July 2002
Volume 43, Issue 7
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Physiology and Pharmacology  |   July 2002
Interaction of GABA Receptor/Channel ρ1 and γ2 Subunit
Author Affiliations
  • George M. Ekema
    From the Division of Molecular Medicine, Harbor-UCLA Medical Center, School of Medicine, University of California Los Angeles, Torrance, California.
  • Wei Zheng
    From the Division of Molecular Medicine, Harbor-UCLA Medical Center, School of Medicine, University of California Los Angeles, Torrance, California.
  • Luo Lu
    From the Division of Molecular Medicine, Harbor-UCLA Medical Center, School of Medicine, University of California Los Angeles, Torrance, California.
Investigative Ophthalmology & Visual Science July 2002, Vol.43, 2326-2333. doi:https://doi.org/
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      George M. Ekema, Wei Zheng, Luo Lu; Interaction of GABA Receptor/Channel ρ1 and γ2 Subunit. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2326-2333. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine whether protein–protein and functional interactions can occur between γ-aminobutyric acid (GABA)A receptor/channels γ2 subunit and the retina-specific GABAC ρ1 subunit.

methods. Protein–protein interaction was characterized by immunocoprecipitation of these subunits in brain and spinal cord with anti-γ2 subunit antibody and by Western blot analysis with anti-ρ1 subunit antibody. The ρ1 and γ2 subunits were detected in the adult rat brain and spinal cord lysates that had been previously precipitated with the specific antibodies against the ρ1 and γ2 subunits, respectively. A two-microelectrode voltage clamp was used to measure GABA-induced currents in oocytes. In addition, a yeast two-hybrid system was used to detect the interactions of these subunits in vivo.

results. Based on yeast transformed with the N-terminal fragment of the γ2 subunit (γ2-N′), the N-terminal fragment of the ρ1 subunit (ρ1-N′), and the full-length ρ1 subunit, the protein–protein interaction of the GABAA γ2 subunit and the GABAC ρ1 subunit was found in yeast grown in triple-dropout medium (deficient in Leu, Trp, and His) and expressing the LacZ reporter gene. Interaction of the ρ1 and γ2 subunits was investigated by functional studies in which γ22-N′ with 837 bp) and ρ1 cRNAs were coinjected in Xenopus oocytes. In studies of the functional interaction, after injection of the γ2 subunit mutant cRNA containing a N-terminal fragment, GABA-induced ρ1 originated currents declined to 16% of the control level of homooligomeric ρ1 current. This inhibitory effect of coexpressing γ2 subunit mutants with ρ1 subunit on the ρ1-originated current in oocytes was dose dependent. In addition, coexpression of the GABA ρ1 and γ2 subunits in oocytes altered pharmacologic properties of the homooligomeric receptor/channel formed by ρ1 or γ2 subunits. Further evidence was provided by results obtained with specific antibodies showing that the ρ1 subunit was coimmunoprecipitated with the γ2 subunit from the retina, brain, and spinal cord.

conclusions. The results indicate that protein–protein and functional interactions can occur between the GABAA γ2 subunit and the GABAC ρ1 subunit. Therefore, the functional role of GABA receptor/channels in the brain, retina, and spinal cord is more diversified because of the possible assembly between the GABAA γ2 subunit and GABAC ρ1 subunit.

