the ER and Golgi, on the basis of immunofluorescence transiently staining expressing the protein [21]. In contrast, we found no colocalization of mouse RNF13 with either of two ER chaperones or with golgin 97, whether RNF13 is expressed transiently or stably. Consistent with our results, AtRMR1 and GRAIL are both present in endosomes under steady-state conditions. The plant family member AtRMR1 localizes to protein storage vacuoles [10] and mouse GRAIL to recycling endo- somes [15], although rat Goliath has been reported to be present in mitochondria [53]. Because, unlike GRAIL, RNF13 was not found in early endosomes, as determined by the lack of costaining with the early endosome tether EEA1, the two mouse proteins have overlapping but different steady-state locali- zations.
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Proteolysis regulates the half-life of RNF13. Stably expressed protein is barely detectable unless cells are treated with protease inhibitors such as MG132 or the We have determined that endosomal membranes pos- sess an E3 ubiquitin ligase that can be released into the cytoplasm by proteolysis. The PA-TM-RING pro- tein RNF13 is synthesized as an integral endosomal membrane protein, but post-translational proteolysis solubilizes the C-terminal half of this type I membrane protein. The role of ubiquitin addition in receptor
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Proteolytic regulation of RNF13
proteasome inhibitor epoxomicin [54] prior to harvest. MG132 has been used similarly to stabilize and so aid visualization of biosynthetic forms of other integral membrane proteins, including Notch [55], epidermal growth factor receptor [55], growth hormone receptor [56], and APP [49]. The regulation of other PA-TM- RING proteins by proteolysis has not been described, although multiple forms of h-Goliath have been detected by in vitro translation [57].
functions independent of cytoplasm suggests that RNF13 is enriched in the Triton X-114 phase when cellular membranes are extracted with this nonionic detergent [21]. Incorporation of the RING domain into an integral membrane protein allows the E3 activity to be spatially regulated. Presumably, the TM anchor enables the enzymatic RING domain to be targeted to the same cellular site as its substrate(s). Proteolytic pro- cessing adds a potential for temporal control of enzy- matic activity, but it could also alter the cellular site of the E3 activity. The E3 enzyme could target endosomal proteins or a substrate distant from the membrane of the endosome. Thus, solubilized RING domain could the full- potentially exert length, endosomally localized protein.
By expressing RNF13 that was engineered with dis- tinct epitope tags at its N-terminus and C-terminus, we were able to determine the origin of its proteolytic fragments. Our detection of the C-terminal domain that RNF13 may within the undergo regulated intramembrane proteolysis, like APP and Notch [58]. The endogenously generated fragment comigrated on polyacrylamide C-terminal gels with the ectopically expressed C-terminal half of RNF13 (residues 206–381), indicating that the former is essentially the entire cytoplasmic tail of RNF13. Thus, cleavage must occur within or very near the TM sequence. We have found that this ICD fragment can mediate E3 ubiquitin addition in vitro, as can pancre- atic RNF13 [21]. Interestingly, a splice variant of human RNF13 (NM183383) has been identified that is equivalent to this portion of RNF13.
Ectodomain shedding must precede proteolytic cleavage within the membrane. The soluble HA-tagged RNF13 fragment detected within microsomes, which is approximately the size of the PA domain, is probably primarily degraded in lysosomes, but it could be secreted if the endosomes fuse with the plasma mem- brane. A gene product equivalent to the PA domain has been identified in mammalian cells (hPAP21), but its physiological function is unknown [59]. The locali- zation of RNF13 to MVBs or late endosomes sug- gested that its N-terminal proteolysis might be mediated by lysosomal enzymes. However, isoforms of RNF13 were only slightly stabilized by the addition of reagents that inhibit lysosomal proteases. Thus, other cellular proteases must be primarily responsible for mediating the turnover of RNF13.
