A Novel Mammalian ER-located J-protein, DNAJB14, Can Accelerate ERAD of Misfolded Membrane Proteins

Abstract

Misfolded proteins in the endoplasmic reticulum (ER) are dislocated out of the ER to the cytosol, polyubiquitinated, and degraded by the ubiquitin-proteasome system in a process collectively termed ER-associated degradation (ERAD). Recent studies have established that a mammalian ER-localized transmembrane J-protein, DNAJB12, cooperates with Hsc70, a cytosolic Hsp70 family member, to promote the ERAD of misfolded membrane proteins. Interestingly, mammalian genomes have another J-protein called DNAJB14 that shows a high sequence similarity to DNAJB12. Yet, very little was known about this protein. Here, we report the characterization of DNAJB14. Immunofluorescence study and protease protection assay showed that, like DNAJB12, DNAJB14 is an ER-localized, single membrane-spanning J-protein with its J-domain facing the cytosol. We used co-immunoprecipitation assay to find that DNAJB14 can also specifically bind Hsc70 via its J-domain to recruit this chaperone to ER membrane. Remarkably, the overexpression of DNAJB14 accelerated the degradation of misfolded membrane proteins including a mutant of cystic fibrosis transmembrane conductance regulator (CFTRΔF508), but not that of a misfolded luminal protein. Furthermore, the DNAJB14-dependent degradation of CFTRΔF508 was compromised by MG132, a proteasome inhibitor, indicating that DNAJB14 can enhance the degradation of a misfolded membrane protein using the ubiquitin-proteasome system. Thus, the mammalian ER possesses two analogous J-proteins (DNAJB14 and DNAJB12) that both can promote the ERAD of misfolded transmembrane proteins. Compared with DNAJB12 mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more weakly, being most abundant in testis, implying its specific role in this tissue.

Introduction

The heat shock protein 40 (Hsp40)/J-protein family is a protein group conserved in all kingdoms of life. The members of this family play important roles in various cellular events such as protein synthesis and maturation, protein translocation across membranes, and degradation of misfolded proteins (Walsh et al., 2004; Qiu et al., 2006). J-proteins share a conserved signature J-domain that mediates the binding of the J-protein to a heat shock protein 70 (Hsp70) family member. The binding of a J-protein to Hsp70 activates the ATPase activity of Hsp70, allowing the active Hsp70 to recognize and fold client proteins (Jiang et al., 2007; Kampinga and Craig, 2010). In eukaryotic cells, J-proteins are distributed in many subcellular compartments, such as cytosol, nucleus, mitochondria and endoplasmic reticulum (ER) (Walsh et al., 2004; Qiu et al., 2006; Vos et al., 2008).

The ER is a cellular compartment where newly-synthesized secretory and membrane proteins are folded and modified (Hebert and Molinari, 2007). However, when proteins fail to attain their correctly folded states, they are recognized, dislocated from the ER to the cytosol, and polyubiquitinated for degradation by the proteasome in the cytosol. This process is collectively called ER-associated degradation (ERAD) (Wiertz et al., 1996; Tsai et al., 2002; Vembar and Brodsky, 2008; Hegde and Ploegh, 2010).

DNAJB12 is an ER-localized type II transmembrane J-protein having its J-domain in the cytosol. We and others have shown that DNAJB12 cooperates with Hsc70, a cytosolic Hsp70, to promote the ERAD of misfolded membrane proteins (Yamamoto et al., 2010; Grove et al., 2011).

Recent search of human and mouse genomes has identified the DNAJB14 gene that encodes a putative J-protein having a high sequence similarity with DNAJB12 (Qiu et al., 2006; Vos et al., 2008; Hageman and Kampinga, 2009; Kampinga and Craig, 2010). However, the basic character and function of this putative protein remained entirely unknown. Here, we show that DNAJB14 is also an ER-localized, type II transmembrane protein, having its J-domain in the cytosol. Moreover, we present data that DNAJB14 can accelerate the proteasome-dependent degradation of misfolded transmembrane proteins. These findings suggest that mammalian cells have two analogous ER transmembrane J-proteins that can promote the degradation of mis-folded membrane proteins.

Materials and Methods

RNA extraction, cDNA amplification and plasmid construction

Eight-week old male mice were sacrificed and used to prepare tissues. The dissected tissues were lysed in RNAiso (Takara) using Mixer Mills MM300 (Retsch) and total RNA was extracted from the lysate following the instructions provided by the manufacturer. Total RNA preparation from mouse fibroblast cell line, NIH3T3, followed the same instructions. All first strand cDNA was synthesized using Superscript™ II Reverse Transcriptase (Invitrogen) and Oligo-dT primer.

