RNA Isolation, Gene Isolation, and Phylogenetic Analysis

Two months old guayule plants were ground in liquid nitrogen using mortar and pestle, and 100 mg of powder was mixed with 1 mL of Trizol (Invitrogen), and total RNA was extracted according to the manufacturer’s protocol. First strand synthesis of cDNA was performed using 1 μg of the extracted RNA (leaf and stem) as template, combined with the M-MuLV reverse transcriptase (NEB) and anchored oligo dT₂₂ (IDT). The synthesis of cDNA was performed according to NEB’s provided protocol. PaCPT1 was amplified from guayule cDNA by primers 1/2 and cloned into the pGEMT-easy vector (Promega) using TA/blunt ligase mix (NEB). PaCPT2/3 and PaCBP were synthesized after yeast codon optimization (SynPaCBP, MF688939; SynPaCPT2, MF688937; SynPaCPT3, MF688939). Phylogenetic and molecular evolutionary analyses were conducted using MEGA ver. 6 (Tamura et al., 2013). All sequences were retrieved from TAIR or NCBI database. The CPT sequences are from AtCPT1 (At2g23410), AtCPT2 (At2g23400), AtCPT3 (At2g17570), AtCPT4 (At5g60510), AtCPT5 (At5g60500), AtCPT6 (At5g58780), AtCPT7 (At5g58770), AtCPT8 (At5g58782), AtCPT9 (At5g58784), CeCPT (NP_001023351), EcCPT (WP_077899378), HbCPT1 (BAB92023), HbCPT2 (BAB83522), HsCPT (NP_995583), LsCPT1 (XP_023749459), LsCPT2 (XP_023743551), LsCPT3 (XP_023762616), PaCPT1 (ATD87115), PaCPT2 (ATD87118), PaCPT3 (ATD87116), RER2 (NP_009556), SaCPT (CEH25875), SlCPT1 (NP_001234633), SlCPT2 (AFW98426), SlCPT3 (AFW98427), SlCPT4 (AFW98428), SlCPT5 (AFW98429), SlCPT6 (AFW98430), SlCPT7 (AFW98431), SRT1 (NP_013819), TbCPT1 (AGE89403), TbCPT2 (AGE89404), TbCPT3 (AGE89405). The CBP sequences are from AtLew1 (At1g11755), CeCBP (NP_495928), HaCBP (OTG12218), HbCBP (XP_021659156), HtCBP (GHBN01046996), LsCBP1 (AIQ81186), LsCBP2 (AIQ81187), NogoBR (NP_612468), NUS1 (NP_010088), PaCBP (ATD87120), PtCBP (XP_002305421), SlCBP (NP_001333078), ToCBP1 (DY820019), ToCBP2 (DY832694), VvCBP (XP_003631635). The guayule sequences were deposited at NCBI: PaCBP (MF688938), PaCPT1 (MF688933), PaCPT2 (MF688936), PaCPT3 (MF688934).

Yeast Complementation Assay and in vitro Biochemical Assay

Details of yeast complementation, in vitro enzyme assay, and reverse-phase thin layer chromatography were previously described (Kwon et al., 2016). All primer sequences are given in Supplementary Table S1. PaCPT1-3 were amplified using primers 3/4, 5/6 or 7/8, respectively, and the amplicons were cloned into p414-GPD using SpeI/SalI, SpeI/XhoI or BamHI/ClaI sites, respectively. PaCBP was cloned into p415-GPD using SpeI/XhoI sites. The cloned vectors were introduced to a yeast strain deficient in both copies of CPTs, rer2Δ::HIS3 srt1Δ::KanMX [pRS316-RER2], described in Kwon et al. (2016). This strain is referred to as rer2Δ srt1Δ hereafter, and Ro lab yeast stock number is ROY13. The constructed PaCBP/CPT pairs were transformed in this yeast strain. To serve as a positive control of complementation, p414-GPD ScRER2-TRP1 (previously cloned in Kwon et al., 2016) with empty p415-GPD was also transformed in the rer2Δ srt1Δ strain. The transformants were streaked on both SC-Leu-Trp-Ura-His plates (positive control) and SC-Leu-Trp-His plates containing 5FOA (5-fluoroorotic acid) and uracil to allow for counter-selection of pRS316 ScRER2-URA3. Growth on 5FOA/Ura-containing media indicates functional complementation of PaCBP/CPTs in place of yeast CPTs. Growth on both media was observed after a 7-day incubation period at 30°C.

