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Originally published In Press as doi:10.1074/jbc.M110443200 on December 17, 2001

J. Biol. Chem., Vol. 277, Issue 11, 9307-9317, March 15, 2002
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N4WBP5, a Potential Target for Ubiquitination by the Nedd4 Family of Proteins, Is a Novel Golgi-associated Protein*

Kieran F. HarveyDagger §, Linda M. Shearwin-WhyattDagger §, Andrew FotiaDagger , Robert G. Parton, and Sharad KumarDagger ||

From the Dagger  Hanson Center for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia and the  Institute for Molecular Bioscience, Center for Microscopy and Microanalysis and Department of Physiology and Pharmacology, University of Queensland, Queensland 4072, Australia

Received for publication, October 31, 2001, and in revised form, December 5, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect (homologous to E6-AP Carboxyl terminus)domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ubiquitin-mediated protein modification regulates a variety of cellular processes including protein turnover and trafficking, endocytosis, transcriptional regulation, activation of transcription factors, and virus budding (reviewed in Refs. 1 and 2). The conjugation of ubiquitin to a protein substrate is a multistep process (1, 2). In an initial ATP-requiring step, a thioester is formed between the carboxyl terminus of ubiquitin and an internal cysteine residue of a ubiquitin-activating enzyme (E1).1 Activated ubiquitin is then transferred to a specific cysteine of one of several ubiquitin-conjugating enzymes (E2). E2 enzymes may donate ubiquitin directly to protein substrates, resulting in branched protein conjugates in which the carboxyl terminus of ubiquitin is linked by an isopeptide bond to specific internal lysine residues of target proteins. Substrates are also recognized by associated substrate-recognition proteins known as E3 proteins or ubiquitin-protein ligases, which play a major role in defining the substrate specificity of the ubiquitin system (1, 2). Hect (homologous to E6-AP Carboxyl terminus) domain containing proteins, which include members of the Nedd4 family, are a major class of E3s (3, 4).

Nedd4 was originally identified as a developmentally regulated gene that is highly expressed in the mouse embryonic central nervous system (5). Further analysis revealed that the expression of Nedd4 is not restricted to the embryonic central nervous system, and that it is expressed at varying levels in several embryonic and adult tissues (6). The Nedd4 protein has three types of protein domains, an amino-terminal C2 domain (involved in Ca2+-dependent binding of membrane phospholipids and proteins), three or four WW protein-protein interaction domains, and a Hect ubiquitin-protein ligase domain at the carboxyl terminus. WW domains consist of ~35 amino acids and are often found in proteins in multiple copies (7). They bind to several target sequences including the PY motif (PPXY), PPLP motif, and phosphoserine and phosphothreonine residues (8-11).

The Nedd4 family consists of a number of proteins that share the same modular structure as Nedd4 (12). Although the function of the majority of the Nedd4 family members is not known, these proteins have been implicated in a variety of cellular processes including endocytosis, TGF-beta signaling, virus budding, transcription, and protein trafficking (reviewed in Refs. 12-14). Nedd4 homologues exist in both Saccharomyces cerevisiae and Schizosaccahromyces pombe. The S. cerevisiae protein, Rsp5 is the best studied of all Nedd4 family members. Rsp5 is an essential protein in yeast that regulates the ubiquitin-mediated endocytosis and/or turnover of several membrane proteins (reviewed in Refs. 13 and 14). Additionally, Rsp5 is believed to play a role in minichromosome maintenance (15), protein sorting and trafficking (16-18), transcriptional regulation (19), and regulated proteasome-dependent processing of SPT23 transcription factor (20, 21). Although mammalian Nedd4, a widely expressed protein, has been implicated in various cellular processes, much less is known about the specific pathways and proteins regulated by Nedd4. The best characterized targets of Nedd4 and its close homologue Nedd4-2/KIAA0439 are the epithelial Na+ channel (ENaC) subunits, which interact with Nedd4 WW domains via the PY motifs present in the cytosolic carboxyl termini of each subunit (22-28). As the widespread expression of Nedd4 suggests that it may have additional targets, we carried out a far-Western protein expression screen using the WW domains of Nedd4 as a probe and identified eight new Nedd4 WW domain-binding proteins from mouse embryo cDNA libraries (29). Of these, one encoding a novel protein, which we named N4WBP5, was represented in 25% of clones obtained from the screen (29). In this paper, we have further characterized N4WBP5 and report that it is a novel evolutionarily conserved protein with a potential role in Golgi structure and function.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence Retrieval and Homology Analysis-- Hydrophobicity of the N4WBP5 protein was estimated using the Kyte-Doolittle program, and transmembrane domains were predicted using the TMPRED program. Multiple sequence alignments and construction of phylogenetic trees were carried out using bionavigator software packages Protpars and Protml at the Australian National Genome Information Services server.

Northern Blot Analysis-- A full-length mouse N4WBP5 cDNA was used as a probe for RNA blot analysis using a multiple tissue Northern blot containing poly(A)+ RNA from mouse tissues (CLONTECH). A beta -actin cDNA probe (CLONTECH) was used as a loading control probe. A full-length human N4WBP5 cDNA probe was used to probe a human multiple tissue expression array (CLONTECH) containing poly(A)+ RNA samples normalized against eight housekeeping mRNAs.