To generate the cDNA of mouse DNAJB14, total RNA from testis, brain and muscle was used for the first strand cDNA synthesis. The resulting cDNA was amplified by PCR using a forward primer, mDnaJB14-F (5′-TTAGAATTCATGGAGGGCAACCGC-GACGAG-3′), and a reverse primer, mDNAJB14-R (5′-CCG-GAATTCTTATCCCCCCTTGTAGAGAC-3′); the underline indicates an EcoRI site; the italic letters indicate the start or stop codon. The amplified cDNA was cloned into the EcoRI site of a mammalian expression vector, pCAGGS, to obtain pCAGGS-mDNAJB14. To construct pCAGGS-HA-mDNAJB14 encoding an HA-tagged mouse DNAJB14, the same cloning strategy was used except that the forward primer was replaced with mDNAJB14-N-HA-F (5′-TAAGAATCCATGTATCCATATGATGTGCCTGATTATGCTG-GCTCCATGGAGGGCAACCGCGACGAGGCG-3′); the italic letters encode the first methionine and HA tag.

To construct pCAGGS-mDNAJB12 encoding the non-tagged mouse DNAJB12, DNAJB12 cDNA was amplified by PCR using pCAGGS-HA-DNAJB12-Flag (Yamamoto et al., 2010) as a template. The primers used were mDNAJB12-F (5′-GCAGAAT-TCATGGAATCCAACAAGG-3′) and mDNAJB12-R (5′-ATA-GAATTCCTATCCATGCAGGGAGG-3′); the underline indicates an EcoRI site; the italic letters indicate the start or stop codon. The resulting fragment was cloned into the EcoRI site of pCAGGS to obtain pCAGGS-mDNAJB12.

To construct pCAGGS-mDNAJB14-D138N that expresses the DNAJB14 D138N mutant, a substitution mutation was introduced into pCAGGs-mDNAJB14 by site-directed mutagenesis using two primers: mDNAJB14-D138N-F (5′-TTTCATCCAAACAAAAA-CCACGCCCCTGG-3′), and mDNAJB14-D138N-R (5′-GTGGT-TTTTGTTTGGATGAAACTTCAAAGC-3′); the underlines indicate the substitution mutation; this change alters the HPD motif of DNAJB14 to HPN.

The expression plasmids, pEGFP-ΔF508-CFTR (Johnston et al., 1998), and pcDNA3.1-TCRα (Yu et al., 1997), were kindly provided by R.R. Kopito (Department of Biological Sciences, Stanford University, Stanford, CA, USA). Construction of pcDNA3.1-NHK has been described (Yamamoto et al., 2010).

To compare the levels of DNAJB14 and DNAJB12 mRNA in mouse tissues, the first strand cDNA was synthesized using 1 μg of total RNA prepared from mouse tissues. The resulting cDNA was then used as a template for PCR to evaluate the level of each mRNA. Primers used were: mJB14-3′-F (5′-CGTACTCCTTA-TACCCCAGATCTGG-3′), and mJB14-3′-R (5′-TCCTTGTAG-AGACTGGTCAGCCG-3′) for DNAJB14; mJB12-5′-F (5′-ATC-CAACAAGGATGAAGCCGAGCG-3′) and mJB12-5′-R (5′-TGC-TTGACCCTTTTCACAGCTG-3′) for DNAJB12; and hβ-Actin-F (5′-AACTGGAACGGTGAAGGTGACA-3′) and hβ-Actin-R (5′-ACTGGTCTCAAGTAGTGTACAGG-3′) for β-actin. The following cycle (94°C for 45 sec, 55°C for 30 sec, and 72°C for 30 sec) was repeated 32 times for the detection of DNAJB14 or DNAJB12 mRNA and 25 times for the detection of β-actin mRNA.

In the experiment comparing the levels of DNAJB14 and DNAJB12 mRNA in NIH3T3 cells under heat or ER stress, the following primers were also used: mHSP47-F (5′-ACATCCTC-CTGTCACCCTTG-3′) and mHSP47-R (5′-AACCTCTCATCCC-AGTGTGG-3′) for HSP47; mXBP1-F (5′-GAGAACCAGGAGT-TAAGAACACG-3′) and mXBP1-R (5′-GAAGATGTTCTGGG-GAGGTGAC-3′) for XBP1; and mHSPA5-F (5′-ATGATGAAGT-TCACTGTGGTGG-3′) and mHSPA5-R (5′-GAAGGGTCATTC-CAAGTGCG-3′) for BiP. The numbers of PCR cycles used are: 22 cycles for Hsp47; 24 cycles for BiP and β-actin; 27 cycles for DNAJB12; 33 cycles for DNAJB14 and XBP1.