A major inhibitory neurotransmitter in the vertebrate central nervous system (CNS) is γ-aminobutyric acid (GABA). Its cognate receptors have been classified into three distinct subtypes based on their pharmacologic properties. The GABAA receptor/channel is bicuculline sensitive, the GABAB receptor is baclofen sensitive, and the GABAC receptor/channel is insensitive to either bicuculline or baclofen. However, the current classification of GABA receptors does not completely describe GABA receptor/channel heterogeneity, based on pharmacologic profiles of diseases of the GABAergic system, GABA receptor/channel subunit distributions in the CNS, and electrophysiology of the GABA receptor/channel in vivo. 1 2 3 4 5 6 7 8 GABA receptor/channel heterogeneity in the mammalian CNS is due to interactions among at least 21 different subunits, including α1 to α6, β1 to β4, γ1 to γ4, δ, ω, π, ζ, and ρ1 to ρ3. 9 10 11 12 13 14 15 16 17 18 19 20 Composition of different subunits in the receptor/channel determines the pharmacologic and electrophysiological properties of GABA receptors-channels. 10 12 20 21 22 Analysis of molecular structure of GABA receptors-channels reveals that subunits assemble in a putative pentameric heterooligomeric combination to form the receptor/channel. 23 The GABAC ρ1 subunit, cloned from the human retina, forms functional homooligomeric receptor channels when expressed in Xenopus oocytes. 11 The ρ1 subunit was initially reported to be a retina-specific GABA receptor/channel, exclusively localized in the retina. 24 However, distribution of the ρ1 subunit is not limited to the retina; it has also been reported in the CNS 25 in the hippocampus, 26 27 cerebellum, 28 29 anterior pituitary, 1 dorsal root ganglia, 30 and optic tectum. 31  
The molecular structure of GABAA receptor/channel complex is thought to be a heteropentomeric glycoprotein of approximately 275 kDa composed of combinations of multiple peptide subunits. 32 This has been deduced from Western blot, immunoprecipitation, immunoaffinity chromatography, and in situ hybridization. Topologic structure of GABA receptor/channel is predicted from the structure of another ligand-gated receptor/channel. 33 The functional heterogeneity of GABAA receptors in neurons arises, not only from the multiple GABA receptor/channel subunit genes and splice variants, but also from the combinatorial mixing of different GABA receptor/channel polypeptides to form heterooligomeric receptor/channels. 16 34 35 36 37 38 Various GABAA receptor/channel subunits are differentially expressed during specific developmental stages and under varying physiological conditions in different regions of the body. 11 39 40 These considerations may contribute to the diversity seen in GABA receptor/channel pharmacology and function. If heterooligomeric receptors were formed by random combinations, it would create numerous GABA receptor/channel isoforms. In fact, functional GABAA receptor/channels always exist in preferred subunit combinations, as mentioned earlier. 33 GABA α1β2/3γ2 and α2β2/3γ2 receptor/channels represent two major GABAA receptor subtypes contributing to 75% to 85% of the diazepine-sensitive GABAA receptors. 10 It has been shown that different subunits do not have the same assembly behavior. 35 41 GABAC ρ1 or ρ2 subunits can form homooligomeric receptors and express robust currents in oocytes. 11 42 However, sole expression of a single GABAA receptor subunit in oocytes results in a much smaller GABA-induced current compared with heterooligomeric expression, suggesting a low efficiency for GABAA subunits to form homooligomeric channels in oocytes. 35 41 GABA receptor/channel assembly involves several posttranslation maturation steps and is driven by noncovalent protein–protein interaction. 43 There are specific recognition sites on the individual subunit that guides their proper assembly into a functional receptor/channel. However, structural elements that determine the compatibility for the subunit assembly are still unknown. 
Recently, electrophysiological studies of recombinant GABA receptor/channels in Xenopus oocytes suggest that GABA γ2 and ρ1 subunits may form a functional receptor/channel in oocytes. 44 45 46 However, there is no direct evidence that demonstrates the assembly of a heterooligomeric receptor/channel between GABA γ2 and ρ1 subunits at protein–protein interaction level in vitro and in vivo. In the present study, we report that both protein–protein and functional interactions can occur between the retinal specific GABAC ρ1 subunit and GABAAγ2 subunit. This determination is based on our results in studies in which immunoprecipitation, the yeast two-hybrid system, and electrophysiological measurements in the oocyte expression system were used. The first approach indicates that ρ1 and γ2 subunits can interact, because it was possible to immunoprecipitate the ρ1 subunit from the retina, brain, and spinal cord lysates with an antibody selective for the γ2 subunit. Further evidence for protein–protein interaction is that in the yeast two-hybrid system ρ1 and γ2 interaction occurred based on elicited gene expression. Such an interaction is reflective of a very specific assembly process occurring in vivo. Furthermore, coexpression of the ρ1 subunit along with an N-terminal fragment of the γ2 subunit occurred in Xenopus oocytes, because increases in γ2 subunit expression inhibited homooligomeric ρ1-originated currents in a dose-dependent pattern. 
Materials and Methods
Immunoprecipitation and Western Blot Analysis
The protocol of the study adhered to the provisions of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Whole brain, retina, and spinal cord from adult albino rats were lysed with lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM NaF, 1 mM Na-orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 250 μM p-nitrophenylphosphate, 10 μg/mL aprotinin, and 10 μg/mL leupeptin. Cell lysates were centrifuged at 5000 rpm for 5 minutes, and the supernatant was transferred to a new tube containing anti-γ2 antibody (polyclonal IgG from rabbit against the N terminus of the γ2 subunit) plus protein a affinity medium (Protein A Sepharose beads; Sigma, St. Louis, MO) and incubated overnight at 4°C. 47 Final dilution of the anti-γ2 antibody was 1:15,000. Cell lysates were centrifuged at 5000 rpm for 5 minutes, and the precipitate was boiled in an equal volume of SDS-PAGE sample buffer for 5 minutes. Aliquots were loaded on SDS-PAGE gels followed by Western blot analysis, using a 1:10,000 dilution of anti-ρ1 antibody (polyclonal IgG from guinea pig against the N terminus of the ρ1 subunit) for the detection. 47 Briefly, an equal volume of 2× Laemmli buffer was mixed with the immune complex and boiled for 5 minutes. After resolution by SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) and probed with antibodies against specific subunits. Membranes were then incubated with secondary antibodies conjugated with alkaline phosphatase. Secondary antibodies were detected with a Western blot detection kit (Phototope-Star; New England Biolabs, Beverly, MA). 
Yeast Two-Hybrid cDNA Construction and Transformation
