Structural characterization and subcellular localization of Drosophila organic solute carrier partner 1
© Huu et al.; licensee BioMed Central Ltd. 2014
Received: 3 February 2014
Accepted: 13 June 2014
Published: 18 June 2014
Organic solute carrier partner 1 (OSCP1) is known to facilitate the transport of various organic solutes into cells and reported to play a role in cell growth and cell differentiation. Moreover, OSCP1 is known as a tumor suppressor gene that is frequently down-expressed in nasopharyngeal carcinomas and acute myeloid leukemia. However, the underlying mechanisms of action remain unclear and the subcellular localization of OSCP1 has yet to be determined in detail.
Drosophila contains a single orthologue of OSCP1 (dOSCP1) that shares 58% homology with its human counterpart. To study the expression pattern and subcellular localization of dOSCP1, we prepared a specific antibody. Subcellular localization analyses of dOSCP1 with these revealed localization in the plasma membrane, endoplasmic reticulum, Golgi apparatus and mitochondria, but no detection in cytosol. dOSCP1 signals were also detected in the nucleus, although at weaker intensity than in plasma membranes and subcellular organelles. In addition, native polyacrylamide gel electrophoresis analysis with and without β-mercaptoethanol treatment revealed that recombinant dOSCP1 forms dimers and trimers in solution. The dimer form of dOSCP1 could also be detected by Western immunoblot analyses in third instar larval extracts.
The data revealed that dOSCP1 localizes not only in the plasma membrane but also in the nucleus, ER, Golgi apparatus and mitochondria. It is therefore conceivable that this protein may interact with various partners or form multimeric complexes with other proteins to play multiple roles in cells, providing clues to understanding the functions of dOSCP1 during Drosophila development.
KeywordsOrganic solute carrier partner 1 Drosophila Subcellular organelle
Transporter proteins play important roles in absorption and elimination of numerous endogenous molecules, as well as exogenous substances and their metabolites, from cells. Moreover, identification and characterization of transporter proteins are important for drug discovery and development . The mechanisms involved in cellular transport of organic anions and cations have been studied and reviewed elsewhere [2–4]. Transporter proteins belong to two major superfamilies: solute carrier (SLC) and ATP-binding cassette (ABC) . The SLC transporters include two families consisting of the organic anion transporting polypeptides (OATPs) and the SLC22A group, which contains the organic cation transporters (OCTs) and the organic anion transporters (OATs) . In general, transporters are designed to recognize a single substance or a group of very similar substances, although some carrier proteins such as OATPs show broad substrate specificities [6, 7].
Recently, organic solute carrier protein 1 (OSCP1) was identified in mammals as a polyspecific solute carrier protein [8–11] and likely novel member of the SLC transporters. When expressed in Xenopus laevis oocytes, OSCP1 mediated high affinity transport of p-aminohippurate (PAH), tetraethylammonium, and a wide range of structurally diverse organic compounds including prostagladin E2, prostaglandin F2α, estron sulfate, glutarate, L-leucine, L-ascorbic acid and tetracycline [8–11]. These results suggest that OSCP1 mediates transport of various organic solutes into cells.
On the other hand the OSCP1 gene, also named as Oxidored-nitro domain-containing protein 1 (NOR1), has been identified as a tumor suppressor gene that is frequently down-expressed in nasopharyngeal carcinomas (NPCs) [12–16]. Moreover, the OSCP1 (NOR1) promoter region has been found to be frequently methylated in acute myeloid leukemia (AML) patients . These data indicate that OSCP1 may play a role in genesis of NPC and AML, although the underlying mechanisms are not fully understood.
Most SLC22A super family proteins are predicted to consist of 12 transmembrane domains, with intracellular amino and carboxy-termini [18, 19], although organic solute transporter alpha (OSTα) and beta (OSTβ) contain 7 and 1 transmembrane domains, respectively . However, the structure of OSCP1 has not yet been biochemically and physically characterized. In the present study, we therefore examined the structure of Drosophila melanogaster OSCP1 (dOSCP1). Native polyacrylamide gel electrophoresis analysis with and without β-mercaptoethanol treatment revealed that the recombinant dOSCP1 forms dimers and trimers in solution. The dimer form of dOSCP1 was further confirmed by Western immunoblot analyses with third instar larval extracts.
Subcellular localization of OSCP1 is controversial. Although it has been reported to localize in plasma membranes of human trophoblast cells and mouse Sertoli cells [9, 11], cytoplasmic localization has also been reported in mouse cerebral neuronal cells [8, 21] and human HeLa cells . Therefore, in this study, we examined subcellular localization of dOSCP1 and revealed its presence in the plasma membrane, endoplasmic reticulum, Golgi apparatus, mitochondria and nucleus of cells. The data indicate that dOSCP1 plays not only in the transport of organic solutes through the cell membrane, but also into the organelles and nucleus, and consequently it may be involved in regulation of apoptosis, differentiation and/or proliferation.