fractionation. The validity of
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RING E3s are commonly cytosolic, nuclear or peripheral membrane proteins. The initial study of RNF13 [18] concluded that the protein is localized in the nucleus, on the basis of both immunofluorescent the staining and cell fractionation protocol utilized has been questioned [1], however, and the tmpred algorithm predicts that RNF13 has a TM sequence. Consistent with this, our analysis of cellular membranes washed with high-salt solution establishes that RNF13 is indeed an integral membrane protein. This is consistent with data showing There is increasing precedent for proteolysis as a means of mobilizing dormant transcription factors [60]. On the basis of their detection of RNF13 in chicken brain cell nuclei, Tranque et al. [18] suggested that RNF13 might modulate transcription in embry- onic chicken brain. The solubilization of the ICD of RNF13 and the presence of the putative NLS on the C-terminal side of the TM domain are consistent with the possibility that the ICD, released from the mem- brane by proteolysis, has a function in the cell nucleus. We have, however, been unable to date to detect the RNF13 ICD in the nucleus. There are at least three pos- sible explanations for the absence of detectable RNF13 staining in the nucleus: (a) the PEST domain adjacent to the RING domain is likely to ensure that the cytoplas- mic tail, and thus the RNF13 E3 activity, has a short half-life that is tightly regulated by ubiquitination and proteasome degradation; (b) RNF13 may need a specific signal to move into the nucleus either on its own or with the help of an adaptor protein that facilitates entry into or retention in the nucleus – it is possible that the adap- tor protein is more abundant in certain cell types, or is upregulated in response to an external stimulus, and thus RNF13 cannot effectively move into the nucleus in cultured cells; and (c) alternatively, RNF13 may not go to the nucleus and instead may function as an E3 ligase in the cytoplasm. Precedents exist for all three possibili- ties. The ICDs of proteins such as Notch and APP are notoriously difficult to detect in nuclei, as they represent a small percentage of the whole protein and they are very labile, so it remains possible that we are simply unable to detect the small amount of ICD that might localize to the nucleus constitutively. The APP ICD must form a complex with the nuclear adaptor Fe65 and the histone acetyltransferase Tip60 in order to target to the nucleus [61,62]. Other ICDs function exclusively in the cytoplasm. The adhesion protein N-cadherin under- goes processing by c-secretase to generate an epsilon cleavage product, N-Cad ⁄ CTF2, which forms a complex
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Proteolytic regulation of RNF13
Pierce/Thermo Fisher Scientific (Rockford, IL, USA). Flu- orsave was from Calbiochem/EMD Chemicals (Gibbstown, NJ, USA). Purified ubiquitin was a gift from J. McCarville (UNC-CH). Rabbit E1 enzyme and human UbcH5a, UbcH5c and UbcH6 E2 enzymes were from Boston Biochemicals (Cambridge, MA, USA).
in the cytoplasm with the transcription factor CBP, pro- moting CBP ubiquitination, proteasomal degradation, and repression of CBP ⁄ CREB transcription [63].
Antibodies
The PA-TM-RING proteins GRAIL [15], Goliath- related E3 ubiquitin ligase 1 [64], h-Goliath [57] and RNF13 [21] have been established to be E3 ubiquitin ligases. Plant AtRMR1 has not been shown to have this enzymatic activity. Conserved recognition motifs on E3 target substrates have not been defined, making detection of E3 substrates difficult. Only two substrates of a member of the PA-TM-RING family have been identified; RhoGDI [65] and CD40 ligand [66] have been shown to be ubiquitinated by GRAIL. Identifica- tion of the RNF13 substrate(s) will allow us to deter- mine whether proteolysis is a positive or negative regulator of RNF13 E3 activity.
[67],
recognition molecules
Affinity-purified polyclonal rabbit anti-RNF13 serum was prepared by GenScript Corp (Piscataway, NJ, USA). using the peptide CPNGEQDYNIANTV, the 14 C-terminal resi- dues of mouse RNF13. Mouse and rabbit anti-FLAG IgG sera, horseradish peroxidase (HRP)-conjugated anti-FLAG M2 IgG1 serum and HRP-conjugated anti-HA IgG1 serum were from Sigma-Aldrich. Sheep anti-mouse HRP-conju- gated serum was from Amersham/GE Healthcare (Chalfont St Giles, UK). Mouse anti-LAMP2 IgG2A and anti-CD63 IgG1 sera were from the Developmental Studies Hybrid- oma Bank, University of Iowa. EEA1 IgG1 antibodies were a gift from J. Trejo (University of California, San Diego, CA, USA). Mouse anti-human Golgin 97 IgG1 serum, mouse anti-human MPR Ig2A serum, donkey anti-mouse AlexaFluor 488 IgG serum, donkey anti-rabbit Alexa- Fluor 488 IgG serum and goat anti-mouse AlexaFluor 568 IgG serum were from Invitrogen. Goat anti-rabbit Texas from Jackson Immunoresearch (West Red serum was Grove, PA, USA). Anti-calnexin IgG serum was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-PDI IgG2A serum from Affinity Bioreagents/ Thermo Fisher Scientific (Rockford, IL, USA).
In addition to PA-TM-RING proteins, only one other family of mammalian integral membrane RING E3 ubiquitin ligases has been identified that does not localize to the ER. These proteins, members of the membrane-associated RING-CH family are immune evasion proteins human homologs of viral that downregulate cell surface glycoproteins, including immune [68]. They possess two TM domains and are structurally unrelated to the PA-TM-RING proteins.