cDNAs of the N termini (γN′ and ρN′, see the Results section) were subcloned into the yeast expression vector (pACT2; Clontech, Palo Alto, CA) encoding a GAL4 transcriptional activation domain (AD) of the yeast Saccharomyces cerevisiae. Full-length ρ1 cDNA was subcloned into a yeast expression vector (pAS2-1; Clontech) encoding the GAL4 DNA-binding domain (BD). The orientation and alignment of the reading frame for the subcloned cDNAs were confirmed by DNA sequencing. The yeast two-hybrid system (Matchmaker; Clontech) was used to determine interaction of the GABAA receptor/channel γ2 subunit with the GABAC receptor/channel ρ1 subunit. Reporter yeast (strain Y190; Clontech)-competent cells were prepared by the lithium acetate method. Yeast cells were cultured overnight at 30°C with shaking at 250 rpm in 50 mL of medium (YPD; Clontech) and diluted in 300 mL of the medium to an optical density at 600 nm (OD600) of 0.2. Yeast cells were centrifuged and rinsed in water at room temperature. They were resuspended in 1.5 mL of TE (Tris-EDTA) buffer/lithium acetate (TE/LiAC; Clontech). Full-length ρ1 (2.05 kbp) in pAS2-1 was simultaneously transformed into the Y190 cells with each of the N termini γ2 (γN′, 0.84 Kbp) and ρ1 (ρN′, 0.80 kbp) in the pACT2 vector. Each plasmid DNA (100 μg) was added to 100 μg herring testes carrier DNA. Yeast cells were heat shocked for 15 minutes in a 42°C water bath, spun down, and resuspended in 0.5 mL TE buffer. Transformation mixture was spread on 100-mm plates containing a synthetic dropout (SD) selection medium without leucine, tryptophan, and histidine (−Leu/−Trp/−His), supplemented with 25 mM 3-amino-1,2,4-triazole (+3-AT; Sigma, St. Louis, MO). Plates were incubated at 30°C for 2 to 4 days. For LacZ selection, only fresh colonies (3 days in culture) on SD/−Leu/−Trp/−His/+25 mM 3-AT agar plates were assayed. Assayed colonies were transferred onto filter paper and then submerged in liquid nitrogen for 10 seconds each time, followed by 1 minute of thawing. Each filter was placed, colony side up, on a presoaked filter and incubated at 30°C. 
Construction of γ2 and ρ1 N-Terminal Domains
Construction of N-terminal γ2 and ρ1 subunits was based on analysis of the full-length DNA subunit sequences. 11 18 48 49 N-terminal fragments were amplified by PCR from full-length sequences inserted into a vector (pBluescript SK(−; Stratagene, La Jolla, CA) vector. PCR primers were designed as follows: 5′-GGA TCCGGCGAGAGGAAAAAAAAGCG-3′ with an introduced BamHI restriction enzyme site (γ2 sense); 5′-GATCTGAGCAGAAGAATGGGGCTCGAG-3′ with an introduced XhoI restriction enzyme site (γ2 antisense); 5′-GGATCCCCATGTTGGCTGTCCCA-3′ with an introduced BamHI restriction enzyme site (ρ1 sense); 5′-ccccgctaccctgatggtcatgGGATTC-3′ with an introduced EcoRI restriction enzyme site (ρ1 antisense). Each N-terminal fragment was subcloned into the multicloning site (MCS) of a PCR3 vector. 
In Vitro Transcription and Microinjection of Oocytes
Fragments of subunit cDNAs for in vitro transcription were subcloned into the multiple cloning site of a PCR3 expression vector in an orientation of T7 to SP6. Plasmid constructs with inserts were linearized with PstI (New England Biolabs). A T7 in vitro transcription kit (Invitrogen, San Diego, CA) was used in all transcription reactions. Transcription reactions were performed in the presence of the cap analogue diguanosine triphosphate, and were catalyzed by T7 RNA polymerase. The cRNA was quantified by agarose gel electrophoresis and photospectroscopy (Pharmacia, Piscataway, NJ), and then dissolved in sterile RNase-free water to a final concentration of 5 μg/μL. Xenopus oocytes were prepared from frogs by using a method described previously. 47 Stage 5 to 6 oocytes were selected and incubated at 18°C in MBS (Modified Barth’s Solution) for 24 hours, followed by injection of cRNA. Microinjection of cRNA into oocytes was performed by positive displacement with a 10-μL micropipette (Drummond Scientific Inc., Broomall, PA). 
Voltage Clamp
Two-microelectrode voltage-clamp recordings were performed at room temperature with continuous perfusion (10 mL/min). The glass microelectrodes (resistance ∼2.0 MΩ) were made with a horizontal puller (PD-5; Narishige USA, Greenvale, NY), and were backfilled with 3 M KCl. The perfusion bath was connected to the voltage-recording amplifier (Axoclamp 2A; Axon Instruments, Burlingame, CA) by an Ag-AgCl-Agar-3 M KCl bridge. Data were filtered with a four-pole Bessel filter at 500 Hz. Data acquisition was performed with the pCLAMP software (Axon Instruments). 
Statistical Analysis
Data were presented as original values, or as the mean ± SE. Significant differences were determined by using the paired t-test or ANOVA and the Tukey post-hoc range test at the confidence interval indicated. 
Results
Construction of N-Terminal Domain of the ρ1 and γ2 Subunits
It has been suggested that the N-terminal part of ligand-gated channels has a large extracellular domain containing ligand-binding sites. 50 51 Its C-terminal part is believed to traverse the cell membrane and to form four transmembrane segments with a large intracellular loop between the third and fourth transmembrane segments. The second transmembrane region is likely to form the lining of the channel pore. The intracellular loop contains specific sites for channel regulation by protein kinase-induced phosphorylation. In the present study, N-terminal constructs of the ρ1 and γ2 subunits were composed of 292 and 271 amino acids, respectively (Fig. 1) . These N-terminal domain peptides were also used to produce fusion proteins in bacteria, by using a glutathione S-transferase (GST) fusion system. N-terminal domain fusion proteins were then used to raise specific antibodies against the ρ1 and γ2 subunits that demonstrate tissue and functional specificity. 47  
Immunoprecipitation of the Retinal ρ1 Subunit from the Rat Brain and Spinal Cord
Immunoprecipitation experiments were performed to determine a protein–protein interaction between GABA γ2 and ρ1 subunits in the mammalian CNS. Brain and spinal cord lysates from adult rats were obtained and incubated with specific antibodies against the GABA γ2 subunit. Brain and spinal cord proteins that were pulled down by the anti-γ2 subunit antibody were analyzed in a polyacrylamide gel. A 50-kDa band was recognized by the anti-ρ1 subunit antibody in the brain and spinal cord, but not in the control lanes in which brain and spinal cord proteins were immunoprecipitated with an anti-Erk (mitogen-activating protein [MAP] kinase) antibody (Fig. 2A) . Western blot analysis was used to detect γ2 subunits in brain and spinal cord lysates that had been immunoprecipitated with the selective γ2 antibody. A 48-kDa band was detected by the anti-γ2 antibody in the brain and spinal cord tissues, but not in lysates immunoprecipitated in the control experiment with the anti-Erk antibody (Fig. 2B) . In addition, the retinal lysate from adult rats was used to incubate with the antibody against the GABA γ2 subunit. Western blot analysis demonstrated that there was a 50-kDa band in the retina identified by the anti-ρ1 antibody (Fig. 2C) . Protein bands with molecular weights of 48 and 50 kDa identified in Western blot by anti-γ2 and anti-ρ1 antibodies are consistent with the sizes of GABA γ2 and ρ1 subunits, respectively. These results suggest that there is indeed interaction and assembly of the γ2 and ρ1 subunits in the CNS. 