OSCP1 is an evolutionary conserved protein across species
Expression and purification of recombinant dOSCP1 protein
Structural analysis of dOSCP1 by native PAGE
To evaluate the possible multimer formation of dOSCP1, we performed native-PAGE analysis of recombinant dOSCP1 protein, treated with or without 4% SDS and 10% β-mercaptoethanol with or without heating. The apparent dimer form of dOSCP1 was detected at the position corresponding to 70 kDa when compared with the standard maker (Figure 2B, lanes 1 and 2). In addition a more slowly migrating band corresponding to 100 kDa was also detected that could represent a trimer form of dOSCP1 under the native PAGE conditions. In any event, by this method, we first established that dOSCP1 can form multimers in solution.
Subcellular localization of dOSCP1 protein in various Drosophila larval tissues
Localization of dOSCP1 protein on plasma membranes
Localization of dOSCP1 protein in subcellular organelles
In immunostaining with salivary gland, we observed dOSCP1 signals in a mesh-like structure in the cytoplasm (Figure 5). As a first step to clarify the nature of this dOSCP1-positive structure, regarding the precise localization of the OSCP1 protein in the cytoplasm, double-immunostaining of the salivary gland with anti-dOSCP1 and specific antibodies for each organelle was performed.
To further evaluate the Golgi apparatus localization of dOSCP1, double immunostaining with anti-dOSCP1 and anti-GM130 antibodies was performed. The anti-GM130 antibody detects a peripheral membrane protein GM130 that binds to the Golgi complex . The data showed overlapping signals (Figure 8E-H), further confirming the Golgi apparatus localization of dOSCP1.
OSCP1 has been reported to mediate the transport of various organic solutes such as organic anions and cations across the plasma membrane in a pH-dependent and sodium-independent manner [8–10] . However, the underlying mechanisms of OSCP1 function have been unclear. Up to now, there has been no report on the structure of OSCP1 protein. Previous studies demonstrated that oligomerization of many transporter proteins plays an important role in movement of materials across cells [37–39]. Thus, oligomerization allows proteins to form large structures without increasing genome size and provides stability, while the reduced surface area of monomers in a complex can offer protection against denaturation [18, 40, 41]. Moreover, many proteins are known to self-assemble into oligomers to perform their biological functions . In this study we found that dOSCP1 could form dimers and trimers in solution, which may be required for its transporter functions. Regarding membrane transporter proteins, dimerization and higher order oligomerization have actually been proposed as important factors that modulate their activity . Therefore, further investigation of the dimerization of dOSCP1 would appear necessary to ascertain the underlying mechanisms of function.
In addition, several membrane transporters such as OATs are predicted to have a 12 membrane spanning domain , while the exceptions of OSTα and OSTβ predicted to have seven and one, respectively . We used bioinformatics tools to gain insight into whether or not Drosophila OSCP1 carries a potential transmembrane domain(s). However, Kyte-Doolittle hydropathy analysis method indicated that Drosophila OSCP1 carries no potential transmembrane domain (http://gcat.davidson.edu/DGPB/kd/kyte-doolittle.htm). The same prediction was obtained with the SMART program (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) and the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html). Furthermore, a previous study predicted that mouse OSCP1 does not carry any potential transmembrane domain . These observations indicate that dOSCP1 may be not imbedded in the membrane but associated with some membrane bound proteins and consequently indirectly mediate and facilitate the transport of organic solutes into the cells. It is well established that many proteins associated with the cell membrane are either directly or indirectly involved in membrane transport [45–47]. Multimeric complex formation was also reported for L-type amino acid transporter 1 with the 4EF2 heavy chain , and between OSTα and OSTβ . Taken together these observations suggest that dOSCP1 may associate with other transporters to assist their functions. Future investigations should focus on whether OSCP1 acts as a regulator or a constituent of the functional transporter system in cells.
Various membrane transporters have been detected in different tissues including the blood–brain barrier, lung, heart, intestine and kidney . The broad tissue distribution of human, mouse and rat OSCP1 has been well characterized, brain, lung, trachea, spinal chord and nasopharynx being included as shared sites [9, 11, 13, 14]. Therefore, the present detection of dOSCP1 in various Drosophila larval tissues including brain lobes, eye discs, wing discs, leg discs, fat body and salivary glands suggests that the broad distribution pattern of OSCP1 is conserved among multiple species.