Bacterial Expression Plasmids and Production of GST Fusion Proteins-- The expression constructs used to generate the various WW domain proteins fused to glutathione S-transferase (GST) have been described previously (24, 27). The constructs for N4WBP5-GST fusion proteins that were used as probes in far-Western assays and for antibody production were generated by PCR amplification of the N4WBP5 cDNA encoding the amino-terminal 113 residues and cloning into BamHI/EcoRI sites of pGEX-2TK (Amersham Biosciences, Inc.). N4WBP5 PY motif mutants in which the Tyr residue was replaced by Ala were generated by PCR mutagenesis and verified by automated sequencing (PerkinElmer Life Sciences). GST fusion proteins were produced and purified as described previously (24).

Antibody Production and Affinity Purification-- The purified N4WBP5-GST fusion protein was used to inoculate two rabbits (0.5 mg of protein/rabbit). Rabbits were boosted three times at three-week intervals with 0.5 mg of protein/boost/rabbit. Sera were collected and tested on recombinant protein. The serum sample that showed the highest reactivity with N4WBP5 was affinity-purified. Initially polyclonal antibodies were passed through a column of GST coupled to cyanogen bromide-activated Sepharose 4 (Amersham Biosciences, Inc.) to remove GST-specific antibodies. Flow through from this step was affinity-purified against N4WBP5-GST coupled to cyanogen bromide-activated Sepharose 4. Bound proteins were eluted into Tris-HCl, pH 8.6, with 100 mM glycine, pH 2.5, and dialysed against phosphate-buffered saline. Affinity-purified antibodies were diluted in 50% glycerol and stored at -20 °C.

SDS-PAGE and Far-Western Analysis-- 32P-Labeled protein probes were produced by directly labeling the appropriate GST fusion protein using protein kinase A (New England Biolabs). Glutathione beads containing bound fusion protein were incubated with protein kinase A and [gamma -32P]ATP in a buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 12 mM MgCl2, and 1 mM dithiothreitol for 60 min at 4 °C. Beads were washed five times in phosphate-buffered saline, and labeled protein was eluted with glutathione buffer. To prepare WW domain protein filters, ~1 µg of each induced GST fusion protein lysate was resolved on SDS-PAGE gels and transferred to nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked in Hyb75 (30) and then hybridized with 32P-labeled N4WBP5 protein probes with both PY motifs intact, PY motif 2 intact or with PY motif 1 intact for 4 h at 4 °C in Hyb75. Membranes were washed three times in Hyb75 and exposed to x-ray film.

Mammalian Expression Plasmids-- pcDNA3-N4WBP5-FLAG was generated by PCR2 amplification of the N4WBP5 open reading frame with a FLAG tag engineered at the carboxyl terminus and cloning into the EcoRI site of pcDNA3 (Invitrogen). pcDNA3-N4WBP5-FLAG with PY motifs mutated was generated by PCR mutagenesis and verified by automated sequencing. pCXN2-Nedd4 was generated by relieving the open reading frame of Nedd4 using HaeII and Sap I (New England Biolabs) end filling using T4 DNA polymerase and cloning into pCXN2 (31). The pCXN2-Nedd4 cysteine mutant was produced by subjecting a pBluescript-Nedd4 construct to Kunkel mutagenesis (32). Mutant clones were verified by sequencing, and then the mutagenized open reading frame of Nedd4 was subcloned into pCXN2. pcDNA3-Nedd4 WW domains (N4WBP5/Nedd4 WW) were generated by PCR amplification of the region of Nedd4 encoding for the WW domains and cloning into BamHI/EcoRI sites of pcDNA3. The HA-ubiquitin expression plasmid was kindly provided by Dr. Dirk Bohmann (Aab Institute of Biomedical Sciences, Rochester, NY).

Cell Lines, Transfections, Immunoprecipitations, and Western Blotting-- All cell lines with the exception of HEK 293T were grown in Dulbecco's modified Eagle's medium, 10% fetal calf serum at 37 °C with 5% CO2. HEK 293T cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Transfections were performed using FuGENE6 (Roche Molecular Biochemicals) according to manufacturer instructions. Immunoprecipitations were performed by harvesting cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM EDTA, complete protease inhibitor mixture (Roche Molecular Biochemicals) and incubating lysates for 2 h at 4 °C with the appropriate antibody and then for 2 h at 4 °C with protein G-Sepharose (Amersham Biosciences, Inc.). Immunoprecipitates were washed three times in lysis buffer and once in phosphate-buffered saline and then subjected to SDS-PAGE.

Immunoblots were performed using the following primary antibodies anti-FLAG M2 (Sigma) at 1/1000, Nedd4ab1 (1/4000) (6), anti-N4WBP5 (1 µg/ml), and anti-beta -actin (Sigma) at 1/2000 and secondary antibodies anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Biosciences, Inc.). For the anti-N4WBP5 immunoblot, cells were harvested in lysis buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM EDTA, complete protease inhibitor mixture) and disrupted by freeze thaw. An equivalent amount of protein from each cell type was subjected to SDS-PAGE. Detection of bound antibody was achieved using ECL (Amersham Biosciences, Inc.).