Next, we tested whether an interaction between the γ2 and ρ1 subunits occurs as a posttranslational subunit–subunit interaction. To probe this possibility, GST-ρ1 fusion protein containing the N-terminal part of the ρ1 subunit was purified using a glutathione-Sepharose matrix. Lysates of the brain and spinal cord from adult albino rats were incubated with GST-ρ1 fusion proteins that were bound to GST affinity matrix. Oocytes expressing ρ1 subunit were also incubated with GST-ρ1 fusion proteins. After the fusion protein and affinity matrix were rinsed with cold PBS, GST-ρ1 fusion proteins were eluted from the affinity matrix with a glutathione elution buffer and subjected to Western blot analysis. The anti-ρ1 antibody was used to probe for interaction of the GST-ρ1 fusion protein and native ρ1 subunit from the brain and spinal cord (Fig. 3A) and the GST-ρ1 fusion protein and expressed ρ1 subunit from cRNA-injected oocytes (Fig. 3B) . In addition, lysates from brain and spinal cord tissues, and from cRNA-injected oocytes were immunoprecipitated with anti-Erk antibodies and probed with the anti-ρ1 antibody for the control, respectively. Western blot analysis demonstrated that GST-ρ1 fusion proteins used in these precipitation experiments were detectable, but native ρ1 subunit peptides were not detected. These results imply that γ2 and ρ1 subunit interaction or assembly in the CNS is unlikely to be a posttranslational event. 
Protein–Protein Interaction of γ2 and ρ1 Subunits in the Yeast
To further confirm protein–protein interaction between GABA γ2 and ρ1 subunits in vivo, a high-fidelity yeast two-hybrid expression system was used. In the reporter yeast strain Y190, expression of the His-3 and LacZ reporter genes requires the DNA BD and AD of the GAL4 to be colocalized upstream from the activation sequence of GAL4. In addition, we fused full-length ρ1 to the DNA BD, whereas γ2N′ and ρ1N′ terminal fragments were fused to the transcriptional AD of the yeast GAL4 transcriptional activator. The colocalization is mediated in yeast by the specific interaction of these fusion proteins that were connected to the AD and BD. In addition, 3-amino-1,2,4-triazole (3-AT) was added to the triple-dropout medium to prevent leaky His-3 activity, thereby ensuring the fidelity of the system. Thus, activation of transcription of the His-3 and LacZ reporter genes in our two-hybrid assay was due to the specific interaction of the γ2 or ρ1 N′-terminal fragment and ρ1 subunit that were fused to the AD and BD, respectively. The 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) reaction (detection of LacZ reporter gene expression) was also used as the other indication of the interaction between AD and BD fusion proteins. The X-gal reaction ensures that survival of transformed yeast grown in SD−Leu/−Trp/−His selection medium is due to transcription of the His-3 reporter gene, not to external histidine contamination. 
Y190 cells were simultaneously transformed with ρ1-BD cDNA and each of the γ2N′-AD and ρ1N′-AD cDNA, in turn, and the transformation mixture was plated on SD−Leu/−Trp/−His/+25 mM 3-AT agar medium. Results shown in Table 1A indicate that in both transformations there was significant growth of Y190 primary colonies. The ρ1N′/ρ1 transformation was used as the positive control, because their interaction has been extensively characterized. Control experiments were also performed by simultaneously transforming yeast with antisense ρ1-BD cDNA and γ2N′-AD or by ρ1N′-AD cDNA and by plating transformation mixture on SD−Leu/−Trp/−His/+25 mM 3-AT medium (Table 1A) . No growth of Y190 was observed in these plates. To further ensure that neither BD nor AD could independently activate transcription of the reporter genes, the reporter yeast strain Y190 was transformed with either BD or AD vector. The transformation mixture was plated on SD−Leu/−Trp/−His/+25 mM 3-AT agar medium (Table 1A) . There was no growth of Y190, again showing that the activation of transcription of the His-3 gene was due to specific interaction between the ρ1 and γ2 subunits. 
The affinity for protein–protein interaction is indicated in the yeast by the number of primary colonies on SD medium agar plates. There was no significant difference in the number of primary colonies from the ρ1-BD with ρ1N′-AD transformation and ρ1-BD with γ2N′-AD transformation (Table 1B) . This suggests that there is comparable protein–protein affinity between these subunits. The auxotrophic integrity of the Y190 cells was also determined, because the integrity of the reporter yeast strain (Y190) determines the fidelity of the yeast two-hybrid assay. Before each experiment, Y190 cells were transformed with either BD or AD vector, and the transformation mixture was plated on either SD−Trp or SD−Leu, respectively (Table 1C) . Specific growth on these media showed that the reporter yeast strain maintained its auxotrophic integrity. 
Functional Interaction of the γ2 N Terminus with the ρ1 Subunit in Xenopus Oocytes
It has been suggested that there are specific peptide motifs in the N-terminal domain that mediate protein–protein interaction for both homooligomeric and heterooligomeric interactions. 52 The competitive inhibition studies in Xenopus oocytes were designed by coinjecting cRNAs of full-length subunits and C-terminal, part-truncated subunits. These C-terminal, part-truncated subunits are mutant receptor/channels that do not have the ability to conduct chloride ions. If there is an assembly occurring among full-length and mutant subunits, the population of functional receptor/channel in the membrane is decreased. Coexpression of the C-terminal, part-truncated mutant of the ρ1 subunit (ρN′) with the full-length ρ1 subunit in oocytes resulted in a cRNA concentration-dependent reduction in GABA-induced whole-cell current (Fig. 4A) . Whether the γ2 subunit interacts with the ρ1 subunit was investigated by coexpression of the C-terminal, part-truncated mutant of the γ2 subunit (γN′) with the native ρ1 subunit in Xenopus oocytes. Coinjection of the ρ1 subunit cRNA with the γN′ mutant in oocytes demonstrated a concentration-dependent inhibition of GABA-induced whole-cell current (Fig. 4B) . The reduction in GABA-induced whole-cell current of the ρ1 subunit suggests that the N terminus of the γ2 subunit contains motif(s) for heterooligomeric interaction with the ρ1 subunit. 
Altered Pharmacologic Properties of the Recombinant GABAH Receptor/Channel