In addition to the dotted or mesh-like structures of dOSCP1 signals in the cytoplasm of the salivary glands, we detected some nuclear dOSCP1 signals in the proximal regions of the glands. In contrast, no nuclear signal was detectable in the distal region. It is known that most of cells in salivary glands of late stage of third instar larvae have completed polytenization in the distal region, but not in the proximal region . Therefore dOSCP1 may play a role in nuclei of endoreplicating cells. Although dOSCP1 does not contain a canonical nuclear localization signal (NLS), the PSORT II tool based on the amino acid sequence predicts nuclear localization of dOSCP1 with 76.7% reliability (http://psort.hgc.jp/form2.html). However, since we cannot exclude the possibility that the antibody may be more accessible to the proximal region, further analyses are necessary to confirm these observations.
In the present study, we could show that dOSCP1 localizes not only in the plasma membrane but also in the nucleus, ER, Golgi apparatus and mitochondria. It is therefore conceivable that this protein may interact with various partners or form multimeric complexes with other proteins to play multiple roles in cells. Whatever the case, the present data should help in furthering understanding of the roles of the dOSCP1 gene during Drosophila development.
Sequence comparison of the OSCP1 gene
The OSCP1 homologue sequence from the GeneBank database was analyzed in different species: (Homo sapiens) OSCP1 (NP_659484.4), (Pan troglodytes) OSCP1 (JAA38622.1), (Macaca mulatta) OSCP1 (XP_002802362), (Mus musculus) OSCP1 (NP_766289.2), (Canis lupus) OSCP1 (XP_53256.3), (Bos Taurus) OSCP1 (NP_001039369.1), (Gallus gallus) OSCP1 (XP_001233001.1), (Rattus norvegicus) OSCP1 (NP_001025094.1), (Danio rerio) OSCP1 (XP_003201654.1), (Caenorhabditis elegans) R10F2.5 (NP_497640.1), (Drosophila melanogaster) CG13178 (AFF58577.1). Sequence alignment was performed using ClustalW2 software.
For construction of pCold-dOSCP1, the following oligonucleotides were synthesized.
To construct the plasmid pCold-dOSCP1, the full length cDNA of dOSCP1 was amplified by PCR with primers carrying XhoI and EcoRI sites and the amplified fragment was inserted into the plasmid pColdI (Takara, Japan) using an Infussion HD Kit (ClonTech, Japan)
Preparation of the recombinant dOSCP1-His fusion protein and production of anti-dOSCP1 antibody
The amplified full length dOSCP1 cDNA carrying XhoI and EcoRI sites was cloned into the pColdI vector (Takara, Japan) to carry a C terminal His tag fusion sequence. After the nucleotide sequence was confirmed by sequencing, the plasmid was transformed into E. coli BL21. Expression of His-dOSCP1 fusion protein was carried out with a cold shock expression system (Takara, Japan). Cell lysates were prepared by sonication in PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% SDS, then the supernatant and the pellets were separated by centrifugation at 12,000 g for 20 min at 4°C. The supernatant was diluted with binding buffer (500 mM NaCl, 50 mM NaH2PO4, pH 8.0, 5 mM Imidazol, 1 mM PMSF) and purified with Ni-NTA agarose (QIAGEN, USA). After purification, the His-dOSCP1 fusion proteins were used to immunize a Guinea pig (SLC, Inc. Japan). Production of polyclonal antibodies was checked by Western blotting.
Fly stocks were maintained at 25°C on standard food containing 0.7% agar, 5% glucose and 7% dry yeast. The transgenic fly line carrying glass minimal response element GMR-GAL4 on X chromosome was as described previously . Fly lines carrying UAS-dOSCP1IR 466–609 were obtained from the Vienna Drosophila RNAi Center.
Flip out experiments
RNAi clones in Drosophila larval salivary gland were generated with a flip-out system . Female flies with hs-flp; Act5C > FRT y FRT > Gal4, UAS-GFP were crossed with male flies carrying UAS-dOSCP1IR 466–609 . Clones were evaluated by the presence of green fluorescent protein (GFP) expressed under control of the Act5C promoter. Flip-out was induced by heat shock (60 min at 37°C) at 24–48 hours after egg laying.