Pulse-chase Experiments-- For pulse-chase experiments, COS cells were transfected in triplicate with 1 µg of pcDNA3-N4WBP5-FLAG alone or with 1 µg of pcDNA3-N4WBP5-FLAG and 3 µg of pCXN2, pCXN2-Nedd4 (Nedd4), or pCXN2-Nedd4 cysteine mutant and incubated for 24 h. Cells were incubated in methionine and cysteine-free medium (ICN) for 20 min and labeled for 2 h in the same medium containing 0.1 mCi/ml [35S]L-Met (ICN). Cells were washed in Dulbecco's modified Eagle's medium, 10% fetal calf serum and chased in cold Dulbecco's modified Eagle's medium, 10% fetal calf serum for up to 24 h. Where indicated, culture medium was supplemented with 10 µM lactacystin, 50 µM MG132, or 0.4 mM chloroquine. Cells were harvested in lysis buffer, and lysates immunoprecipitated with the anti-FLAG antibody and protein G-Sepharose were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and exposed to x-ray film. Band intensities were quantitated using Image QuaNT version 4.2a (Molecular Dynamics). N4WBP5 half-lives were calculated using SigmaPlot 4.0 software assuming first order decay.

Immunofluorescence-- N18 cells grown on coverslips were fixed for 15 min in 2% paraformaldehyde and permeabilized in 0.2% Triton X-100 for 2 min. Where indicated, cells were treated with 5 µg/ml of brefeldin A (BFA) at 37 °C for 45 min prior to fixing. The fixed cells were stained with affinity-purified anti-N4WBP5 polyclonal antibody (7.25 µg/ml) and anti-GM130 monoclonal (BD Transduction Laboratory) at 6.25 µg/ml for 30 min at room temperature followed by rhodamine-conjugated anti-rabbit antibody at 10 µg/ml (Chemicon) and fluorescein isothiocyanate-conjugated anti-mouse antibody at 10 µg/ml (Chemicon) for 30 min at room temperature. All antibodies were diluted in phosphate-buffered saline, 2% fetal bovine serum.

Transient Transfection Experiments-- Baby hamster kidney cells were transfected with pcDNA3-NWBP5-FLAG. After an overnight incubation, cells were treated with cycloheximide for 4 h to prevent new protein synthesis. Cells were fixed and double labeled with antibodies to the FLAG (data not shown) or the affinity-purified N4WBP5 antibody together with antibodies to GM130 as above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of N4WBP5-- Full-length cDNA for N4WBP5 was identified in a far-Western screen of a day 16 embryonic mouse cDNA library (29). N4WBP5 is a 221 amino acid protein with a predicted size of 26 kDa containing two PPXY (PY) motifs within the amino-terminal region (Fig. 1A). N4WBP5 contains three hydrophobic regions in the carboxyl-terminal region that correspond to three predicted transmembrane domains using the TMPRED software. A TBLASTN search of GenBankTM revealed a human counterpart of mouse N4WBP5 (GenBankTM hypothetical protein MGC10924 similar to Nedd4 WW-binding protein 5) and highly related homologous proteins in mammals that we have termed N4WBP5A (GenBankTM accession number AAL05872). N4WBP5A shares 52% identity and 79% similarity to N4WBP5 (Fig. 1B). N4WBP5A proteins also contain the two conserved PY motifs, which correspond to the PY motifs in N4WBP5. A homologue of N4WBP5 and N4WBP5A in Drosophila melanogaster (CG8056, GenBankTM accession number AAF49316) indicates that these proteins form an evolutionarily conserved family (Fig. 1C). An amino acid sequence alignment of mouse, human, and Drosophila N4WBP5 and mouse and human N4WBP5A is shown (Fig. 1B). A single PY motif exists in Drosophila N4WBP5. The three predicted transmembrane domains are conserved between each of the proteins. The construction of a phylogenetic tree indicated that the Drosophila protein is more closely related to N4WBP5 than N4WBP5A and suggests that mammalian N4WBP5 and N4WBP5A are likely to have evolved from the Drosophila N4WBP5 gene. An analysis of the human genome sequence in public data bases suggests that N4WBP5 and N4WBP5A are not linked and are located on chromosomes 5q24 and 13q23, respectively.


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  		Fig. 1.   The N4WBP5 forms part of an evolutionarily conserved family of transmembrane proteins. A, hydrophobicity of the N4WBP5 protein was estimated using the Kyte-Doolittle program, and transmembrane domains (TM) were predicted using the TMPRED program. B, multiple sequence alignments of N4WBP5 and its homologues. Identical residues are boxed in black, similar residues are boxed in gray, PY motifs are underlined by thick bars, whereas transmembrane domains are underlined with thin bars. The sequences for N4WBP5 and their homologues were derived by sequencing of expressed sequence tag clones or were obtained from the GenBankTM data base. C, a phylogeny tree showing N4WBP5 and N4WBP5A as distinct proteins that probably evolved from a common ancestral protein in Drosophila.

N4WBP5 Transcript Is Widely Expressed-- Northern blot analysis of RNA derived from mouse tissues suggested that N4WBP5 has two transcripts of 2.1 and 1.2 kb (Fig. 2A). Expression is highest in the adult liver, brain, kidney, and heart with the larger transcript predominating in these tissues. Lower level expression is apparent in the adult lung and testis, and in the testis a similar amount of both transcripts is present. A human multiple tissue expression array (CLONTECH) was probed with a full-length human N4WBP5 cDNA probe to determine the sites of expression in the human. The N4WBP5 transcript is detected in most adult tissues (Fig. 2, B and C). High levels of expression are seen in adult brain tissues including the left and right cerebellum, pituitary and thalamus. High expression is also seen in the kidney, liver, testis, salivary gland, and placenta of the adult and in the brain, kidney, and lung of the fetus. However, very low or no expression can be detected in a range of human cell lines (Fig. 2, B and C, column 10).