The yeast strains, rescued from the 5FOA selection in the complementation assay, were cultured overnight and inoculated into 100 mL of SC-Leu-Trp-His media. The cells were harvested at late logarithmic phase by centrifugation at 3,000 × g for 3 min. The pelleted cells were then resuspended in 1 mL of TES-B buffer (0.6 M sorbitol, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.4) and homogenized by bead-beating. The yeast lysate (1 mL) was centrifuged at 10,000 × g for 10 min to remove large cellular debris. The supernatant was diluted with 10 mL of TES-B and centrifuged again similarly. The final supernatant was centrifuged at 100,000 × g for 1 h, 4°C. The pellet was re-suspended in TG buffer [50 mM Tris-HCl and 20% (v/v) glycerol, pH 7.4] to a concentration appropriate for in vitro biochemical assays.

The in vitro biochemical assays of CPB/CPT were performed in 50 μL reaction mixtures containing 50 μg of prepared microsomal proteins, 20 μM FPP, 80 μM of IPP (¹⁴C-IPP PerkinElmer, 50–60 mCi mmol^-1), 50 mM HEPES, 5 mM MgCl₂, 2 mM NaF, 2 mM Na₃VO₄, and 2 mM DTT, pH 7.5. The reaction mixture was incubated at 30°C for 2 h. For each reaction, the extraction of polyisoprenoid products was performed in 1 mL of chloroform/methanol (2:1, v/v) and 0.4 mL of 0.9% NaCl, by vortexing for 20 s followed by centrifuged at 10,000 × g for 1 min at room temperature. The chloroform phase was transferred to a new tube and washed twice with 0.5 mL of methanol/water/chloroform mixture (48:47:3, v/v/v) by vortexing and centrifuging as above to remove unincorporated IPP. The lower chloroform phase was transferred to a glass vial and allowed to evaporate. Dephosphorylation of the product to polyprenol was carried out by incubating the dried product in 300 μL of 1 M HCl at 85°C for 1 h, followed by benzene extraction. The concentrated products in benzene were used for thin layer chromatography (TLC). The concentrated products were spotted on a C18 reverse-phase silica plate (Analtech cat. P90011, 20 × 20 cm) and separated by acetone/water (39:1, v/v) mobile phase. The TLC plate was then exposed to a BAS Storage Phosphor Screen (GE Healthcare) and the screen was scanned by a phosphorimager (Bio-Rad, Molecular Imager FX). The R_f value of known RER2 products (Brasher et al., 2015) was confirmed in our TLC system, and the R_f values were used to determine the carbon numbers.

