Báo cáo khoa học: 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.pdf (Scientific reports)

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 neuroblastoma 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 membrane. 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 temporal regulation that has not been previously described for an endosomal E3 ubiquitin ligase. Proteins of the PA-TM-RING family have a proteaseassociated (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 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. 1860 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS J. P. Bocock et al. families [3], including the transferrin receptor, a catalytically inactive protease, prostate-speciﬁc membrane antigen [4], the human Golgi ⁄ endosomal signal peptidase peptidase-like proteins SPPL2a and SPPL2b [5], and streptococcal C5a peptidase [6]. PA domains have been proposed to serve as substrate or ligand recognition domains [1] or as protease regulatory regions [2], yet they have been functionally characterized only in plant proteins. The BP-80 receptor, which targets proteases 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]. 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 interactions [8]. Many RING ﬁnger proteins are E3 ubiquitin ligases [9]. The ubiquitination system functions in a variety of cellular processes, including protein degradation and protein trafﬁcking. PA-TM-RING proteins that combine these two domains have been identiﬁed in plants, Xenopus, Drosophila and mammals, but not in yeast. The Arabidopsis thaliana PA-TM-RING receptor homology region–transmembrane domain–RING-H2 motif protein (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 storage 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. 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 conﬁrmed 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 protein upregulated when chicken embryo brain cells were treated with the extracellular matrix component tenascin-C [18]. The protein was also upregulated in basilar Proteolytic regulation of RNF13 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 immunoﬂuorescence microscopy and nuclear fractionation experiments, Tranque et al. [18] reported that RNF13 is a nuclear protein, even though the tmpred algorithm [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 adenocarcinoma tissues, suggesting that the protein may participate in pancreatic cancer development [21]. We show that RNF13 is synthesized as an endosomal 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 subject to unexpected proteolysis that releases both the PA domain and the RING domain from the membrane, providing a biochemical basis for understanding the regulation of this family of multimodular endosomal membrane E3 ubiquitin ligases. Results Domain structure of RNF13 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 predicted by the algorithm signalp v.3.0 [23] to function as a transient signal peptide, suggesting that the newly synthesized polypeptide is translocated across the endoplasmic reticulum (ER) membrane cotranslationally. 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, residues 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 protein, near the membrane. psort [26] also predicts that RNF13 has an NLS (PVHKFKK) but at residues 227–233, a site C-terminal to that identiﬁed by predictnls. Residues 240–292 form a RING-H2 domain. Contiguous with the RING-H2 domain is a FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1861 Proteolytic regulation of RNF13 J. P. Bocock et al. PA TM NLS RING PEST Ser-Rich 307 284 240 182 162 56 3XFLAG HA 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 (residues 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 protein 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 serine residues, similar to transcription factor activation domains. Multiple phosphorylation sites are predicted in the cytoplasmic half of the protein both by netphosk1.0 [29] and by group-based phosphorylation scoring (GPS) 1.1 [30,31]. Sequence alignment of RNF13 with other PA-TM-RING proteins Three PA-TM-RING proteins, plant AtRMR1, mouse GRAIL and mouse RNF13, exhibit little overall 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 RING ﬁnger sequences frequently mediate ubiquitin ligase activity [9]; however, at least three distinct roles have been described for RING domains [33]. We therefore investigated whether the RING domain in the cytoplasmic half of RNF13 was capable of catalyzing polyubiquitination. The cytosolic domain 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 mutation was designed to inactivate E3 ubiquitin ligase 1862 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 contained 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 approximately 18 residues are missing from the C-terminus. As the RING domain of RNF13 is composed of residues 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. When RNF13D1–205 was added to an in vitro ubiquitination reaction mixture including ubiquitin, puriﬁed 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 suggest 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 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS J. P. Bocock et al. Proteolytic regulation of RNF13 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 cysteine 