The plant-derived convulsant bicuculline is a potent antagonist at GABAA receptor/channels. Application of 100 μM bicuculline resulted in a more than 75% reduction of GABA-induced current in oocytes expressing heterooligomeric receptor/channel of the GABAA α1, β2, and γ2 subunits, but showed no effect on homooligomeric receptor/channels of the GABAC ρ1 subunits (data not shown). Application of 100 μM bicuculline showed no effect on recombinant GABAH receptor/channels (assembled by the γ2 and ρ1 subunits) in Xenopus oocytes (Fig. 5A) . In contrast, I4AA is an antagonist of GABAC receptor/channels in the retina but is an agonist at recombinant GABAC receptor/channels in Xenopus oocytes. 3 Application of 100 μM I4AA showed neither agonist nor antagonist effects at recombinant GABAH receptor/channels in Xenopus oocytes (Fig. 5B) . The pharmacologic studies provided additional evidence of the assembly of the γ2 subunit with the ρ1 subunit in Xenopus oocytes indicating the importance of subunit composition in the diversity of the GABAergic system. 
Discussion
Subunit composition determines the pharmacologic and electrophysiological properties of the GABA receptor/channels in the brain. Because the retina is a window of the brain, this study is important as well for understanding the GABA receptor/channels in the retina. The native GABAA receptor/channel is a heterooligomeric and pentameric protein containing γ subunits. 52 The GABAC receptor/channel ρ1 subunit has been expressed in Xenopus oocytes and forms a homooligomeric receptor/channel with a high affinity for GABA and a large ionic conductance of current. 4 5 In addition, pharmacologic and electrophysiological profiles of the GABAC receptor/channel are distinct from GABAA receptor/channels. Recently, pharmacologic and electrophysiological studies on some GABA receptor/channels have revealed characteristics different from other GABA receptor/channels. These differences could be due to novel GABA receptor/channels formed by unidentified subunits, splicing variants of cloned subunits, or subunit compositions forming novel hybrid receptor/channels. Considering that some drugs modify CNS responses through their interaction with specific subunits of GABA receptor/channels, a better understanding of their mode of action depends on additional characterization of GABAergic system diversity. Such an understanding can be obtained through further studies on subunit composition of GABA receptor/channels. We undertook in the present study to determine the protein–protein interaction of the GABA γ2 and ρ1 subunits in vivo. 
In vivo expression determined by the yeast two-hybrid and the Xenopus oocyte systems is ideal for studying protein–protein interaction and subunit assembly, because subunits are very likely to be expressed in their native conformations in these systems, as opposed to in vitro translation systems. The other advantage of these systems is that they allow examination of the subunit assembly at the translation level. Because C-terminal, part-truncated mutant subunits can be expressed in these systems, there is an additional advantage of using such systems to determine the specific peptide domain(s) that mediate interaction and assembly of these subunits. The affinity for protein–protein interaction in the yeast two-hybrid system can be determined by counting the number of primary colonies grown in the triple-dropout medium. We found, by using the two-hybrid system, that the γ2 subunit indeed interacts with the ρ1 subunit and that the affinity of this interaction is similar to the ρ1-to-ρ1 subunit interaction to form a homooligomeric receptor/channel. This suggests in neurons expressing both the γ2 and ρ1 subunit mRNAs that there may be equal possibilities for homooligomeric ρ1 assembly or heterooligomeric ρ12 assembly. 
Despite our evidence for assembly and interaction, it is also possible that the reduction in whole-cell current may be due to disruption of translation of the full-length ρ1 cRNA by the N-terminal cRNA. If this decrease in whole-cell current were due to disruption of translation, one would expect a similar reduction in whole-cell current on coexpressing full-length ρ1 cRNA with the cRNA of the N-terminal–truncated mutant of the ρ1 subunit. Coexpression of the N-terminal–truncated ρ1 mutant cRNA with the full-length ρ1 cRNA, however, showed no effect on the whole-cell current, compared with homooligomeric ρ1 expression (data not shown). In fact, increasing the amount of coexpressed γN′ and ρN′ mutants competitively inhibited GABA-induced currents of the homooligomeric ρ1 subunit in Xenopus oocytes (Fig. 4) . There are two possible explanations for the competitive inhibition: (1) Coexpression of a truncated mutant with a wild-type subunit in Xenopus oocytes resulted in a population of mutant receptor/channels that may not be translocated to the membrane, or (2) if these mutant receptor/channels were translocated to the membrane, they may not be able to form functional chloride channels because of the absence of channel-forming domains. 
The interaction of γ2 and ρ1 subunits in both the Xenopus oocyte and yeast two-hybrid systems suggests, but does not necessarily imply, that there is assembly of these subunits in the retina and CNS. We therefore used immunoprecipitation to show that there is assembly of these subunits in the retina and CNS. The fidelity of an immunoprecipitation reaction largely depends on whether there are specific and nonspecific posttranslation interactions in the protein clumping. Our data showed that subunit interaction is a cotranslation process rather than a posttranslation process, by using a fusion protein and tissue lysate incubation assay. In addition, we found that the γ2 subunit was immunoprecipitated with the ρ1 subunit when specific antibodies were used, providing strong indication of assembly of these two subunits. Incubation of GST-ρ1 fusion protein with brain and spinal cord lysates and with the homogenate from Xenopus oocytes expressing the ρ1 subunit showed that there was no posttranslation subunit–subunit interaction. More evidence in favor of cotranslation subunit interaction comes from coexpressing full-length ρ1 subunit with ρ1 mutants that do not have the N-terminal signal sequence in Xenopus oocytes, by using the voltage recording of GABA-induced whole-cell currents (data not shown). In addition, if interaction of subunits could be a posttranslation process, one would expect the mediation of chaperone proteins, which have never been shown for the ionotropic GABA receptor channel subunits. Cotranslation interaction of these subunits indicates that the subunits assemble in the ER or Golgi to form the receptor/channel complex, which is then translocated to the membrane as a complete unit. 
In summary, in our study, we showed, by the yeast two-hybrid expression system, that the γ2 subunit interacted with the ρ1 subunit, and we also showed, by the Xenopus oocyte expression system, the assembly of these two subunits. Most important, we showed that the ρ1 and γ2 subunits assembled in the retina and CNS to form a novel hybrid GABA receptor/channel. It is very likely that this hybrid receptor/channel possesses pharmacologic and electrophysiological properties that are distinct from those of GABAA and GABAc receptor/channels. Considering that subunit composition defines GABAergic system properties, additional studies are warranted to explore in the CNS the electrophysiological and pharmacologic profiles of these hybrid GABA receptor/channels. 
 