Split Ubiquitin Membrane Yeast Two-Hybrid (MYTH) and β-Galactosidase Assay

MYTH assays were performed according to the published methods (Gisler et al., 2008; Snider et al., 2010). The bait plasmid (PaCBP-Cub-LexA-VP16) was generated by ligating the PaCBP amplified with primers 9/10 into pAMBVα using StuI/NcoI sites. For the prey plasmids, PaCPT1-3 were amplified with primers 11/12, 13/14 or 15/16, and cloned into pPR3C-NubG in SpeI/BamHI (PaCPT1) or BamHI/ClaI (PaCPT2 and 3) to yield PaCPT-HA-NubG constructs. PaCPT1-3 were also amplified with primers 17/18, 19/20 or 21/22 and cloned into pPR3N-NubG in BamHI/SalI (PaCPT1) or BamHI/ClaI (PaCPT2 and 3) to generate NubG-HA-PaCPT constructs. The bait and prey plasmid pair was co-transformed into the Y2H reporter yeast strain, THY.AP4. The control prey vectors pOST1-NubG and pOST1-NubI were also co-transformed with the bait vector. Individual transformed-yeast colonies were cultured in 30°C until OD₆₀₀ reached to 3.0–3.5. The cell densities were then normalized to 0.05 (OD₆₀₀ value) by dilution. The normalized cells were diluted to produce dilution of 1/10 and 1/100. Ten μL of cells was spotted on SC-Leu-Trp (SC-LT) and on SC-Leu-Trp-Ade-His (SC-LTAH) containing 3-amino-1,2,4-triazole (3-AT) at concentrations of 5, 10, or 25 mM. Spotted plates were incubated for 3 days at 30°C to observe cell growth.

The strength of protein interactions was quantified using β-galactosidase assay with O-nitrophenyl-β-D-galactoside (ONPG) as the substrate. From SC-LTAH selection plates, the positive colonies were cultured overnight in SC-LTAH medium without 3-AT. Cells from the overnight culture were inoculated to 4 mL of SC-LTAH medium and cultured for 5 h. The cells were harvested at 9,000 × g for 3 min at 4°C and resuspended in 1 mL of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO_4, and 50 mM β-mercaptoethanol, pH 7.0), 20 μL of chloroform and 20 μL of 0.1% SDS. After vortexing, the mixture was pre-incubated for 5 min at 28°C. The reaction was initiated by adding 0.2 mL of ONPG (4 mg mL^-1 in Z buffer) and terminated by the addition of 1 M of Na₂CO₃. The mixture was centrifuged at 20,000 × g for 10 min, and the absorbance at 420 nm and 550 nm were measured to determine the Miller units (Miller, 1972).

Cloning and in vitro Translation of PaCPT1-3 and PaCBP

DNA constructs for in vitro translation were cloned into pT7CFE1-3xFLAG (constructed using pT7CFE1-NHa) for PaCPT1-3 and pT7CFE1-NHa (Thermo Fisher Scientific) for PaCBP. pT7CFE1-3xFLAG was generated by replacing HA tag in pT7CFE1-NHa with 3xFLAG. The 3xFLAG (atggactacaaagaccatgacggtgattataaagatcatgatatcgattacaaggatgacgatgacaag) was amplified using primer sets 23/24, 23/25, and 23/26 sequentially using the IRES region in pT7CFE1-NHa as a template. The final PCR product containing partial IRES sequence and 3xFLAG was digested by KpnI and BamHI and ligated to corresponding restriction sites in pT7CFE1-NHa. Synthetic PaCPT1 and PaCPT2 (GenScript, Piscataway, NJ, United States) were ligated to BamHI and XhoI sites in pT7CFE1-3xFLAG. PaCPT3 was amplified from P. argentatum leaf cDNA using a primer set, 27/28. The PaCPT3 PCR product was cloned into pT7CFE1-3xFLAG via BamHI and XhoI restriction sites. Synthetic PaCBP (GenScript, Piscataway, NJ, United States) was amplified using a primer set 29/30 and digested with BamHI and XhoI, followed by ligation to corresponding sites in pT7CFE1-NHa. A control construct, pT7CFE1-NHa-GFP for co-immunoprecipitation described below, was generated by cloning GFP sequence into pT7CFE1-NHa. GFP was amplified from pCFE-GFP (Thermo Fisher Scientific) using a primer set 31/32, containing BamHI and XhoI, respectively, followed by ligation to corresponding sites in pT7CFE1-NHa. The purified plasmid preparations of all IVT constructs were cleaned up further by ethanol precipitation, followed by resuspension in nuclease-free water to a concentration of 500 ng μL^-1 prior to IVT. IVT was carried out using 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific), following the manufacturer’s procedure except for reducing the reaction to half of the recommended amounts. Immediately following incubation of the IVT reaction mixture at 30°C for 4 h, 1 μL of the 12.5 μL IVT mixture was saved as input for Western blotting by adding SDS-PAGE loading buffer and storage in -20°C until use. The remaining IVT mixture was used immediately for immunoprecipitation as described below.