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  		Fig. 2.   mRNA expression analysis of N4WBP5 in mouse and human. A, a mouse multiple tissue Northern blot (CLONTECH) containing 2 µg of each poly(A)+ RNA was hybridized to a mouse N4WBP5 cDNA probe (upper panel). The N4WBP5 gene was expressed as two transcripts, ~2.1 and 1.2 kb in size. In the lower panel in A, the same blot was hybridized to a human beta -actin control probe (CLONTECH). B, a full-length human N4WBP5 cDNA probe was hybridized to a human multiple tissue expression array (CLONTECH). C, a key indicating various RNA samples loaded on the human multiple tissue expression array.

The PY Motifs of N4WBP5 Interact with the WW Domains of Nedd4-- We have previously shown that either individually or together the WW domains of mouse Nedd4 can interact with the PY motifs of N4WBP5 (29). The mutation of PY motif 1 of N4WBP5 greatly reduces the interaction between the 32P-labeled GST fusion protein containing all three WW domains of Nedd4 and N4WBP5, whereas the mutation of PY motif 2 had little effect. Individually, each WW domain of Nedd4 was also able to interact with the PY motifs of N4WBP5. The mutation of either PY motif of N4WBP5 had little effect on the interaction with WW domain 1, suggesting that this weak interaction is not mediated via the PY motifs. However, the mutation of PY motif 1 greatly reduced the interaction between WW domains 2 and 3 of Nedd4 and N4WBP5 (29). To further characterize the interaction between Nedd4 and N4WBP5, we carried out far-Western analysis in which the WW domains of mouse Nedd4 expressed as GST fusion proteins were run on SDS-PAGE gels (Fig. 3A) and probed with 32P-labeled GST fusions containing both PY motifs of N4WBP5. The PY motifs of N4WBP5 interacted with wild type WW domains 1-3 of mNedd4 or mutants in which one WW domain was mutated (Fig. 3B). The mutation of PY motif 1 of N4WBP5 greatly reduced these interactions, whereas the mutation of PY motif 2 has little effect, indicating that PY1 has a much higher affinity for the WW domains of Nedd4 than PY2 (Fig. 3, C and D). When individual WW domains of mouse Nedd4 were expressed as GST fusions, wild type N4WBP5 interacted strongly with WW domains 2 and 3 but not with WW1 (Fig. 3B). Again, the mutation of PY1 of N4WBP5 almost completely abolished this interaction, whereas the mutation of PY2 somewhat reduced this interaction (Fig. 3, C and D). Although PY1 is clearly the main contributor to WW domain binding, because the PY1 mutant showed small but significant binding to Nedd4 WW2, both PY motifs of N4WBP5 may contribute to Nedd4 binding, albeit with significantly different affinities.


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  		Fig. 3.   The PY motifs of N4WBP5 interact with the WW domains of Nedd4 with varying affinity. A, Coomassie Blue-stained gel of GST WW domain fusion proteins. The first lane contains GST alone, and molecular mass markers in kDa are indicated on the right-hand side of the gel. The lanes marked mWW1-3 contain GST fusion proteins containing all three WW domains of mouse Nedd4. In the lane marked wt, all WW domains were in wild type configuration. In the following three lanes one of the WW domains (shown as 1*, 2*, and 3*) was mutated leaving two intact WW domains. The last six lanes contain GST fusion proteins of individual WW domains in wild type (1, 2, and 3) or mutant (1*, 2*, and 3*) configuration. B-D, far-Western blots of the gel in A or identical gels probed with a 32P-labeled N4WBP5-GST protein comprising the amino-terminal 113 amino acids of N4WBP5 fused to the carboxyl-terminal of GST. This region of N4WBP5 contains both PY motifs. In B, the blot was probed with the fusion protein containing both PY motifs. In C, the blot was probed with the fusion protein in which the first PY motif, PPPY (Fig. 1A, PY1), was mutated to PPPA leaving the second PY motif (PY2) intact. In D, the blot was probed with the fusion protein in which the PY2 motif, PPSY, was mutated to PPSA leaving PY1 motif intact. A N4WBP5-GST probe with both PY motifs mutated did not bind to any of the WW domains of Nedd4 (data not shown).

Nedd4 Interacts with N4WBP5 in Transfected Cells-- To investigate the possible interaction between N4WBP5 and Nedd4 in vivo, coimmunoprecipitation experiments were performed in transfected COS cells. Cells were cotransfected with pCXN2-Nedd4, and either pcDNA3 or pcDNA3-N4WBP5-FLAG (N4WBP5) or pcDNA3-N4WBP5-FLAG with both PY motifs mutated and immunoprecipitated with preimmune serum or rabbit polyclonal antiserum to Nedd4 (N4). In cells cotransfected with Nedd4 and N4WBP5, the proteins were coimmunoprecipitated with anti-Nedd4 antiserum, indicating that the proteins interact in vivo (Fig. 4A). Only a very weak interaction was seen when N4WBP5 lacking functional PY motifs was coexpressed with Nedd4, indicating that the association between these proteins in vivo is predominantly mediated via PY-WW interactions. Control immunoblotting experiments showed that both N4WBP5 and N4WBP5 PYm proteins were efficiently expressed in transfected cells (Fig. 4B), and that both sets of transfected cells have roughly equivalent amounts of Nedd4 protein (Fig. 4C).