Figure 1.
 
Putative structures of N-terminal domain of the ρ1 and γ2 subunits. The cDNA fragments encoding 292 and 271 amino acids (aa) of ρ1 and γ2 subunits, respectively, were amplified with PCR, using the full-length cDNA of ρ1 and γ2 subunits.
Figure 1.
 
Putative structures of N-terminal domain of the ρ1 and γ2 subunits. The cDNA fragments encoding 292 and 271 amino acids (aa) of ρ1 and γ2 subunits, respectively, were amplified with PCR, using the full-length cDNA of ρ1 and γ2 subunits.
Figure 2.
 
Determination of interaction between GABA γ2 and ρ1 subunits in the brain and spinal cord. (A) Immunoprecipitation of the ρ1 subunit from rat brain (BR) and spinal cord (SC) using anti-γ2 antibody. Whole-brain and spinal cord lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates, with anti-ρ1 antibody. (B) The γ2 subunit (∼48 kDa) was detected in Western blot analysis of the immunoprecipitates from rat brain and spinal cord, with the same anti-γ2 antibody that was used to pull down the precipitate. (C) Immunoprecipitation of the ρ1 subunit from rat brain and retina (RT), with anti-γ2 antibody. Whole-brain and retinal lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates using anti-ρ1 antibody. In these experiments, an anti-Erk antibody (polyclonal IgG from rabbit) was used for the negative control. This antibody did not pull down any proteins that could be detected by the anti-ρ1 or the anti-γ2 antibody.
Figure 2.
 
Determination of interaction between GABA γ2 and ρ1 subunits in the brain and spinal cord. (A) Immunoprecipitation of the ρ1 subunit from rat brain (BR) and spinal cord (SC) using anti-γ2 antibody. Whole-brain and spinal cord lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates, with anti-ρ1 antibody. (B) The γ2 subunit (∼48 kDa) was detected in Western blot analysis of the immunoprecipitates from rat brain and spinal cord, with the same anti-γ2 antibody that was used to pull down the precipitate. (C) Immunoprecipitation of the ρ1 subunit from rat brain and retina (RT), with anti-γ2 antibody. Whole-brain and retinal lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates using anti-ρ1 antibody. In these experiments, an anti-Erk antibody (polyclonal IgG from rabbit) was used for the negative control. This antibody did not pull down any proteins that could be detected by the anti-ρ1 or the anti-γ2 antibody.
Figure 3.
 