Co-immunoprecipitation of PaCPT1-3 and PaCBP

For pre-binding of protein G magnetic beads (NEB, ON, Canada) to monoclonal mouse anti-FLAG M2 antibody (Sigma, ON, Canada) or monoclonal rat anti-HA 3F10 antibody (Roche, Laval, QC, Canada), for each co-immunoprecipitation reaction, 10 μL of the bead suspension was washed (invert five times/vortex 5 s) in 100 μL of room temperature PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) four times and incubated with 0.5 μL of the antibody in 100 μL of cold PBST (PBS, pH 7.4, 0.1% Tween 20) at 4°C for 16 h on a Labquake^TM Tube Rotator. The separation of magnetic beads from solution was achieved by applying magnetic field for 30 s on a magnetic stand. To remove unbound antibody, the antibody-immobilized beads were washed (invert five times/vortex 5 s) three times, each in 100 μL of cold IP buffer (150 mM NaCl, 100 mM Tris-HCl pH 7.4, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 0.5% NP-40, 1 mM PMSF). The immobilized beads were resuspended in 500 μL of cold IP buffer, followed by addition of the 11.5 μL of the completed IVT reaction mixture. Co-immunoprecipitation reaction was incubated at 4°C for 1 h on the rotator. To remove unbound proteins, the mixture was washed (invert five times/vortex 5 s) five times, each in 700 μL of cold IP buffer. Immunoprecipitated proteins were eluted from the beads by resuspending in 20 μL of 2X SDS-PAGE loading buffer [100 mM Tris-HCl pH 6.8, 4% (w/v) SDS, 20% glycerol, 0.01% (w/v) bromophenol blue, 200 mM β-mercaptoethanol] and incubating at 95°C for 5 min with intermittent gentle flicks. This elution also liberates anti-FLAG and anti-HA antibodies from the magnetic beads as the heavy (50 kDa) and light (25 kDa) chains of the IgG antibodies. After bringing the eluted mixture to room temperature, the mixture was spun down by a brief centrifugation, followed by transfer of the eluate while on a magnetic stand. Half of the eluate was used to run 12% SDS-PAGE followed by Western blotting with mouse anti-FLAG M2 antibody and the other half with rat anti-HA 3F10 antibody. The IVT reaction mixture (1 μL of 12.5 μL total IVT reaction) saved as input was also divided in half to run in SDS-PAGE and Western blotting with anti-FLAG and anti-HA antibodies. SDS-PAGE gel was transferred to PVDF membrane using a Tris-glycine-methanol buffer system, and the antibody hybridization was performed in 5% skim milk TBST (5% skim milk, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) at 1:2000 for both anti-FLAG and anti-HA antibodies by overnight incubation at 4°C. HRP-conjugated anti-mouse and anti-rat secondary antibodies were also hybridized in 5% skim milk TBST at 1:10,000 at room temperature for 1 h.

Phylogenetic Analysis of PaCPT1-3 and PaCBP

To understand phylogenetic relatedness of PaCPT1-3 to other known eukaryotic and prokaryotic CPTs, a phylogenetic tree was reconstructed using the deduced amino acid sequences from PaCPT1-3 cDNAs. It has been established that two clades of CPTs (clade I and clade II) are present in plants (Figure 1A). Clade I is comprised of chloroplastic CPTs that likely originated from prokaryotic homo-dimeric CPTs, whereas clade II consists of cytosolic/ER CPTs that interact with CBPs to form hetero-protein complexes, which are then recruited to ER. The CPT clade II is further divided into two sub-clades – one type of CPT for dolichol biosynthesis in the primary metabolism and the other type for NR biosynthesis in the specialized secondary metabolism.