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  		Fig. 4.   Nedd4 interacts with N4WBP5 in transfected cells. COS cells were cotransfected with pCXN2-Nedd4 and either pcDNA3 vector (V), pcDNA3-N4WBP5-FLAG (N4WBP5) or pcDNA3-N4WBP5-FLAG with both PY motifs mutated (N4WBP5 PY m) and harvested in lysis buffer 36 h posttransfection. A, lysates were immunoprecipitated with either preimmune serum (PI) or Nedd4ab1 rabbit polyclonal antiserum (N4), subjected to SDS-PAGE, and immunoblotted with an anti-FLAG antibody. Lysates were also subjected to SDS-PAGE prior to immunoprecipitation to examine the presence of N4WBP5 using the anti-FLAG antibody (B) and Nedd4 using Nedd4ab1 (C).

WW Domains from Several Nedd4 Family Members Interact with N4WBP5-- To test whether N4WBP5 can interact with other proteins of the Nedd4 family, we analyzed the binding of the GST-WW domains from various Nedd4-like proteins with N4WBP5 by far-Western analysis (Fig. 5). Previous studies have shown that the binding between WW domains of Nedd4 family members and their ligands is very specific. For example, ENaC subunits only interact with the WW domains of Nedd4 and Nedd4-2, both of which are known to regulate ENaC, and not to the WW domains of KIAA0322, Smurf1, WWP2/AIP2, AIP4, and Itch (27). Equivalent amounts of GST-WW domain fusion proteins, as determined by SDS-PAGE and Coomassie Blue staining, were electrophoresed by SDS-PAGE (Fig. 5A) and transferred to nitrocellulose membrane. Filters were probed with a 32P-labeled GST-N4WBP5 fusion protein and exposed to x-ray film. Interestingly, N4WBP5 bound to the WW domains of human Nedd4, Nedd4-2, KIAA0322, WWP2/AIP2, AIP4, and mouse Itch with varying affinities but failed to interact with the WW domains of Smurf1 (Fig. 5B). In control experiments, under identical conditions, only Nedd4 and Nedd4-2 WW domains interacted with ENaC subunits (data not shown). These results suggest that in vivo N4WBP5 may interact with a number of Nedd4 family members, not only Nedd4 itself.


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  		Fig. 5.   The PY motifs in N4WBP5 interact with the WW domains of multiple Nedd4 family members. Escherichia coli extracts expressing various GST-WW proteins were blotted and probed with a 32P-labeled N4WBP5-GST protein comprising the amino-terminal 113 amino acids of N4WBP5 fused to the carboxyl-terminal of GST. A, Coomassie Blue-stained gel of GST-WW domain fusion proteins. B, far-Western blot of the GST-WW domain proteins probed with 32P-labeled GST-N4WBP5.

N4WBP5 Is Ubiquitinated-- A portion of overexpressed N4WBP5 migrates as high molecular mass complex when subjected to SDS-PAGE. COS cells were transfected with N4WBP5-FLAG, and lysates were subjected to SDS-PAGE and immunoblotted with anti-FLAG antibody. A short immunoblot exposure time shows a single band corresponding to the N4WBP5-FLAG protein migrating at its expected size of 26 kDa (Fig. 6A, short). Longer exposure times reveal a portion of N4WBP5 migrating as a high molecular mass complex (Fig. 6A, medium and long). To determine whether the high molecular mass complex reflects a pool of ubiquitinated N4WBP5, COS cells were cotransfected with ubiquitin-HA and pcDNA3 or N4WBP5-FLAG, and lysates were immunoprecipitated with a control antibody or anti-FLAG antibody. The blot was probed with anti-FLAG to show immunoprecipitation of N4WBP5 (Fig. 6C) and anti-HA to identify ubiquitinated proteins (Fig. 6B). Where cells were cotransfected with N4WBP5 and ubiquitin-HA, ubiquitinated forms of N4WBP5 could be detected. These higher molecular mass forms of N4WBP5 reflect monoubiquitinated (34 kDa), diubiquitinated (42 kDa), and polyubiquitinated (high molecular mass complexes) forms of N4WBP5 (Fig. 6B).


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  		Fig. 6.   N4WBP5 is ubiquitinated. A, a fraction of N4WBP5 protein migrates as part of a high molecular mass complex in SDS-PAGE gels. COS cells were transiently transfected with pcDNA3-N4WBP5-FLAG, harvested 36 h posttransfection, subjected to SDS-PAGE, and immunoblotted with an anti-FLAG antibody. Short, medium, and long exposures of blots are shown. N4WBP5 is only detected in the lanes with transfected cell lysates (+) as a 26-kDa protein. In longer exposures, a fraction of N4WBP5 appears to migrate in a high molecular mass complex. C, COS cells were cotransfected with either the vector pcDNA3 (-) or pcDNA3-N4WBP5-FLAG (+) and a HA-tagged ubiquitin expression plasmid (+) and harvested 36 h posttransfection. B, lysates were immunoprecipitated with either an isotype matched mouse monoclonal antibody (lanes marked C) or an anti-FLAG antibody (lanes marked F), subjected to SDS-PAGE, and immunoblotted with an anti-HA antibody to detect the presence of ubiquitinated N4WBP5 species. The bands corresponding to monoubiquitinated (monoub), diubiquitinated (diub), and polyubiquitinated (polyub) N4WBP5 species are indicated. In C, lysates were subjected to SDS-PAGE prior to immunoprecipitation to examine the presence of N4WBP5 using the anti-FLAG antibody.