The PCR product was separated by gel electrophoresis, stained with ethidium bromide, and detected with LAS4000 (GE Life Sciences). The band intensities were then quantified using Multiguage (Fujifilm).

Cell culture and transfection

NIH3T3 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum at 37°C in 5% CO₂ air. NIH3T3 and HeLa cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen) and Effectene (Qiagen), respectively. The exception is that we used Lipofectamine 2000 for transfection of HeLa cells with a plasmid in the experiment determining the topology of DNAJB14. For the transfection, we followed the manufacturers’ instructions. For cycloheximide chase experiment, HeLa cells grown on a 6-well culture plate for 24 h were transfected with 300 ng of a plasmid encoding an appropriate ERAD substrate and 5 ng of a plasmid encoding DNAJB14 or DNAJB12. At 24 h after the transfection, cycloheximide was added at a final concentration of 150 μg/ml to start the cycloheximide chase experiment.

Antibodies

The anti-mouse DNAJB14-N antibody which recognizes the N-terminus of mouse DNAJB14 was prepared by immunizing guinea pigs with a polypeptide that has an amino acid sequence corresponding to the N-terminal one hundred amino acid residues of DNAJB14. To prepare the polypeptide, the nucleotide encoding the corresponding region was cloned from pCAGGS-DNAJB14 into an expression vector, pQE30 (Qiagen). The resulting plasmid was used to produce the polypeptide in E. coli Rosetta™ strain (Novagen). The resulting polypeptide was then purified with Prep-foresis (Atto) and used to immunize guinea pigs. The anti-mouse DNAJB14-C antibody which recognizes the C-terminus of mouse DNAJB14 was prepared in the same manner except that a polypep-tide that has an amino acid sequence corresponding to the C-terminal one hundred amino acid residues of DNAJB14 was used to immunize guinea pigs.

Guinea pig anti-mouse DNAJB12 antibody has been described (Yamamoto et al., 2010). The other antibodies used in this study are: rabbit anti-GFP antibody (MBL), mouse anti-HA 12CA5 antibody (Boehringer Manheim), rabbit anti-β-COP antibody (Oncogene Research Products), rabbit anti-calnexin antibody (Stressgen), rat monoclonal anti-Hsc70 antibody (Stressgen), rabbit anti-A1AT antibody (DAKO), and rabbit anti-GAPDH antibody (Cell Signaling). Mouse monoclonal anti-ERp57 antibody was obtained by the following procedure. Briefly, mice were immunized with the membrane fraction from unfertilized eggs of Xenopus laevis (Murray, 1991). Hybridomas producing various antibodies were collected, and we obtained one hybridoma, which secretes a specific monoclonal antibody that recognized Xenopus and mammalian ERp57, referencing authentic antibody. For immunoblotting experiments, appropriate secondary antibodies were used. As secondary antibodies for immunofluorescence microscopy, we used Alexa Fluor 647 goat anti-mouse IgG (highly cross-adsorbed) and Alexa Fluor 488 anti-rabbit IgG (highly cross-adsorbed) (Invitrogen).

Immunofluorescence staining

HeLa cells, grown on a cover slip, were fixed in 1% acetic acid in ethanol, permeabilized with 1% Triton-X100 in phosphate-buffered saline (PBS), and then blocked with PBS containing 3% BSA. The resulting cover slip was incubated with a primary antibody diluted in blocking solution, washed three times with PBS, incubated again with an appropriate secondary antibody diluted in blocking solution, and finally washed three times with PBS. The cover slip was then mounted with Prolong Gold Antifade Reagent (Invitro-gen), and placed on a glass slide. Images were captured using a confocal fluorescence microscope, FV1000 (Olympus).

Immunoprecipitation

HeLa cells overexpressing DNAJB14 were collected with a scraper, lysed in NP-40 lysis buffer [1% (v/v) NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% (w/v) SDS] supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml benzamidine, and 1 mM PMSF) and 20 μM MG132, and subjected to centrifugation at 14,000 rpm, 4°C for 5 min to remove cell debris. The resulting supernatant was incubated with Protein G-Sepharose (GE life sciences) for 30 min at 4°C. After removing the beads by centrifugation at 2,000 rpm, 4°C for 5 min, the supernatant was incubated with anti-DNAJB14 antibody at 4°C for overnight and immunoprecipitated using Protein G-Sepharose. The immunoprecipitates were washed three times with PBS containing 1% Triton X-100, and then eluted by incubation in 2x SDS-PAGE sample buffer at 55°C for 15 min. The proteins were separated by SDS-PAGE and detected by immunoblotting.