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). RNF13 is an endosomal protein 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-TMRING protein has been found in the nucleus or the ER, we performed immunoﬂuorescence experiments to determine the subcellular localization of mouse RNF13 (Figs 2 and 3). A B C D E F G H I Fig. 2. Endogenous, transiently expressed and stably expressed RNF13 all show punctate staining consistent with localization to endosomal–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 stably 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 anticalnexin 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. FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1863 Proteolytic regulation of RNF13 J. P. Bocock et al. A B C D E F G H I Golgin 97 LAMP2 CD63 J K L M N O MPR EEA1 Marker RNF13 Merge RNF13 observed in embryonic mouse cortical neurons 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, epitopetagged RNF13 exhibited punctate staining in PC12 cells, which are derived from a pheochyromocytoma of 1864 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 Golgin 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. 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 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS J. P. Bocock et al. Proteolytic regulation of RNF13 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 confocal microscopy studies indicated that RNF13 is localized in the endosomal–lysosomal system (Fig. 3). RNF13 showed signiﬁcant colocalization with lysosomal-associated membrane protein 2 (LAMP2), which localizes to the membranes of endosomes and lysosomes (Fig. 3D–F). RNF13 also partially colocalized with CD63 (Fig. 3G–I), a tetraspanin that localizes to multivesicular endosomes [35], and with mannose 6-phosphate receptors (MPRs) (Fig. 3J–L), which are enriched in late endosomes. RNF13 failed to colocalize with the early endosomal tether early endosomal antigen 1 (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). Anti-FLAG A kDa 97 - 1 2 No accumulation of RNF13 in the nucleus could be detected at steady-state by immunoﬂuorescence staining of primary neurons or of cells expressing the protein either stably or transiently (Figs 2 and 3). Similarly, nuclear RNF13 was not observed in pancreatic cancer cells transiently expressing RNF13 [21]. RNF13 undergoes extensive post-translational proteolysis To characterize the biosynthetic processing of RNF13, we constructed viral expression vectors 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-speciﬁc bands were not detected by western blot analysis of cells expressing empty vector (Fig. 4A, lane 1). Surprisingly, RNF13-speciﬁc FLAG-positive bands were barely detectable in cell lysate when cells 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 post-translational modiﬁcation was reproducibly Anti-HA B 1 3 C 2 3 4 5 Anti-FLAG 1 2 45” NS 54 1 2 3 37 RNF13 DMSO MG132 – + + RNF13 – + + + + – – + – + + DMSO MG132 + + – – + – + + + + Epoxomicin – – – + + 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 indicated, with dimethylsulfoxide, MG132 or epoxomicin for 10 h. RNF13 was visualized with anti-HA serum. (C) CHO cells transiently expressing 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). FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1865 Proteolytic regulation of RNF13 J. P. Bocock et al. detected (Fig. 4A, lane 3). The pattern included a heterogeneous 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 visualized 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). We next utilized antiserum speciﬁc for the N-terminal 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 stabilized RNF13 (Fig. 4B, lane 5), as did MG132 (Fig. 4B, lane 4). Both the heterogeneous bands at 80 kDa and the group of bands at 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 A 1 2 3 4 5 Transfection – + + + + Endo F – – + + – Tunicamycin – – – – + 65 kDa 1 B 2 ~80 kDa 65 kDa – + Chondroitinase 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 antiserum 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. 1866 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 proteolysis. To determine which of the RNF13 bands is the initial biosynthetic product, we pulsed transiently transfected 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 normal serum as a negative control (Fig. 4C, lane 2). RNF13 acquires carbohydrate modification 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 that RNF13 possesses 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 oligosaccharides [36]. The 65 kDa region resolved, on this gel, into four distinct proteins in the absence of endoglycosidase treatment (Fig. 5A, lane 2). After endoglycosidase treatment, the two upper protein bands disappeared, with a concomitant increase of the lowest band. Identical results were obtained with drug preparations 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 dolicholmonophosphate [37], thus blocking the synthesis of asparagine-linked oligosaccharide chains on glycoproteins. 