Expressed sequence tags, cloning, and site-directed mutagenesis
Experimental procedures
RNF13 was previously identified in a screen for proteins whose ectopic expression stimulates neurite outgrowth of PC12 cells cultured on collagen [22]. In further support of a role for RNF13 in neuronal devel- opment, we have determined by quantitative real-time RT-PCR analysis that endogenous RNF13 is upregu- lated upon induction of neurite outgrowth in B35 neu- roblastoma cells. The RNF13 gene is thus responsive to signaling cascades that result in cell differentiation and neurite outgrowth. Although RNF13 homologs have been identified in humans, dog, chicken, fruit fly, mosquito, tobacco and rice, no PA-TM-RING protein has been identified in yeast, suggesting that this E3 activity may be necessary to modulate a function unique to multicellular organisms, such as cell–cell interaction or tissue development.
inserted after
Bovine fibronectin, dibutyryl-cAMP, chondroitinase ABC, MG132 and bafilomycin A1 were obtained from Sigma- Aldrich (St Louis, MO, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA) and FuGENE 6 transfection reagent was from Roche Diagnostics (Indiana- polis, IN, USA). Bicinchoninic acid reagents were from
Image clone 4317972 was obtained from ATCC and deter- mined to encode complete RNF13 by sequencing. For mammalian transient expression, RNF13 was cloned into pCDNA3 (Invitrogen) and pSG5X (Stratagene, Cedar Creek, TX, USA), pSG5 modified to contain an expanded multiple cloning region. Mutagenesis was performed using a QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Transiently expressed RNF13 was mutated to possess a 1· FLAG epitope inserted after residue 377. This site was initially chosen because mouse RNF13 terminates with a valine, and C-terminal valines have been shown to modulate tar- geting of some integral membrane proteins [69–71]. Stably expressed RNF13 was subsequently engineered to express a C-terminal 3· FLAG tag added by cloning into p3· FLAG vector (Sigma-Aldrich). The expression and target- ing of the protein with the 3· FLAG epitope appeared to be indistinguishable from those of the protein modified with a 1· FLAG epitope residue 377 (Fig. 2). The oligonucleotides described in Table S1 were
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Reagents
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Proteolytic regulation of RNF13
was added for 24 h, and bafilomycin A1 (1 lm) for 8 h. When specified, RNF13 was immunoprecipitated from cells and resolved on polyacrylamide gels as described previously [46].
purchased from Integrated DNA Technologies (Coralville, IA, USA), Invitrogen, and the Nucleic Acids Core Facility, UNC-CH. Automated sequencing was performed at the UNC-CH Genome Analysis Facility.
High-mannose sugar addition was inhibited by treatment of cells with tunicamycin (Roche) at 1.5 lgÆmL)1 for 14 h. N-linked sugar was removed by treating immunoprecipi- tates with PNGase F (Roche and NE BioLabs, Ipswich, MA, USA) according to the manufacturers’ instructions.
Bacterial expression and purification
eluted
protein
from resin
BL21DE3 bacteria were transformed with pET3E-His vectors encoding mouse RNF13D1–205 or RNF13D1–205 with a C266A point mutation. Concentrations of purified protein were determined using a bicinchoninic acid assay according to the manufacturer’s instructions. Purified protein was resolved on both 12% and 15% polyacrylamide gels to assess purity; 15% gels were analyzed by Coomassie stain, and 12% gels were western blotted with anti-6· His serum. Relative amounts of protein were determined by (kodak 1d, version 3.6.2, using densitometry software Eastman Kodak, Rochester, NY, USA).
Chondroitinase
Immunoprecipitated in SDS ⁄ PAGE loading buffer was divided into two equal parts. After addition of pH 7 sodium acetate to a final con- centration of 50 mm, chondroitinase ABC (0.2 U) or an equivalent volume of SDS ⁄ PAGE buffer was added as specified. Samples were incubated at 37 (cid:2)C overnight. Dith- iothreitol (50 mm) and bromophenol blue were added to each tube, and proteins were resolved by 12% SDS ⁄ PAGE and visualized by western blotting as indicated.
In vitro ubiquitin ligase assay
To perform the ubiquitin ligase assay, we used a reaction mixture consisting of 50 mm Tris ⁄ HCl (pH 7.6), 2 mm MgCl2, 2 mm ATP, 1 mm dithiothreitol, 100 nm rabbit E1 enzyme, 1 lm E2 enzyme, 100 ngÆlL)1 purified ubiquitin, and 0.1 lgÆlL)1 purified RNF13. These mixtures were incu- bated for 2 h at 25 (cid:2)C. Samples were resolved on 12% polyacrylamide gels, and western blots were probed with anti-ubiquitin serum.