Protein preparation, and immunoblotting

Cells were lysed in NP-40 lysis buffer, and centrifuged to remove cell debris. The supernatants was diluted 3:4 with 4x SDS-PAGE sample buffer containing 6 M Urea, and incubated at room temperature for 30 min. The proteins were separated by SDS-PAGE, blotted onto an Immobilon-P PVDF membrane (Millipore), incubated with appropriate primary and secondary antibodies, and detected with ECL Plus Western Blotting Detection Reagents (GE Health-care) and X-ray film. The signals were then analyzed by image analysis program ImageJ (NIH).

Microsome preparation and topology assay

To prepare microsomes, HeLa cells overexpressing DNAJB14 were harvested by centrifugation, and homogenized by 27-guage syringe in HES buffer (10 mM HEPES-KOH [pH 7.4], 1 mM EDTA, 0.25 mM sucrose and 0.008% digitonin) supplemented with proteasome inhibitors. The lysate was centrifuged at 14,000 rpm, 4°C for 5 min to remove cell debris. The microsomes were collected from the supernatant by centrifugation at 44,000 rpm for 2 h at 4°C, and re-suspended in HES buffer without digitonin. The resulting microsomes were then subjected to topology assay using proteinase K. For the assay, proteinase K and Triton X-100 were used at final concentrations of 10 μg/ml and 1%, respectively. The enzyme reaction was carried out at 4°C for 40 min.

Results

DNAJB14 mRNA, encoding a paralogue of DNAJB12, is expressed in mouse tissues

Genomic studies on mammals have predicted the existence, in their genomes, of the DNAJB14 gene that encodes a protein highly homologous to DNAJB12 (Qiu et al., 2006; Vos et al., 2008; Hageman and Kampinga, 2009; Kampinga and Craig, 2010). The deduced amino acid sequence of mouse DNAJB14 shows 50% identity to that of mouse DNAJB12 (Fig. 1A). The sequence comparison suggests that both DNAJB14 and DNAJB12 share a very similar domain organization: a J-domain is followed by a glycine/phenylalanine-rich region and a transmembrane segment (Fig. 1A, B). Particularly conserved are these three domains. Notably, the amino acid sequence of mouse DNAJB14 is much closer to that of human DNAJB14 (93% identity) than to that of mouse DNAJB12 (50% identity). Thus, DNAJB14 has evolved as a paralogue of DNAJB12. The orthologues of DNAJB14 and DNAJB12 are widely found among vertebrates (National Center for Biotechnology Information protein database; www.ncbi.nlm.gov/protein).

Fig. 1

Comparison of DNAJB14 and DNAJB12 from mouse and human. (A) Sequence alignments of DNAJB14 and DNAJB12 from mouse (Mus musculus) and human (Homo sapiens). The J-domain (red box), HPD motif (red asterisks), a glycine/phenylalanine-rich region (green box) and transmembrane domain (TM) are indicated. (B) Predicted membrane topology of DNAJB14. J-domain, red; a glycine/phenylalanine-rich region, green; transmembrane domain, black; amino acids, aa. (C) Tissue distribution of DNAJB14 mRNA examined by RT-PCR. Total RNA from the indicated tissues was subjected to RT-PCR with primers specific to DNAJB14, DNAJB12 and β-actin, and separated by agarose gel electrophoresis. The band intensities of DNAJB14 and DNAJB12 were recorded and normalized by the intensities of β-actin, and plotted onto a graph on panel D. Control RT indicates RT-PCR using total RNA from testis, performed without synthesizing the first strand cDNA. To analyze the expression of DNAB12 and DNAJB14 mRNA, the same volume of samples obtained after RT-PCR was loaded in each lane.

To examine whether DNAJB14 is transcribed in vivo, we amplified a 1,140-base pair cDNA corresponding to the coding sequence of DNAJB14 by performing RT-PCR. The nucleotide sequence of the cDNAs, obtained from mouse testis, brain and muscle, completely matched with the predicted sequence of the mouse DNAJB14 mRNA, confirming that DNAJB14 is actually transcribed in mouse tissues.

We next studied the expression of DNAJB14 in mouse tissues by amplifying the 5′-coding region of DNAJB14 mRNA with RT-PCR. In contrast to DNAJB12 mRNA that was ubiquitously expressed, DNAJB14 mRNA was relatively abundant in testis (Fig. 1C). The semi-quantitative analysis of the intensities of the bands from RT-PCR suggested that the levels of DNAJB14 mRNA were generally smaller than those of DNAJB12 mRNA in most tissues (Fig. 1D).