Nedd4 May Regulate the Stability of N4WBP5-- Pulse-chase experiments were carried out to assess the stability of N4WBP5-FLAG in cells cotransfected with Nedd4 or a catalytically inactive cysteine mutant of Nedd4. COS cells were cotransfected with N4WBP5-FLAG and pCXN2-Nedd4 or the catalytically inactive cysteine mutant of Nedd4 pCXN2-Nedd4 cm. Following metabolic labeling, cultures were chased for up to 24 h in the absence of the label. N4WBP5 was immunoprecipitated with the anti-FLAG antibody. Initial pulse-chase experiments revealed that the half-life of N4WBP5 was ~10 h in COS cells. When cotransfected with the vector control, ~25% N4WBP5 remained by 24 h. In comparison, when cotransfected with pCXN2-Nedd4, only 5% N4WBP5 remained by this time point. Cotransfection with the catalytic cysteine mutant of Nedd4 led to ~20% of labeled N4WBP5 remaining by 24 h (Fig. 7, A and B). Consistent with the results in Fig. 7, A and B, the calculated half-lives of N4WBP5 under various conditions were 10.3 ± 1.4 h for N4WBP5 + vector, 5.4 ± 0.4 h for N4WBP5 + Nedd4, and 8.2 ± 0.9 h for N4WBP5 + Nedd4 cm. Thus, cotransfection with the active form of Nedd4 significantly increased the turnover rate of N4WBP5, suggesting that N4WBP5 is a target for the ubiquitin-ligase activity of Nedd4. These results were also supported by immunoblot analysis of cells cotransfected with N4WBP5 and Nedd4, Nedd4 cm, or the WW domains of Nedd4. Reduced levels of N4WBP5 protein were seen when coexpressed with catalytically active but not inactive Nedd4 (data not shown).


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  		Fig. 7.   Stability of N4WBP5 protein may be regulated by Nedd4. COS cells were transfected with 1 µg of N4WBP5-FLAG and 3 µg of pCXN2, pCXN2-Nedd4 (Nedd4), or pCXN2-Nedd4 cysteine mutant (Nedd4 cm). Cells were metabolically labeled and chased for up to 24 h, and N4WBP5-FLAG immunoprecipitated with an anti-FLAG antibody. A, Example of a pulse-chase experiment showing the stability of N4WBP5 in cells cotransfected with pCXN2, pCXN2-Nedd4, or pCXN2-Nedd4 cm. B, data showing quantification of pulse-chase experiments. Each value is expressed as a percentage of the amount of N4WBP5 at 0 h. Data were from three independent experiments, each done in triplicate, and error bars represent mean ± S.E. C and D are representative examples of pulse-chase experiments carried out in the presence of proteasome inhibitors lactacystin (lact) or MG132 (C) or the lysosomal inhibitor chloroquine (chlor) (D). In D, U indicates untransfected cells.

We also carried out pulse-chase experiments in the presence of the proteasome inhibitors lactacystin and MG132 or the lysosomal inhibitor chloroquine to determine whether N4WBP5 is degraded via the proteasomal or lysosomal pathway. Treatment with proteasomal inhibitors had a moderate but reproducible effect on N4WBP5 stability with higher levels of N4WBP5 protein remaining after 18 h in the presence of the inhibitors (Fig. 7C). Similarly, the use of the lysosomal inhibitor chloroquine moderately decreased the turnover rate of N4WBP5 protein (Fig. 7D). These data suggest that both proteasomal and lysosomal/endosomal pathways are involved in the turnover of N4WBP5.

N4WBP5 Is a Golgi-associated Protein-- Polyclonal antisera to N4WBP5 were generated by inoculation of rabbits with the amino-terminal domain of N4WBP5 expressed as a GST fusion protein. An affinity-purified antiserum was used to detect endogenous N4WBP5 protein in a range of cell lines. In immunoblot experiments, moderate amounts of N4WBP5 were detected in the mouse neuroblastoma cell line N18 and human embryonic kidney cell line HEK 293T, whereas very low levels of expression were seen in mouse NIH-3T3 fibroblasts and primate COS cells (Fig. 8A). The N4WBP5 protein in HEK 293T and COS cells comigrates with overexpressed N4WBP5, whereas the N4WBP5-specific band in N18 and NIH-3T3 appears to migrate with a slightly higher molecular mass (Fig. 8A). The reason for this finding is currently unclear.


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  		Fig. 8.   N4WBP5 is an integral Golgi protein. A, upper panel, immunoblot analysis of equivalent amounts of protein from a range of cell lines probed with affinity-purified anti-N4WBP5 antibody. Lower panel, immunoblot as shown above was probed with monoclonal anti-beta -actin antibody. B-J, the columns show N18 cell labeling for N4WBP5 (panels B, E, and H) or GM130 (panels C, F, and I) with the merged images (panels D, G, and J) showing both N4WBP5 (red) and GM130 (green). N18 cells were double labeled by immunofluorescence with antibodies to N4WBP5 and the Golgi matrix protein GM130. N4WBP5 was detected with rhodamine-conjugated anti-rabbit secondary antibody. GM130 was detected with fluorescein isothiocyanate conjugated-anti-mouse secondary antibody. B-D show images of untreated cells, whereas E-J are images of cells treated with BFA for 45 min prior to fixation and immunostaining. Note that BFA treatment alters the localization of both N4WBP5 and GM130. Scale bar = 5 µm.