Cell fractionation
Cell fractionation was performed as described previously [46], with addition of 10 mm sodium fluoride, 10 mm sodium vanadate and 10 mm sodium pyrophosophate to the homogenization buffer. Soluble cytoplasmic proteins and microsomes were separated by centrifugation for 1 h at 60 000 g and 4 (cid:2)C in a Beckman TL-100 centrifuge. The pellet or microsome fraction contained all cellular mem- branes. Microsomes were lysed, and the peripheral proteins were stripped from the membranes by resuspending intact microsomes in carbonate buffer containing 100 mm sodium carbonate (pH 11.5), 100 mm KCl, and 5 mm EDTA. Frac- tions were resuspended in SDS ⁄ PAGE buffer for analysis using a Novex 4–12% Bis–Tris gel (Invitrogen) in Mops running buffer (Invitrogen).
Viral expression
For stable expression, cDNA encoding epitope-tagged RNF13 was cloned into the lentiviral FG12 vector [72]. B35 rat neurons [51], PC12 rat adrenal medulla pheochy- romocytoma cells, human HeLa cells or CHO cells were infected as previously described [72]. Green fluorescent pro- tein-expressing cells were selected by fluorescence-activated cell sorting by the Flow Cytometry Facility, UNC-CH, to lines CHO-RNF13-HAF, PC12-RNF13- generate the cell HAF, HeLa-RNF13-HAF and B35-RNF13-HAF, which stably express RNF13, or CHO-Con (control) and B35- Con, which stably express empty vector.
SDS ⁄ PAGE and immunoblotting
Cell monolayers were harvested by scraping into hot gel loading buffer. Protein concentrations were determined using a bicinchoninic acid assay according to the manufac- turer’s instructions. Equal quantities of cellular protein were routinely loaded in each lane of a given gel, as speci- fied. Proteins were resolved on 12 · 14 cm 1-mm-thick SDS-containing polyacrylamide gels of the percentage indi- cated. Immunoblots were blocked with 1% nonfat dry milk at either room temperature for 1 h or at 4 (cid:2)C overnight, and then probed with primary antibodies, either HRP-con- jugated anti-FLAG M2 serum or HRP-conjugated anti-HA serum. Western blots were developed using enhanced chemiluminescence.
Mouse cortical neurons, prepared as previously described [73], were a gift from L. Brennaman, UNC-CH. HeLa and CHO cells were transfected using Lipofectamine 2000, and COS cells were transfected using FuGENE 6, according to the manufacturers’ instructions. When specified, cells were treated for 8–12 h with dimethylsulfoxide (0.1%), the peptide aldehyde MG132 (5 lm in dimethylsulfoxide), or epoxomicin (500 nm in dimethylsulfoxide). NH4Cl (10 mm)
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Immunofluorescence was performed as described previously [74]. Confocal images were obtained with a Zeiss LSM 210; images were assembled using adobe photoshop ele- ments 2.0 and adobe illustrator.
Confocal immunofluorescence microscopy
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Carolina Animal Clinical Chemistry and Gene Expres- sion Laboratories, UNC-CH, which is under the direc- tion of H.-S. Kim. Cell sorting was performed at the UNC-CH Flow Cytometry Facility, which is under the direction of L. Arnold. This work was supported by research grant MCB-0235680 (to A. H. Erickson) from the National Science Foundation. S. Carmicle is supported by NIH SPIRE Postdoctoral Fellowship GM000678. Quantitative real-time RT-PCR analysis of RNF13 mRNA expression
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We wish to thank V. Mauro, The Scripps Research Institute, CA, and M. Lomax, University of Michigan Medical School, MI, for generously sharing reagents that were helpful in the initial stages of this work. L. Brennaman kindly provided primary mouse neu- rons. Microscopy was performed at the University of North Carolina in the Microscopy Services Labora- tory, Department of Pathology and Laboratory Medi- cine, under the direction of C. Robert Bagnell Jr, or in the Microscopy Facility, Department of Biology, under the direction of T. Perdue. Quantitative real-time RT-PCR was performed at the University of North
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The following supplementary material is available: Fig. S1. tcoffee alignment [32] of murine RNF13 (mRNF13) (NCBI Locus AAH58182) with PA-TM- (NCBI RING proteins murine GRAIL (mGRAIL) Locus NP_075759) and A. thaliana (AtRMR1) (NCBI Locus AAF32326). Fig. S2. RNF13 has ubiquitin ligase activity in vitro. Table S1. Oligonucleotides used for cloning RNF13 into vectors, introducing mutations and deletions, and adding epitope tags. Table S2. Oligonucleotides used for quantitative real- time RT-PCR. Table S3. Statistical analysis of RNF13 expression data quantitated by real-time RT-PCR. This supplementary material can be found in the online version of this article.
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