Neither heat stress nor ER stress affects the expression of DNAJB14 mRNA

To further study the expression of DNAJB14, we examined the effect of heat stress on the expression of DNAJB14 mRNA in NIH3T3 by incubating the cells at 42°C for 45 min and then 37°C for another 2 h. The level of Hsp47 mRNA, encoding a typical ER-resident heat shock protein (Nagata et al., 1986; Takechi et al., 1994), was significantly increased after the treatment, confirming that the cells were indeed stressed with heat (Fig. 2A). However, this treatment failed to affect the levels of DNAJB14 and DNAJB12 mRNAs, suggesting that their expression was not induced by heat shock. We next studied the effect of ER stress on the expression of DNAJB14 mRNA by treating the culture of NIH3T3 cells with 5 mM dithiothreitol (DTT) for 3 h. This treatment triggered the splicing of XBP1u mRNA, a cellular response to the ER stress, confirming that the treated cells indeed experienced this stress (Fig. 2B) (Yoshida et al., 2001). Consistently, upon DTT treatment, the level of BiP mRNA, encoding an ER-stress inducible protein, was increased. However, the same treatment failed to affect the levels of DNAJB14 and DNAJB12 mRNAs (Fig. 2B). Thus, the expression of DNAJB14 and DNAJB12 is insensitive to both heat and ER stresses.

Fig. 2

Expression of DNAJB14 mRNA is insensitive to heat or ER stress. (A) Effect of heat stress on the level of DNAJB14 mRNA. NIH3T3 cells were subjected to heat stress by incubating the cells at 42°C for 45 min, and then at 37°C for 2 h before preparing total RNA. RT-PCR was used to compare the levels of Hsp47, DNAJB12, DNAJB14 and β-actin mRNA before and after the stress. The RT-PCR products were then analyzed by agarose gel electrophoresis (left panel). The band intensities of Hsp47, DNAJB12 and DNAJB14 were quantified and normalized by the intensities of β-actin. The obtained values were then plotted onto bar graphs (right panels). (B) Effect of ER stress on the level of DNAJB14 mRNA. Total RNAs were prepared from NIH3T3 cells that had been treated with 5 mM DTT for 3 h and processed as described above. RT-PCR was also used to detect the splicing of XBP1 mRNA, which occurs upon ER stress. To calculate the splicing efficiency of XBP1 mRNA, the intensity of the spliced band was divided by the sum of the intensities of the spliced and unspliced bands. Error bars indicate standard deviations from the average of triplicate experiments.

DNAJB14 is an ER-localized, type II transmembrane protein having its J-domain in the cytosol

To examine the subcellular localization of DNAJB14, we overexpressed DNAJB14 having an HA tag at its N-terminus (HA-DNAJB14) in HeLa cells and visualized the protein by immuno-staining with anti-HA antibody. As shown in Fig. 3A, HA-DNAJB14 (HA-JB14) was detected in a pattern that overlapped with Calnexin (CNX), an ER marker, indicating that this protein is located at the ER.

Fig. 3

DNAJB14 is an ER-localized, type-II transmembrane protein that binds cytosolic Hsc70. (A) Subcellular localization of DNAJB14 was studied by immunostaining HeLa cells expressing HA-DNAJB14 with antibodies specific to HA (left) and to Calnexin (CNX; an ER marker) (middle). The two images were merged on the right. Bars indicate 5 μm. (B) Membrane topology of DNAJB14. Microsomes, extracted from HeLa cells expressing DNAJB14, were treated with proteinase K. The digestion products were then separated by SDS-PAGE and detected by immunoblotting using anti-DNAJB14-N (left), anti-DNAJB14-C (middle), and anti-ERp57 antibodies. Where indicated, Triton X-100 was added to break microsomes before the digestion reaction. (C) Interaction of DNAJB14 with Hsc70. Cellular extracts were prepared from HeLa cells expressing the wild-type DNAJB14 or a J-domain mutant (JB14 D138N). The lysates were subjected to immunoprecipitation by anti-DNAJB14-C antibody and the resulting immunoprecipitates were analyzed by immunoblotting using the indicated antibodies to study the interaction between DNAJB14 and Hsc70.

As DNAJB14 has a putative transmembrane segment, we also studied the membrane topology of DNAJB14 by proteinase K protection assay using microsomes prepared from HeLa cells overexpressing DNAJB14. As a first step towards this end, we prepared two kinds of antibodies that are specific to DNAJB14. One of them is anti-DNAJB14-N antibody, which recognizes the N-terminus of DNAJB14. This antibody specifically detected a band that runs at around 42-kDa on an immuno-blot (Fig. 3B, lane 1). The same band was also detected by anti-DNAJB14-C antibody that recognizes the C-terminus of DNAJB14 (Fig. 3B, lane 4). Thus, this band at 42-kDa represents the full-length DNAJB14.