The subcellular localization of endogenous N4WBP5 was assessed in N18 cells by immunohistochemistry and subcellular fractionation. Endogenous N4WBP5 has a perinuclear localization with a Golgi-like appearance (Fig. 8B). Costaining with an antibody to the Golgi matrix protein GM130 (33) indicates that N4WBP5 is indeed localized in Golgi structures (Fig. 8, C and D). Localization in the vesicular fraction was confirmed by subcellular fractionation (results not shown). In some cells, the anti-N4WBP5 antibody also stained nuclear components. However, cell fractionation results and anti-FLAG staining of cells overexpressing N4WBP5-FLAG did not indicate that N4WBP5 localized to the nucleus (data not shown). Therefore, we believe that the nuclear staining is likely to reflect the cross-reactivity of the antibody. The subcellular localization of N4WBP5 protein and the predicted transmembrane domains within the carboxyl-terminal region of N4WBP5 suggest that N4WBP5 is an integral Golgi membrane protein. BFA, an inhibitor of the endoplasmic reticulum-Golgi transport of proteins, has the unique property of effecting a rapid increase of Golgi cisternae volume and a subsequent loss of a recognizable Golgi apparatus in treated cells (34). Therefore, we tested whether BFA treatment affects N4WBP5 localization. As shown in Fig. 8, E-J, BFA treatment of N18 cells resulted in an altered localization of both GM130 and N4WBP5, confirming that N4WBP5 is associated with the Golgi complex.

Ectopic Expression of N4WBP5 Disrupts Golgi Structure-- To further study the subcellular localization of N4WBP5, we used FLAG-tagged N4WBP5 expression constructs. Transfection of pcDNA3-N4WBP5-FLAG into baby hamster kidney cells followed by the detection of the protein with anti-FLAG antibody (data not shown) or affinity-purified N4WBP5 antibody together with antibodies to GM130 showed that in some cells N4WBP5 was associated with the Golgi (Fig. 9, J-L, upper cells). However, the ectopic expression of the protein also resulted in its mislocalization (Fig. 9, D-L). Note that cells were treated with cycloheximide for 4 h prior to fixation to block protein synthesis. This treatment generally decreased endoplasmic reticulum staining but had no detectable effect on the general labeling pattern or on Golgi markers (data not shown). In many cells, N4WBP5 characteristically showed a punctate labeling with partial overlap with GM130 (Fig. 9, A-C). Interestingly, most of the N4WBP5-transfected cells showed a disruption of the GM130 staining, which was more punctate or tubular than in the majority of untransfected cells (Fig. 9, A-L). This was particularly evident in the higher expressing cells as shown in Fig. 9, J-L, in which both GM130 and NWBP5 show a tubular staining pattern in the transfected cells. These results indicate that the ectopic expression of N4WBP5 causes a disruption of the Golgi structure and that the altered localization of N4WBP5 and GM130 in N4WBP5-transfected cells is probably to be attributed to the disruption of the Golgi complex. As overexpression of several Golgi-associated proteins, such as giantin and GCP60, has been shown to disrupt Golgi structure (35-37), our results suggest that N4WBP5 is involved in the structural maintenance of the Golgi complex. Consistent with our prediction noted above, no significant nuclear localization of N4WBP5 was apparent in transfected cells.


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  		Fig. 9.   Ectopic expression of N4WBP5 disrupts Golgi structure. Baby hamster kidney cells transfected with N4WBP5 were treated with cycloheximide to prevent new protein synthesis and double labeled with affinity-purified N4WBP5 antibody together with antibodies to GM130. The columns show labeling for NWBP5 (panels A, D, G, and J) or GM130 (panels B, E, H, and K) with the merged images (panels C, F, I, and L) showing both N4WBP5 (green) and GM130 (red). NWBP5 characteristically shows a punctate labeling with a partial overlap with GM130 (small arrows in panels A and B). Note that most of the transfected cells show a disruption of the GM130 staining that is more punctate or tubular than in the majority of untransfected cells (large arrows in panels B, E, and K). This is particularly evident in the higher expressing cells as shown in panels G-L in which both GM130 and N4WBP5 show a tubular staining pattern (small arrows) in the transfected cells. Scale bar = 5 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have described the identification of a novel protein, N4WBP5, associated with the Golgi complex that is a potential target for ubiquitination by the Nedd4 family of proteins. In mammals, a closely related protein, which we have named N4WBP5A, also exists. Whereas two such proteins are present in mammals, a single related protein of unknown function is present in Drosophila that is likely to be a common ancestor for the mammalian proteins. Although there are no obvious orthologues of N4WBP5/N4WBP5A in yeast, the conservation of these proteins from insects to mammals suggest that they play important roles in cell function.

N4WBP5 and N4WBP5A are characterized by three potential transmembrane domains, but they do not share any significant similarity or specific domains with the known proteins in the sequence data base. The presence of transmembrane domains suggest that N4WBP5 and N4WBP5A are likely to be integral membrane proteins. The localization of N4WBP5 in the Golgi complex is consistent with this prediction. N4WBP5 and N4WBP5A contain two PY motifs, both of which are conserved in human and mouse proteins. Both these PY motifs are located toward the amino termini of the proteins. We predict a membrane topology of these proteins in which the amino termini of the proteins face the cytosolic side enabling them to interact with cytosolic Nedd4-like proteins and the carboxyl termini would face the luminal side. In vitro experiments suggest that both PY motifs are able to interact with WW domains 2 and 3 of mouse Nedd4 but not with the WW domain 1. However, the strongest interaction is between PY motif 1 and WW2, and thus it is likely that in vivo, N4WBP5 binds Nedd4 predominantly via PY motif 1.