For immunohistochemistry, third instar larvae were dissected and fixed in 4% paraformaldehyde/PBS for 15 min at 25°C. After washing with PBS/0.3% Triton X-100 (PBS-T), the samples were blocked with PBS containing 0.15% Triton X-100 and 10% normal goat serum for 20 min at 25°C and incubated with primary antibodies for 16 h at 4°C. The following antibodies were used: anti-dOSCP1 (1:100), mouse anti-Dlg (1:400) (Developmental Studies of Hybridoma Bank, DSHB), mouse anti-KDEL (1:800) (Enzo Life Sciences), mouse anti-KDEL receptor (1:300) (Abcam), rabbit anti-GM130 (1:200) (Abcam), mouse anti-HSP60 (1:200) (Enzo Life Sciences). For negative control, 10 μg of Guinea pig IgG dOSCP1 antibody was incubated with 15 μg of the purified His-dOSCP1 fusion protein for 16 h at 4°C and used as primary antibody (1:100). After washing with PBST, samples were incubated with secondary antibodies labeled with either Alexa 594 or Alexa 488 (1:400; Invitrogen, Tokyo, Japan) for 3 hours at 25°C. Following further washing with PBS/0.3% Triton X-100 and with PBS, the samples were stained with DAPI (0.5 μg/ml)/PBS/0.1% Triton X-100. After washing with PBST and PBS, the samples were mounted and observed under a confocal laser scanning microscope (OLYMPUS Fluoview FV10i).
Two μg and 4 μg of His-dOSCP1 fusion protein were prepared in a non denaturing sample buffer (62.5 mM Tris–HCl pH 6.8, 25% Glycerol, 1% bromophenol blue) on ice  and denaturing sample buffer (10% β-mercaptoethanol, 4% sodium dodecyl sulfate, 20% glycerol, 0.25% bromophenol blue, 0.125 M Tris–HCl pH 6.8) in boiling water for 5 min . Proteins were separated on polyacrylamide gels containing 10% acrylamide without SDS at a constant voltage of 100 V for 6 hours at 4°C . The gels were then stained with Coomassie Brilliant Blue G-250 (CBB).
SDS-PAGE and Western immunoblot analysis
Eye imaginal discs of third instar larvae of wild type and transgenic flies carrying GMR-Gal4; UAS-dOSCP1IR; + were dissected in PBS and homogenized in an extraction buffer containing 50 mM Tris–HCl (pH7.5), 5 mM MgCl2, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 0.1% NP-40, 10 μg/ml each of aprotinin, leupeptin and pepstatin A and 1 μg/ml each of antipain, chymostatin and phosphoramidon. Homogenates were centrifuged and total protein quantified by the Bradford method . 30 μg of proteins were boiled at 100°C for 5 min, electrophoretically separated on SDS-polyacrylamide gels containing 10% acrylamide and then transferred to polyvinylidene difluoride membranes (Bio-Rad, USA). The membranes were blocked in TBS-0.05% Tween 20 containing 5% skim milk for 1 hour at 25°C, followed by incubation with mouse monoclonal anti-His antibody (DSHB) at 1:1500 or Guinea pig polyclonal anti-dOSCP1 antibody at 1:1500 dilution or mouse monoclonal anti-α-tubulin antibody (DSHB) at 1:5000 for 16 hours at 4°C. After washing 5 times with TBS, the membranes were incubated with HRP-conjugated rabbit anti-Guinea pig IgG (H + L) (Invitrogen, USA) at 1:10,000 dilution or stabilized peroxidase-conjugated goat anti-mouse (H + L) (Thermo Scientific, USA) at 1:5,000 dilution for 1 hour at 25°C. Detection was performed with ECL Western blotting detection reagents (GE healthcare, USA). Images were captured by AE-9300H Ez–Capture MG system (ATTO corporation, Japan) and analyzed with Image Saver6 software (ATTO corporation, Japan).
Availability of supporting data
The data sets supporting the results of this article are available in the LabArchves. The unique persistent identifier and hyperlink to data sets in https://mynotebook.labarchives.com/share/Masamitsu%2520Yamaguchi/MzIuNXwzOTQ2OS8yNS9UcmVlTm9kZS8xNjQ2ODMxOTAxfDgyLjU= (Additional file 1: Figure S1).
https://mynotebook.labarchives.com/share/Masamitsu%2520Yamaguchi/MzYuNHwzOTQ2OS8yOC9UcmVlTm9kZS8yODcxNjUyNTgyfDkyLjQ= (Additional file 2: Figure S2).
Organic solute carrier partner 1
Organic anion transporting polypeptides
Organic anion transporters
Oxidored-nitro domain-containing protein 1
Acute myeloid leukemia
Nasopharyngeal carcinomas, signal
Coomassie brilliant blue G-250
Nuclear localization signal
Upstream activation sequence
Heat shock protein 60
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Double strand RNA.
We thank Dr. A. Plessis for supplying fly lines, and Dr. M. Moore for comments on the English in the manuscript. This study was partially supported by a scholarship and grants from the KIT, JST and and the JSPS Core-to-Core Program, B. Asia-Africa Science Platforms.
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