Next, we proceeded to determine the membrane topology of this protein using these antibodies. For this purpose, the microsomes were treated with proteinase K, and analyzed by immuno-blotting with both antibodies. Upon treatment with proteinase K, only anti-DNAJB14-C antibody detected a proteolytic fragment of 15-kDa (Fig. 3B, lane 5). The failure of the anti-DNAJB14-N antibody to detect this fragment (Fig. 3B, lane 2) and the size of the protected fragment indicate that proteinase K digested the N-terminus of DNAJB14 including the J-domain. Disruption of the barrier function of membrane by treating the microsomes with Triton X-100, a detergent, caused the disappearance of this fragment, indicating that the C-terminus of DNAJB14 resides in the luminal side of the microsomes (Fig. 3B, lane 6). The right panel of Fig. 3B shows a control experiment demonstrating that a luminal protein, ERp57, was efficiently degraded only in the presence of Triton X-100 (compare lane 9 with lanes 7 and 8), which indicates the integrity of the purified microsomes. These immunofluorescence staining and protease protection assay indicate that DNAJB14, like DNAJB12, is an ER-localized type II transmembrane protein with its J-domain facing the cytosol.

J-proteins interact with Hsc70 family members to perform their functions. We and others have shown that DNAJB12 interacts with Hsc70, a cytosolic Hsp70 family member, via its J-domain, to exert its function (Yamamoto et al., 2010; Grove et al., 2011). Since, like DNAJB12, DNAJB14 has its J-domain in the cytosol, we surmised that DNAJB14 may also interact with Hsc70 in the cytosol using its J-domain. To examine the interaction between DNAJB14 and Hsc70, we purified DNAJB14 from HeLa cells overexpressing DNAJB14 by immunoprecipitation using anti-DNAJB14-C antibody and tested whether Hsc70 was co-purified with DNAJB14. As shown in Fig. 3C, lane 2, Hsc70 was co-immunoprecipitated with DNAJB14. This binding requires an intact J-domain of DNAJB14, because the interaction was lost when we mutated the conserved HPD motif of DNAJB14 to HPN (D138N) (Fig. 3C, lane 3). This finding is consistent with the general notion that HPD motif of J-domain is important for the recognition of Hsp70 by J-protein (Qiu et al., 2006; Jiang et al., 2007; Kampinga and Craig, 2010). We, thus, concluded that, like DNAJB12, DNAJB14 uses its J-domain to interact with Hsc70 in the cytosol.

DNAJB14 can promote the degradation of CFTRΔF508 by the proteasome

DNAJB12 is involved in the ERAD of misfolded transmembrane proteins (Yamamoto et al., 2010; Grove et al., 2011). Due to the similarity between DNAJB14 and DNAJB12 in their structure and molecular characteristics, we thought that DNAJB14 may also promote the ERAD of proteins. To test this hypothesis, we first looked at the effect of DNAJB14 overproduction on the degradation of CFTRΔF508, a mutant of CFTR (cystic fibrosis transmembrane conductance regulator). CFTR is a polytopic plasma membrane chloride channel. The presence of this mutation, ΔF508, in CFTR leads to the development of cystic fibrosis. This mutant is widely used as a model substrate to study the degradation of membrane proteins on the ER due to its diminished folding ability (Ward et al., 1995). In fact, DNAJB12 promoted the ERAD of this protein (Yamamoto et al., 2010; Grove et al., 2011).

To study the effect of DNAJB14 overexpression on the degradation of CFTRΔF508, we performed cycloheximide chase experiment. To do this, we treated HeLa cells expressing CFTRΔF508 with cycloheximide to block the synthesis of new proteins and followed the fate of the existing protein for different chase periods. At the zero time point, CFTRΔF508 was found as an ER-localized form (B-form), which was gradually degraded during chase (Fig. 4A, upper). Strikingly, the overexpression of DNAJB14 (Fig. 4A, lower, and Fig. 4B) enhanced the degradation of CFTRΔF508. Thus, like DNAJB12 (Fig. 4A, middle), DNAJB14 can enhance the degradation of this membrane protein.