Determining interaction of GABA ρ1 subunit fusion protein with GABA γ2 subunit from the brain and spinal cord or from exogenous expression in oocytes. (A) Brain and spinal cord lysates from adult albino rats were pooled and incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was applied to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but native ρ1 protein from the brain and spinal cord was not detected. (B) Homogenates of Xenopus oocytes expressing the GABA ρ1 subunit were incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was used to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but the ρ1 subunit protein expressed in Xenopus oocytes was not detected. Immunoprecipitated lysates from brain and spinal cord tissues (A) and from cRNA injected oocytes (B) with anti-Erk antibodies served as the control in Western blot analysis.
Figure 3.
 
Determining interaction of GABA ρ1 subunit fusion protein with GABA γ2 subunit from the brain and spinal cord or from exogenous expression in oocytes. (A) Brain and spinal cord lysates from adult albino rats were pooled and incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was applied to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but native ρ1 protein from the brain and spinal cord was not detected. (B) Homogenates of Xenopus oocytes expressing the GABA ρ1 subunit were incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was used to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but the ρ1 subunit protein expressed in Xenopus oocytes was not detected. Immunoprecipitated lysates from brain and spinal cord tissues (A) and from cRNA injected oocytes (B) with anti-Erk antibodies served as the control in Western blot analysis.
Table 1.
 
Growth of Y190 Cells on Synthetic Dropout Media
Table 1.
 
Growth of Y190 Cells on Synthetic Dropout Media
Construct −Leu/−Trp/−His+3AT Selection
A.
pAS2-1 ρ1-sense + pACT2 γ N′ +
pAS2-1 ρ1-sense+ pACT2 ρ N′ +
pAS2-1 ρ1-antisense+ pACT2 γN′
pAS2-1 ρ1-antisense+ pACT2 ρ N′
pAS2-1 ρ1-sense
pAS2-1 ρ1-antisense
pACT2 γ N′
pACT2 ρ N′
Construct Colonies (n)
B.
ρ1-BD/ρ1N′-AD 69 ± 21
ρ1-BD/γ2N′-AD 66 ± 17
Construct SD −Leu SD −Trp
C.
pAS2-1 ρ1sense +
pACT2 γ N′ +
pACT2 ρ N′ +
Figure 4.
 
Determination of GABA γ2 and ρ1 subunit functional interaction in Xenopus oocytes. (A) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the ρ1 subunit (ρN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal ρ1 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n = 44). Top: Representative traces from whole-cell current recordings in Xenopus oocytes. (B) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the γ2 subunit (γN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal γ2 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n equals; 40).
Figure 4.
 
Determination of GABA γ2 and ρ1 subunit functional interaction in Xenopus oocytes. (A) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the ρ1 subunit (ρN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal ρ1 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n = 44). Top: Representative traces from whole-cell current recordings in Xenopus oocytes. (B) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the γ2 subunit (γN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal γ2 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n equals; 40).
Figure 5.
 
Effects of bicuculline and I4AA on GABA-induced currents in Xenopus oocytes expressing γ2 and ρ1 subunits. (A) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAA receptor/channel antagonist bicuculline. Bicuculline showed no effect on the GABA-induced whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits. (B) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAC receptor/channel agonist/antagonist I4AA. I4AA (100 μM) showed no agonist or antagonist effect on whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits.
Figure 5.
 
Effects of bicuculline and I4AA on GABA-induced currents in Xenopus oocytes expressing γ2 and ρ1 subunits. (A) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAA receptor/channel antagonist bicuculline. Bicuculline showed no effect on the GABA-induced whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits. (B) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAC receptor/channel agonist/antagonist I4AA. I4AA (100 μM) showed no agonist or antagonist effect on whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits.
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Figure 1.
 
Putative structures of N-terminal domain of the ρ1 and γ2 subunits. The cDNA fragments encoding 292 and 271 amino acids (aa) of ρ1 and γ2 subunits, respectively, were amplified with PCR, using the full-length cDNA of ρ1 and γ2 subunits.
Figure 1.
 
Putative structures of N-terminal domain of the ρ1 and γ2 subunits. The cDNA fragments encoding 292 and 271 amino acids (aa) of ρ1 and γ2 subunits, respectively, were amplified with PCR, using the full-length cDNA of ρ1 and γ2 subunits.
Figure 2.
 
Determination of interaction between GABA γ2 and ρ1 subunits in the brain and spinal cord. (A) Immunoprecipitation of the ρ1 subunit from rat brain (BR) and spinal cord (SC) using anti-γ2 antibody. Whole-brain and spinal cord lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates, with anti-ρ1 antibody. (B) The γ2 subunit (∼48 kDa) was detected in Western blot analysis of the immunoprecipitates from rat brain and spinal cord, with the same anti-γ2 antibody that was used to pull down the precipitate. (C) Immunoprecipitation of the ρ1 subunit from rat brain and retina (RT), with anti-γ2 antibody. Whole-brain and retinal lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates using anti-ρ1 antibody. In these experiments, an anti-Erk antibody (polyclonal IgG from rabbit) was used for the negative control. This antibody did not pull down any proteins that could be detected by the anti-ρ1 or the anti-γ2 antibody.
Figure 2.
 