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FIGURE 1

Phylogenetic analysis of CPT (A) and CBP proteins (B). Phylogenetic trees were created based on the protein sequence similarities between CPTs and CBPs from various prokaryotic and eukaryotic species. Bootstrap values from 1,000 replicates were calculated, and the percentages of replicates are shown in each node only when higher than 50%. Two prokaryotic CPTs (EcCPT and SaCPT) are used as the outgroup and indicated by a gray square. PaCPT1-3 and PaCBP are marked by red boxes. Abbreviations used are: At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Ec, Escherichia coli; Ha, Helianthus annuus; Hb, Hevea brasiliensis; Hs, Homo sapiens; Ht, Helianthus tuberosus; Ls, Lactuca sativa; Pa, Parthenium argentatum; Pt, Populus tremuloides; Sa, Staphylococcus aureus; Sl, Solanum lycopersicum; Tb, Taraxacum brevicorniculatum; To, Taraxacum officinale; Vv, Vitis vinifera; RER2 and SRT1 are yeast CPTs. NUS1 is yeast homolog of CBP. Accession numbers of the sequences used to construct the phylogeny are given in “Materials and Methods.” Arabidopsis CPTs (AtCPT1-9) and tomato CPTs (SlCPT1-7) were numbered according to the published articles (Surmacz and Swiezewska, 2011; Akhtar et al., 2013). Note that Kera et al. (2012) assigned different numbering of the nine Arabidopsis CPTs, and different nomenclature of AtCPTs can be listed in other publications.

In the phylogenetic analysis, all three PaCPTs belong to the cytosolic/ER clade II CPT. At the sub-clade level, with strong statistical supports, PaCPT1 is clustered with LsCPT1 (lettuce), SlCPT3 (tomato), and AtCPT1 (Arabidopsis), all of which are known to synthesize dehydrodolichyl diphosphate (Brasher et al., 2015; Qu et al., 2015; Kwon et al., 2016). On the other hand, PaCPT2 and PaCPT3 form a close cluster with LsCPT3 and TbCPT1-3 (dandelion), known to have implications in NR biosynthesis (Post et al., 2012; Qu et al., 2015). This phylogenetic tree suggested that guayule possesses two sub-clades of diverged CPTs within the CPT clade II. It can be inferred from these results that PaCPT1 is likely to be involved in the dolichol metabolism, while PaCPT2 and PaCPT3 are involved in NR metabolism.

The phylogenetic tree for CBPs was also constructed. CBP is exclusively found in eukaryotes, and thus the phylogenetic structure of CBP shows a monophyletic group (Figure 1B). The duplications of CBP (i.e., two CBP isoforms) are restricted to lettuce and dandelion in the Cichorieae tribe (within Cichorioideae sub-family under Asteraceae family), and other plants with high quality genome sequences encode a single copy of CBP. Interestingly, PaCBP and other single copy CBPs from the Asteroideae sub-family (sunflower HaCBP and Jerusalem artichoke HtCBP) are tightly clustered with the laticifer-specific CBPs (lettuce LsCBP2 and common dandelion ToCBP2), even though there is no developed laticifer in sunflower and artichoke. Thus, the CBP gene duplication event likely occurred prior to the divergence of the two sub-families and the advent of laticifer in Cichorioideae subfamily.