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. As a percentage of certain integral membrane proteins, 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 immunoprecipitated RNF13 was treated with chondroitinase ABC, the intensity of the diffusely staining bands FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS J. P. Bocock et al. Proteolytic regulation of RNF13 at 80 kDa dramatically decreased, whereas the 65– 70 kDa bands increased in intensity (Fig. 5B). This result indicates that at least a proportion of the RNF13 protein is modiﬁed with chondroitin sulfate. Proteolysis releases N-terminal and C-terminal fragments of RNF13 from the membrane 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 that the 36 kDa band is derived from full-length 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 protein suggests that the N-terminal domain of RNF13, like the C-terminal domain, is released by proteolysis from the TM anchor localized approximately in the middle of the protein. A 1 Anti-FLAG 2 3 B Anti-HA 4 5 54 - 38 Ctl Virus ICD RNF13 +DMSO+MG132 RNF13 +MG132 Fig. 6. RNF13 undergoes proteolysis on both sides of its transmembrane 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 dimethylsulfoxide (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. RNF13 is a type I integral membrane protein By isolating microsomes and stripping them of peripheral proteins, we conﬁrmed the prediction of Mahon & Bateman [1] that RNF13 is synthesized as an integral membrane, not a nuclear, protein. We prepared microsomal membranes, by Dounce homogenization in the presence of sucrose to maintain microsome integrity, from a postnuclear supernatant of MG132-treated CHO-RNF13-HAF cells. Proteins in both the postnuclear 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 supernatant ⁄ cytoplasmic fraction (Fig. 7A, lane 1), establishing 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 integral 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-terminal HA-tagged domain (Fig. 7B, lane 3), were present in the microsome fraction, indicating they are either embedded in the microsomal membrane or present inside vesicles. To conﬁrm that RNF13 is an integral, not a peripheral 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 solubilizes 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). 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 vesicles 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), suggesting that the luminal domain is shed within endosomes. With this protocol, an additional HA-positive protein in the RNF13 pattern was reproducibly detect- FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1867 Proteolytic regulation of RNF13 A J. P. Bocock et al. B Lysed microsomes stripped of Cell fractionation C Model for RNF13 Biosynthetic Forms peripheral proteins Anti-FLAG 1 2 97- 1 1 2 2 HA HA HA HA Anti-HA Anti-FLAG Anti-HA 1 2 3 - 95 97 - - 72 54- - 55 Cyto Mb Sol Mb Sol Mb 70 Full-length 63 36 FLAG Mb 80 FLAG FLAG Cyto ~kDa: - 36 Microsomes FLAG 37 - FLAG 37- FLAG 54 - 39-46 36 CTF ICD 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. able in the membrane fraction below 65 kDa (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 biosynthetic form of RNF13 that is stable in intact microsomes (data not shown). 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. Inhibiting the vacuolar ATPase only partially stabilizes RNF13 Since RNF13 localizes to the endosomal–lysosomal membrane system, we treated cells with two inhibitors 1868 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 proteasome inhibitor, it has also been reported to inhibit lysosomal cathepsins [47,48], calpains [47], 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 primarily mediate the turnover of RNF13 in vesicles distinct from mature lysosomes. Ectopically expressed RNF13 ICD does not localize in the nucleus when expressed transiently from a plasmid 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 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS J. P. Bocock et al. Proteolytic regulation of RNF13 A 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 localized to the cytosol (data not shown). n yci Cl 32 filom O 1 S H4 G Ba M N M D + + + + 97 AntiFLAG RNF13 expression is higher in adult than in embryonic tissues 54 B Antiα Tubulin 1 2 3 4 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 bafilomycin A1 (lane 3) for 8 h, or with NH4Cl for 24 h (lane 4). Biosynthetic forms of RNF13 were visualized on a western blot of a 12% polyacrylamide gel with mouse anti-FLAG serum. Migration of prestained 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. therefore transiently expressed RNF13D1–204 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, establishes the speciﬁcity of the anti-FLAG serum. A Genome sequencing suggests that RNF13 is ubiquitously 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 and nervous systems [50]. However, initial northern blot analysis of expression of C-RZF, the chicken homolog of RNF13, showed that the protein was expressed in embryonic heart and brain, but not in liver [18]. We 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 speciﬁcally designed to bind only the full-length RNF13 transcript. Expression of RNF13 in adult heart tissue was similar to that 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 B RNF13 +DMSO C 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, incubated 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. FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1869

Báo cáo khoa học: 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

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