Determination of interaction between GABA γ2 and ρ1 subunits in the brain and spinal cord. (A) Immunoprecipitation of the ρ1 subunit from rat brain (BR) and spinal cord (SC) using anti-γ2 antibody. Whole-brain and spinal cord lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates, with anti-ρ1 antibody. (B) The γ2 subunit (∼48 kDa) was detected in Western blot analysis of the immunoprecipitates from rat brain and spinal cord, with the same anti-γ2 antibody that was used to pull down the precipitate. (C) Immunoprecipitation of the ρ1 subunit from rat brain and retina (RT), with anti-γ2 antibody. Whole-brain and retinal lysates from adult albino rats were incubated overnight at 4°C with a 1:15,000 dilution of anti-γ2 antibody in a protein A–affinity column. The ρ1 subunit (∼50 kDa) was detected in Western blot analysis of both precipitates using anti-ρ1 antibody. In these experiments, an anti-Erk antibody (polyclonal IgG from rabbit) was used for the negative control. This antibody did not pull down any proteins that could be detected by the anti-ρ1 or the anti-γ2 antibody.
Figure 3.
 
Determining interaction of GABA ρ1 subunit fusion protein with GABA γ2 subunit from the brain and spinal cord or from exogenous expression in oocytes. (A) Brain and spinal cord lysates from adult albino rats were pooled and incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was applied to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but native ρ1 protein from the brain and spinal cord was not detected. (B) Homogenates of Xenopus oocytes expressing the GABA ρ1 subunit were incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was used to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but the ρ1 subunit protein expressed in Xenopus oocytes was not detected. Immunoprecipitated lysates from brain and spinal cord tissues (A) and from cRNA injected oocytes (B) with anti-Erk antibodies served as the control in Western blot analysis.
Figure 3.
 
Determining interaction of GABA ρ1 subunit fusion protein with GABA γ2 subunit from the brain and spinal cord or from exogenous expression in oocytes. (A) Brain and spinal cord lysates from adult albino rats were pooled and incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was applied to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but native ρ1 protein from the brain and spinal cord was not detected. (B) Homogenates of Xenopus oocytes expressing the GABA ρ1 subunit were incubated with GST-ρ1 fusion protein that was bound to a GST affinity matrix. After it was raised with PBS, anti-ρ1 antibody was used to detect the 63-kDa GST-ρ1 fusion protein in Western blot, but the ρ1 subunit protein expressed in Xenopus oocytes was not detected. Immunoprecipitated lysates from brain and spinal cord tissues (A) and from cRNA injected oocytes (B) with anti-Erk antibodies served as the control in Western blot analysis.
Figure 4.
 
Determination of GABA γ2 and ρ1 subunit functional interaction in Xenopus oocytes. (A) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the ρ1 subunit (ρN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal ρ1 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n = 44). Top: Representative traces from whole-cell current recordings in Xenopus oocytes. (B) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the γ2 subunit (γN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal γ2 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n equals; 40).
Figure 4.
 
Determination of GABA γ2 and ρ1 subunit functional interaction in Xenopus oocytes. (A) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the ρ1 subunit (ρN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal ρ1 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n = 44). Top: Representative traces from whole-cell current recordings in Xenopus oocytes. (B) Competitive inhibition of GABA-induced ρ1 subunit activation by C-terminal deletion mutants of the γ2 subunit (γN′). There was a proportionate decrease in whole-cell current when the amount of N-terminal γ2 cRNA coexpressed with 25 ng full-length ρ1 cRNA in Xenopus oocytes increased. Data were analyzed by one-way ANOVA (0.05 significance level, n equals; 40).
Figure 5.
 
Effects of bicuculline and I4AA on GABA-induced currents in Xenopus oocytes expressing γ2 and ρ1 subunits. (A) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAA receptor/channel antagonist bicuculline. Bicuculline showed no effect on the GABA-induced whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits. (B) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAC receptor/channel agonist/antagonist I4AA. I4AA (100 μM) showed no agonist or antagonist effect on whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits.
Figure 5.
 
Effects of bicuculline and I4AA on GABA-induced currents in Xenopus oocytes expressing γ2 and ρ1 subunits. (A) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAA receptor/channel antagonist bicuculline. Bicuculline showed no effect on the GABA-induced whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits. (B) The ρ1 and γ2 subunits were coexpressed in Xenopus oocytes, and GABA-induced whole-cell currents were recorded by voltage clamp in the presence and absence of the GABAC receptor/channel agonist/antagonist I4AA. I4AA (100 μM) showed no agonist or antagonist effect on whole-cell currents in oocytes that heteromerically expressed the γ2 and ρ1 subunits.
Table 1.
 
Growth of Y190 Cells on Synthetic Dropout Media
Table 1.
 
Growth of Y190 Cells on Synthetic Dropout Media
Construct −Leu/−Trp/−His+3AT Selection
A.
pAS2-1 ρ1-sense + pACT2 γ N′ +
pAS2-1 ρ1-sense+ pACT2 ρ N′ +
pAS2-1 ρ1-antisense+ pACT2 γN′
pAS2-1 ρ1-antisense+ pACT2 ρ N′
pAS2-1 ρ1-sense
pAS2-1 ρ1-antisense
pACT2 γ N′
pACT2 ρ N′
Construct Colonies (n)
B.
ρ1-BD/ρ1N′-AD 69 ± 21
ρ1-BD/γ2N′-AD 66 ± 17
Construct SD −Leu SD −Trp
C.
pAS2-1 ρ1sense +
pACT2 γ N′ +
pACT2 ρ N′ +
×
×

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