Fig. 4

DNAJB14 enhances the degradation of CFTRΔF508 via the ubiquitin proteasome-dependent pathway. (A) To study the effect of DNAJB14 overexpression on the degradation of CFTRΔF508, HeLa cells were co-transfected with plasmids to express both CFTRΔF508-GFP and DNAJB14 (or DNAJB12). After 24 h, cells were treated with 150 μg/ml cycloheximide to block the synthesis of proteins and chased for 0, 30, 60 and 120 min. Cells were then lysed and the resulting cellular lysates were separated by SDS-PAGE and subjected to immunoblotting with the anti-GFP antibody to detect CFTRΔF508-GFP. The band intensities were quantified and normalized with the intensity at zero time point. Results from three individual experiments were plotted into a graph (B). (C) To study the involvement of the ubiquitin-proteasome system in the DNAJB14-dependent degradation of CFTRΔF508, HeLa cells expressing CFTRΔF508-GFP alone or both CFTRΔF508-GFP and DNAJB14 were processed as described above, except that cells were treated with 20 μM MG132, a ubiquitin-proteasome inhibitor, at zero time point. The data from three individual Western analyses were plotted into a graph on panel D. (E) To study the effect of a mutation in the J-domain of DNAJB14 on the degradation of CFTRΔF508, HeLa cells expressing both CFTRΔF508-GFP and DNAJB14 D138N were processed as described above. Data from three individual experiments were plotted into a graph (F). Error bars indicate standard deviations from the average of three independent experiments. JB12, DNAJB12; JB14, DNAJB14; B, core-glycosylated, ER-localized form of CFTRΔF508-GFP.

To get insight into the degradation enhancing activity of DNAJB14, we examined whether DNAJB14 requires the ubiquitin-proteasome system to promote the degradation of CFTRΔF508. For this end, we studied the effect of MG132, an inhibitor of the proteasome, on the DNAJB14-dependent degradation of CFTRΔF508. Remarkably, treatment of HeLa cells with MG132 greatly retarded the DNAJB14-dependent degradation of CFTRΔF508 (compare the third panel with fourth panel in Fig. 4C, and the red solid line with red dotted line in Fig. 4D), revealing the importance of the ubiquitin-proteasome pathway in the degradation. Consistently, in the presence of MG132, the rate of CFTRΔF508 degradation in DNAJB14-overproducing cells was comparable to that of cells without DNAJB14-overexpression (compare red dotted line with black dotted line in Fig. 4D). Based on these findings, we concluded that DNAJB14 requires the ubiquitin-proteasome pathway to promote the degradation of the misfolded membrane protein.

To further characterize the DNAJB14-dependent degradation of CFTRΔF508, we examined whether this degradation requires the ability of DNAJB14 to interact with Hsc70. For this purpose, we utilized DNAJB14 D138N mutant that was deficient in its interaction with Hsc70 (see Fig. 3C). Intriguingly, this mutation caused only a slight retardation of the DNAJB14-dependent degradation of CFTRΔF508 (Fig. 4E, F), suggesting that DNAJB14 D138N mutant still retained some of its activity. The implications of this finding will be discussed later in the discussion section.

DNAJB14 enhances the degradation of membrane proteins

DNAJB12 enhances the degradation of misfolded membrane proteins. Since DNAJB14 promoted the degradation of CFTRΔF508, we examined whether DNAJB14 can stimulate the degradation of another ERAD substrate, α subunit of T-cell receptor (TCRα). In contrast to CFTR which is a multiple membrane-spanning protein, TCRα is a small single membrane-spanning protein. This protein is misfolded and degraded by ERAD when produced in the absence of its partner subunit (Yu et al., 1997). Under such conditions, TCRα was indeed gradually degraded in HeLa cells (Fig. 5A, B). Importantly, like in the case of DNAJB12, the over-expression of DNAJB14 also accelerated the degradation of TCRα (Fig. 5A, B). These findings indicate that DNAJB14 can promote the degradation of a variety of membrane proteins.

Fig. 5

DNAJB14 accelerates the degradation of TCRα, but not a variant of α1-antitrypsin. HeLa cells expressing both DNAJB14 and one of ERAD substrates, TCRα-HA (A) or null-Hong Kong variant of α1-antitrypsin (A1AT-NHK) (C), were chased with 150 μg/ml cycloheximide for the indicated times. The protein extracts were prepared and analyzed by immunoblotting using anti-HA and anti-A1AT antibodies to detect TCRα-HA (A) and A1AT-NHK (C), respectively. The band intensities of TCRα-HA (A) and A1AT-NHK (C) from three independent experiments were analyzed and plotted onto graphs on panels B and D, respectively.

We next tested whether DNAJB14 can enhance the degradation of an ER luminal protein. α1-antitrypsin protein is a serine protease inhibitor that is secreted into the blood plasma. The null-Hong Kong variant of α1-antitrypsin (A1AT-NHK) cannot fold properly in the ER and, thus, this protein is eliminated from the ER by ERAD (Sifers et al., 1988). Therefore, this protein has been used as a model substrate of the ERAD of an ER luminal soluble protein. The overexpression of DNAJB14 did not affect the stability of this protein (Fig. 5C, D). Thus, like DNAJB12, DNAJB14 can specifically enhance the ERAD of membrane proteins.