Complementation in rer2Δ srt1Δ Yeast

In eukaryotes, dolichol is an indispensable carrier molecule of sugar moieties for post-translational protein modifications. Therefore, all eukaryotes have at least one set of CPT and CBP to synthesize dehydrodolichyl diphosphate (i.e., dolichol precursor), and the absence of either CPT or CBP is lethal. Yeast (Saccharomyces cerevisiae) has one homolog of CBP (NUS1) and two homologs of CPTs (RER2 and SRT1). We previously developed a yeast strain that has double deletions in RER2 and SRT1, in which its lethality is rescued by expressing a plasmid copy of RER2 (Kwon et al., 2016). To examine the ability of PaCPT and/or PaCBP to rescue the dolichol-deficiency of rer2Δ srt1Δ, these guayule genes were first transformed to this strain individually or in PaCPT/PaCBP pairs, and then the URA-selective plasmid containing RER2 was removed by 5-fluoroorotic acid (5FOA) selection (Figure 2). This enabled us to assess the PaCPT activity in the absence of both of the yeast CPTs.

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FIGURE 2

Complementation of rer2Δ srt1Δ yeast by PaCPT1-3 and PaCBP. The yeast strain, rer2Δ srt1Δ is lethal but is maintained by expressing RER2 in URA-selectable plasmid. This strain was used to transform plasmids expressing each PaCPT1-3 and PaCBP. The successful transformants were streaked on 5FOA selection plates to remove RER2 containing URA-plasmid. Yeast growth in 5FOA selection was observed only by PaCPT/PaCBP pairs or by retransformed RER2 in TRP-plasmid. No growth was observed when PaCPT alone or PaCBP alone was expressed.

In the absence of CPT enzymatic activity, the rer2Δ srt1Δ lethal phenotype will not be rescued. In all cases tested, the rer2Δ srt1Δ strain transformed with empty plasmids or a single plasmid that contains either PaCBP or one of the PaCPTs failed to grow upon removal of the RER2-URA plasmid by 5FOA selection. In contrast, co-expression of PaCBP and one of the three PaCPTs could effectively complement the lethality of the yeast CPT-knockouts upon removal of the RER2-URA plasmid. Our results indicate that each PaCPT can produce isoprenoid molecules analogous to dehydrodolichyl diphosphate only in the presence of PaCBP but not together with endogenous yeast CBP, Nus1.

In vitro Enzyme Assays

The surviving yeast strains after 5FOA selection should be able to bring about necessary CPT activities for the yeast survival in the rer2Δsrt1Δ yeast. To assess the guayule CPT activities, microsomes were prepared from the rescued yeast strains from the complementation assay, and radiolabeled ¹⁴C-isopentenyl diphosphate (¹⁴C-IPP) and the priming molecule, FPP, were co-incubated with the isolated microsomes. When the incorporations of ¹⁴C-IPP to hydrophobic polymers were measured, significant IPP-incorporation activities ranging from 14.4 to 50.8% incorporation were measured in 2-h assays (Table 1). Their specific activities were comparable to (in the cases of PaCPT1/PaCBP1or PaCPT3/PaCBP activity) or exceeded (in the case of PaCPT2/PaCBP activity) the CPT activities previously reported from lettuce CPT and CBP (Qu et al., 2015). The CPT activity of the microsomes from the rescued yeast strain transformed with RER2 as a complementation control was substantially lower (4.5- to 15.6-fold) than those from guayule PaCPT/PaCBP co-expression. The lower CPT activity of RER2 control strain is attributable to the imbalance between overexpressed RER2 and its binding partner NUS1, expressed at a native level in yeast.

Table 1

Specific activities of PaCPTs and PaCBP in isolated microsomes.

cDNA expressed in rer2Δ srt1Δ	Activities (pmol IPP μg-1 h-1)	IPP-incorporations (%)
RER2	2.9 ± 0.4	2.9 ± 0.1
PaCPT1/PaCBP	13.0 ± 1.7	14.4 ± 0.5
PaCPT2/PaCBP	45.4 ± 1.3	50.8 ± 5.0
PaCPT3/PaCBP	22.0 ± 0.4	24.6 ± 2.6

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Data are the average ± SD from three independent assays.