We have shown that N4WBP5 can undergo ubiquitination, and that Nedd4 can to a small but significant extent regulate the turnover of the protein, suggesting that N4WBP5 ubiquitination is likely to be mediated by Nedd4 or a Nedd4 family member. N4WBP5 is a relatively long lived protein with a half-life of ~10 h. The stability of N4WBP5 was significantly increased by the overexpression of the Nedd4 WW domains or treatment of cells with proteasome inhibitors lactacystin and MG132 or a lysosomal inhibitor chloroquine, further suggesting the involvement of ubiquitination in the regulation of N4WBP5 protein turnover.

The specificity of interactions between various WW domains and their ligands is governed by both the motifs themselves and the surrounding amino acids (38). The interactions between WW domains and ligands are therefore quite specific. For instance, the PY motifs in the carboxyl termini of ENaC subunits only interact with the specific WW domains of Nedd4 and Nedd4-2, two highly related proteins, but not with the WW domains of other Nedd4 family members (27). Therefore, it is interesting that N4WBP5 can interact with the WW domains derived from several of the Nedd4 family members. This finding suggests that N4WBP5 may interact with multiple members of the Nedd4 family in vivo. It is tempting to speculate that N4WBP5 may act as an adaptor to recruit one or more Nedd4 family of ubiquitin-protein ligases to the Golgi complex. In previous studies (39-42), Nedd4 has been shown to interact with proteins in various subcellular compartments including the plasma membrane, mitochondria, nucleus, cytosol, and lipid rafts. In most cells, the majority of Nedd4 protein is cytosolic, but it can be recruited to various cellular compartments depending upon signaling (6, 39-42). This is consistent with the concept that Nedd4 can target multiple proteins for ubiquitination, thus controlling many cellular processes such as regulation of cell surface channels and transporters, protein trafficking, regulation of cell survival and apoptosis, receptor signaling, virus budding, and transcription (12, 14, 21, 43, 44). Our demonstration that Nedd4 and Nedd4-like proteins can also target a Golgi-associated protein further emphasizes the pleiotropic nature of cellular regulation by the Nedd4 family members.

The Golgi complex plays a key role in modification, sorting, and trafficking of proteins exported from the endoplasmic reticulum (45). Nedd4 and its yeast homologue Rsp5 have been implicated in the regulation of sorting at the trans-Golgi network, trafficking to endosomes, endocytosis, and virus budding (reviewed in Ref. 14). A recently described function of Rsp5 is ubiquitination-dependent trafficking of tryptophan permease Tat2 and general amino acid permease Gap1 (14, 17, 18, 46). Under poor nitrogen nutrient conditions, Tat2 is transported from the Golgi to vacuoles by a pathway that is dependent on Rsp5 and ubiquitin. In contrast to Tat2, Gap1 permease is targeted to the vacuole when nitrogen sources are rich. However, in both cases, Rsp5-dependent ubiquitination determines whether the permeases are transported to the cell membrane or degraded in the vacuole. Gap1 intracellular trafficking is controlled by polyubiquitination mediated by Rsp5 and requires Bul1 and Bul2 proteins, which are believed to act as adaptors to bring together Rsp5 and Gap1 (17). Although no analogous pathways have been identified in mammals, it is possible that N4WBP5 and its relative N4WBP5A are a part of the protein sorting/trafficking apparatus at the Golgi that act as adaptors for various Nedd4-like proteins and their potential targets. Our recent data suggest that N4WBP5A is associated with Golgi and post-Golgi vesicles.3 We are now testing whether N4WBP5 and N4WBP5A are indeed involved in the regulation of ubiquitin-dependent trafficking of the targets of Nedd4-like proteins.

    ACKNOWLEDGEMENTS

We thank Drs. T. Nagase, N. A. Jenkins, and C. A. Ross for the provision of cDNA clones for various Nedd4-like proteins, and D. Bohmann for HA-ubiquitin construct. We are grateful to Amanda Carozzi and Rob Luetterforst for assistance with transfection experiments.

    FOOTNOTES

* This work was supported by grants from the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) N4WBP5A reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AAL05872.

§ Both authors contributed equally to this work.

|| To whom correspondence should be addressed: Hanson Center for Cancer Research, Institute of Medical and Veterinary Science, P. O. Box 14, Rundle Mall, Adelaide, SA 5000, Australia. Fax: 61-8-8222-3139; E-mail: sharad.kumar@imvs.sa.gov.au.

Published, JBC Papers in Press, December 17, 2001, DOI 10.1074/jbc.M110443200

2 Primer sequences are available upon request.

3 L. M. Shearwin-Whyatt and S. Kumar, unpublished data.

    ABBREVIATIONS

The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzymes; E3, ubiquitin-protein isopeptide ligase; ENaC, epithelial sodium channel; PY motif, PPXY sequence; GST, glutathione S-transferase; BFA, brefeldin A; HA, hemagglutinin; HEK, human embryonic kidney.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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