Discussion

Protein quality control systems play important roles in the maintenance of cellular homeostasis by degrading misfolded proteins that arise in the cells (Goldberg and St. John, 1976). When misfolded proteins, such as CFTRΔF508, arise in the ER membrane, they are pulled out of the ER, polyubiquitinated, and degraded by the proteasome in the cytosol (Ward et al., 1995; Vembar and Brodsky, 2008; Houck and Cyr, 2012). DNAJB12 is an ER membrane-localized J-protein having a J-domain in the cytosol. This protein plays an important role in the quality control of membrane proteins by stimulating the degradation of misfolded membrane proteins (Yamamoto et al., 2010; Grove et al., 2011). Here, we presented evidence that the mammalian ER has another membrane-bound J-protein (DNAJB14) that, like DNAJB12, can promote the degradation of misfolded membrane proteins.

Our data indicated that DNAJB14 resembles DNAJB12 in many ways. Firstly, the amino acid sequence of mouse DNAJB14 shares 50% identity with that of mouse DNAJB12 (Fig. 1A). Secondly, like DNAJB12, DNAJB14 is an ER-localized single membrane spanning protein having a J-domain facing the cytosol and uses its J-domain to interact with Hsc70 in the cytosol (Fig. 3). Thirdly, the expression of DNAJB14 and DNAJB12 is insensitive to both heat and ER stresses (Fig. 2). Fourthly, DNAJB14 can also promote the degradation of misfolded ER membrane proteins but not that of a misfolded ER luminal protein (Fig. 4 and Fig. 5). Finally, like DNAJB12, DNAJB14 requires the function of the proteasome to promote the degradation of a membrane protein (Fig. 4).

Then, why does the mammalian ER possess two analogous proteins (DNAJB14 and DNAJB12) that both can promote the degradation of membrane proteins? In contrast to DNAJB12 mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more weakly, being most abundant in testis (Fig. 1C, D). This finding may imply that DNAJB14 has a specific role in this tissue. In addition, the amino acid sequence of mouse DNAJB14 is closer to that of human DNAJB14 than to that of mouse DNAJB12. These observations imply that, during evolution, these two analogous genes have evolved separately to perform distinct functions. Nevertheless, apparently, further studies are needed to clarify this point.

In the ubiquitin-proteasome system, an E3 ubiquitin ligase promotes the poly-ubiquitination of a subset of specific target proteins for degradation. A previous study has shown that, among the E3 enzymes, an ER-membrane-localized E3 enzyme, RMA1, physically interacts and cooperates with DNAJB12 to promote the degradation of CFTRΔF508 (Grove et al., 2011). We have not identified the E3 ubiquitin ligase which physically and functionally interacts with DNAJB14. Yet, in contrast to DNAJB12, DNAJB14 failed to interact with RMA1 in our preliminary co-immunoprecipitation experiment (Sopha et al., unpublished observation). Thus, it may be possible that DNAJB14 binds another ER-resident E3 ligase and thus uses a different mechanism to degrade misfolded membrane proteins.

Our result indicated that, like DNAJB12, DNAJB14 stably associates with Hsc70 (Fig. 3C). This stable association was lost by a mutation in the J-domain of DNAJB14 (Fig. 3C). However, like a J-mutant of DNAJB12 (Yamamoto et al., 2010), this DNAJB14 mutant retained some of its ability to promote the degradation of CFTRΔF508 (Fig. 4E). Thus, as we have already discussed for the J-mutant of DNAJB12 (Yamamoto et al., 2010), it may be possible that DNAJB14 can exert some of its functions without interacting with Hsc70. Alternatively, the DNAJB14 mutant may still be able to weakly interact with Hsc70 in vivo to promote the degradation of membrane proteins.

Acknowledgments

We are grateful to Dr. Kazuhiro Shiozaki for discussions, Dr. Ron Kopito for plasmids, Junko Hashimoto, Naoko Fujimoto, Azumi Wada for technical assistance, and Rie Kurata, and Yoichiro Fukao for mass spectrum analysis. This work was supported by MEXT KAKENHI (19058010 to K.K.), JSPS KAKENHI (24228002 to K.K., 24580141 to H.K.), Noda Institute for Scientific Research (to K.K.), The Uehara Memorial Foundation (to K.K.), Takeda Science Foundation (to K.K.), and Mitsubishi Foundation (to K.K.). P.S. was a recipient of a NAIST International Scholarship, Y.Y. by a fellowship from the Japan Society for the Promotion of Science, and H.K. by an international research fellowship from the Global COE program in NAIST from MEXT of Japan.

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