The resulting ¹⁴C-labeled products were separated by the reverse-phase thin layer chromatography (RP-TLC) optimized for the separation of dolichol-sized polymers. In the RP-TLC analysis, we detected no obvious differences among the radiolabeled isoprene polymers produced from any of the PaCPT and PaCBP pairs (Figure 3A), and their polymer size was slightly smaller than, but still comparable to, the dehydrodolichols produced by RER2 (Figure 3B). We could not find evidence that the cis-isoprene polymer, significantly longer than dolichol, is synthesized by PaCPT and PaCBP in yeast microsomes.

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FIGURE 3

Isoprene product separations by reverse phase thin layer chromatography. Microsomes were prepared from the yeast strains selected on 5FOA in Figure 2, and in vitro enzyme assays were performed using the microsomes and ¹⁴C-isopentenyl diphosphate, followed by TLC (C18 reverse-phase silica) of extracted cis-polyisoprene products using a solvent mixed with acetone and water (39:1), and phosphorimager analysis. (A) The cis-polyisoprene product profiles from yeast RER2 as a size calibration standard. (B) The cis-polyisoprene products from PaCPT/PaCBPs are identical to one another and slightly shorter than those from the RER2-containing microsomes. S indicates the starting point of separation and SF indicates solvent front. R_f values and carbon numbers are shown.

Protein Interactions by Split-Ubiquitin Yeast 2-Hybrid Assay

The complementation of rer2Δ srt1Δ yeast strain by co-expression of PaCPT and PaCBP, but not by a single PaCPT or PaCBP expression, implied that both proteins are required for dolichol metabolism. Thus, possible interactions between each PaCPT and PaCBP were examined by split-ubiquitin yeast 2-hybrid assay developed for protein interactions of membrane-bound proteins (Stagljar et al., 1998). In this assay, the restoration of ubiquitin by N-terminal ubiquitin (Nub) and C-terminal ubiquitin (Cub) allows a release of transcriptional regulator to nucleus to activate the auxotrophic marker genes and the β-galactosidase reporter gene. A single residue mutation (I to G) of natural Nub protein (i.e., NubI to NubG) prevents the auto-assembly of the ubiquitin, unless NubG and Cub binding is mediated by the interaction of the proteins of interest each fused to NubG and Cub. Therefore, interactions between PaCPT and PaCBP proteins translationally fused to Cub and NubG can be assessed by yeast growth on selective medium and β-galactosidase activity.

As a control, co-expression of PaCBP-Cub fusion and OST1 (α-subunit of the oligosaccharyltransferase complex localized on the ER)-NubI fusion allows restoration of ubiquitin, thereby presenting efficient yeast growth and strong β-galactosidase activity (Figure 4A). In contrast, changing NubI to NubG in the same experiment did not permit yeast growth, suggesting PaCBP and OST1 do not interact with each other and cannot bring the two ubiquitin subunits together as expected as a negative control (Figure 4A). Similarly, PaCPTs 1–3 were individually fused to NubG, and each fusion construct was co-expressed with PaCBP-Cub to assess whether PaCPT-PaCBP interaction occurs to restore ubiquitin. The interaction between each of the PaCPTs and PaCBP could be clearly inferred by the efficient yeast growth and the by β-galactosidase activities. Interestingly, the interactions were only observed in one orientation of the PaCPT fusion protein (Figure 4B). The NubG-PaCPT orientation allowed activations of the growth markers and reporter gene, whereas the PaCPT-NubG orientation did not show evidence of protein–protein interactions. Activations of marker genes only when NubG is located in the N-terminus of prey protein have been previously reported (Yao et al., 2017) and have occasionally happened in split-ubiquitin yeast 2-hybrid assays (personal communication with Igor Stagljar, University of Toronto). Wherever interactions were observed, the HIS3 marker gene was activated very efficiently as yeast could grow at up to 25 mM of AT (3-amino-1,2,4-triazole), a competitive inhibitor of HIS3. In addition, β-galactosidase activities were comparable to that from the positive control. Therefore, the split-ubiquitin Y2H provided molecular evidence that each PaCPT and PaCBP interact with each other to form a hetero-protein complex in yeast.

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FIGURE 4

Split ubiquitin yeast 2-hybrid assays to assess the protein interactions between PaCPT1-3 and PaCBP. The bait protein, Cub, was fused at the C-terminus of PaCBP in all assays. Under the selective growth condition, SC-LTAH, yeast growth was observed only when the bait and prey proteins interacted either through NubI-Cub auto-assembly (A: Ost1-NubI, positive control) or through the PaCPT/PaCBP interaction-mediated NubG-Cub assembly (B: NubG-PaCPT3, NubG-PaCPT2, NubG-PaCPT3). PaCPT/PaCBP interactions occurred only when NubG was fused on the N-terminus of PaCPTs. Under the non-selective growth condition, SC-LT, all yeast strains grew regardless of the protein interaction. As an important negative control, NubG-fused Ost1 protein did not interact with Cub-fused PaCBP (A), demonstrating a specific interaction of PaCBP to PaCPT, but not to Ost1. Yeast growth was observed in a wide range of 5–25 mM AT, indicating a strong activation of the HIS3 reporter gene by the ubiquitin-activated transcription factor. The β-galactosidase activity was measured in the corresponding yeast strains that showed protein interactions in the absence of AT. PaCPT1/PaCBP interaction showed the same level of β-galactosidase activity as the Ost1-NubI control while PaCPT2/PaCBP and PaCT3/PaCBP showed lower activities. Ten μL of OD₆₀₀ yeast culture was placed in the first spot, and yeast cells with 10- and 100-times dilution were placed in the subsequent spots. Asterisks indicate statistically significant differences (p-value < 0.05).

Protein Interactions by Co-immunoprecipitation

In addition to the split-ubiquitin Y2H approach, PaCPT1-3 and PaCBP interaction were independently examined by co-immunoprecipitation assays (Co-IP). PaCPT1-3 and PaCBP were tagged by FLAG- and HA-epitope, respectively, and their proteins were prepared by in vitro transcription and translation. Green fluorescent protein (GFP), which has a similar size to PaCBP and the PaCPTs, was used as a negative control in Co-IP experiments. All in vitro co-transcribed and co-translated PaCPT/PaCBP proteins were detectable in input by immunoblotting with either FLAG or HA antibody (Figure 5A,B input; Supplementary Figure 1). Incubation of FLAG-antibody immobilized magnetic beads resulted in the co-IP of FLAG-PaCPTs with HA-PaCBP as detected by FLAG-antibody and HA-antibody immunoblotting, respectively, while HA-GFP control protein could not be pulled down by FLAG-PaCPT2 (Figure 5A and Supplementary Figure 1). Similarly, immunoprecipitation with HA-antibody immobilized magnetic beads resulted in the co-IP of HA-PaCBP with FLAG-PaCPTs, while FLAG-PaCPT2 control protein could not be pulled down by HA-GFP (Figure 5B). These results showed that each of the PaCPT1-3 and PaCBP interact with each other to organize hetero-protein complexes when co-expressed in vitro.

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FIGURE 5

Co-immunoprecipitation of recombinant PaCPT/PaCBP proteins. (A) HA-PaCBP could be co-immunoprecipitated by FLAG-PaCPT1-3, and (B) FLAG-PaCPT1-3 could be co-immunoprecipitated by HA-PaCBP. PaCPT1-3 and PaCBP proteins tagged with FLAG- and HA-epitopes, respectively, and were prepared by in vitro transcription and translation. GFP tagged with an HA-epitope was used as a negative control. The information of the protein mixture is detailed on the top panel with the appropriate epitope-tag. The antibodies used in the immunoblot are indicated beside blots (FLAG, FLAG antibody or HA, HA antibody).

We thank Professor Igor Stagljar (University of Toronto, Canada) for providing the split ubiquitin membrane yeast 2-hybrid system.