The Crg1 N-Terminus Is Essential for Methyltransferase Activity and

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Crg1 N-terminus is essential for methyltransferase activity and Cantharidin resistance in Saccharomyces cerevisiae Pushpendra Kumar Sahu, Sakshi Chauhan, and Raghuvir Singh Tomar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01277 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Biochemistry

Crg1 N-terminus is essential for methyltransferase activity and Cantharidin resistance in Saccharomyces cerevisiae Pushpendra Kumar Sahu1, Sakshi Chauhan2,1, Raghuvir Singh Tomar1*

1Laboratory

of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science

Education and Research Bhopal, 462066, Madhya Pradesh, India. 2Division

of Developmental Biology, National Institute of Child Health and Human Development, NIH,

Bethesda, Maryland, USA (Present Address). *To

whom correspondence may be addressed:

Raghuvir Singh Tomar, Professor, Laboratory of Chromatin Biology, AB-3, Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal - 462066, Madhya Pradesh, India. Email: [email protected], Phone: +91-755- 6691411.

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ABSTRACT Crg1 is an S-adenosine methionine (SAM) dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of Crg1 N-terminus in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of Crg1 N-terminus (crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance as well. The sub-cellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas the wildtype Crg1-daGFP distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants; D44A, D67A, and E105A-D108A also showed the same cytoplasmic aggregates like Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alter the native folding of Crg1 methyltransferase resulting in loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, ER stress, GPI-anchor missorting and cell wall damage. Overall, this study identifies an essential role of the Crg1 N-terminus in methyltransferase activity and cantharidin resistance.


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Biochemistry

INTRODUCTION A large number of methyltransferases have been identified in various organisms, most of which are functionally uncharacterized.1 These methyltransferases recognize a wide range of biomolecules, i.e., DNA, RNA, lipids, and carbohydrates as substrate and regulate various biological functions.2 A few studies have shown that methyltransferases also recognize xenobiotic compounds as substrate and modulate their cytotoxicity via methylation.3, 4 This diversity of substrate specificity has raised a serious concern about drug and antibiotic resistance.5, 6 These methyltransferases modify not only the structure of the small molecules but also alter their targets in host cells.7 Today has to characterize these methyltransferases playing an important role in drug and antibiotic resistance. Budding yeast, Saccharomyces cerevisiae encodes a large number of methyltransferases; mostly uncharacterized by function. Since the release of the whole genome sequence and complete deletion library, enormous numbers of studies have been conducted for functional annotation of the yeast ORFs. These studies helped us to know the function of various uncharacterized genes. A similar study that performed a chemical genetic screening of yeast ORFs identified YHR209W as an essential gene for cantharidin resistance.8 Based on its specificity to cantharidin, YHR209W was named as cantharidin Resistance Gene 1 (CRG1, UniProtKB P3889).8 Comparative sequence analysis categorized Crg1 as a class-1 methyltransferase.9 CRG1 deletion doesn’t show any notable phenotype in the standard growth conditions. Its biological function is unknown, but, its expressed product is shown to methylate a foreign molecule cantharidin in vitro.4 Methylation causes alteration in cantharidin structure, and the molecule becomes toxically inactive.3, 4 Crg1 is believed to perform the same function in vivo as well because its deletion causes sensitive growth with cantharidin treatment.3,

4

Since Crg1 recognizes a small foreign

molecule as a substrate; it can be categorized as small molecule methyltransferase.10 Yeast genome encodes a CRG1 paralogue, TMT1 (Trans-aconitate methyltransferase), a methyltransferase that recognizes trans-aconitate, 3-isopropylmalate, and isopropylmaleate as a substrate, but not the small molecule cantharidin.4 It suggests that both the paralogues have different substrate specificities. CRG1 is also conserved in the pathogenic yeast Candida albicans, and it does the same function as Saccharomyces cerevisiae.3 Crg1 has mammalian homologs as well, METTL7A and METTL7B, the methyltransferases which recognize lipid molecules as substrate.4 Previous studies have characterized the methyltransferase domain of Crg1 in Saccharomyces cerevisiae, and Candida albicans, respectively.3, 4 In Saccharomyces cerevisiae, the Crg1-methyltransferase domain comprises two motifs: (A) Cofactor (SAM) binding motif and (B) Substrate (Cantharidin) binding motif. The critical residues identified for SAM binding are aspartates at 44th and 67th positions. Similarly, the critical residues for substrate binding are glutamate and aspartate at 104th and 108th positions, 3 ACS Paragon Plus Environment

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respectively.4 Over and above the characterization of methyltransferase domain in Crg1, it is difficult to determine the first and last residues of methyltransferase domain in Crg1. Cantharidin is a secondary metabolite and toxin produced by blister beetle. It has been a traditional medicine for the treatment of warts and molluscum-contagiosum in many Asian countries.11 People have also been using this beetle product as an aphrodisiac, tattoo remover, and anti-hepatoma drug for the past few years.12 Recently, enormous studies have been conducted investigating the medicinal property of this small molecule. Many studies describe its anticancer property against a different kind of cancer (hepatoma, leukemia, pancreatic, colorectal, gallbladder, oral, and breast cancer).13-19 The serinethreonine protein phosphatases, PP1, and PP2A are the known targets of cantharidin so far.20, 21 However, some new studies suggest the existence of additional molecular targets.4, 22, 23 A couple of studies have identified two new molecular targets of cantharidin which are independent of PP1 and PP2A.22,

23

Cantharidin alters heat shock response by targeting the promoter binding activity of Hsf1.22 It also impairs GPI-anchored protein sorting by inhibiting the Cdc1 activity in the remodeling process.23 In this study, we characterized the Crg1 N-terminus for the first time. We identified an essential role of the Crg1 N-terminal residues in methyltransferase activity where the truncation of 41 amino acids from the N-terminus resulted in cantharidin sensitivity. In vitro enzyme assay as well as in vivo localization study revealed complete loss of methyltransferase activity in Crg1ΔN. The study uncovered the actual mechanism for loss of methyltransferase activity in Crg1 methyltransferase domain mutants (D44A, D67A, and E105A-D108A) along with Crg1ΔN. MATERIALS AND METHODS Growth conditions, yeast strains, and plasmids Yeast strains of the study were isogenic to either BY4741 (S288C) or JK-3d. The yeast strains, plasmids, and primers are listed in tables, S1-S3, respectively. All the yeast strains were grown in SC-Ura at 30oC excluding some specific conditions. Various chemicals and reagents used in this study were purchased from different sources, i.e., Sigma, Merck, Himedia, Invitrogen, New England Biolabs, Bio-Rad and Applied Biosystems. Side-directed mutagenesis of CRG1 CRG1 ORF was amplified from the construct purchased from Dharmacon, Catalogue# YSC3867202327411. The amplified product and pSF-TEF1-COOH-6xHis vector were digested with BsgI and ligated subsequently. The positive clone pSF-TEF1-CRG1-6xHis was further used as a template for sitedirected mutagenesis. We amplified the plasmid with the respective primers listed in Table S3. The parent 4 ACS Paragon Plus Environment

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Biochemistry

plasmid was degraded by DpnI. The amplified products were transformed into E.coli strain DH5α, and positive colonies were screened and validated by DNA sequencing. The same strategy was followed for pSF-TEF1-COOH-daGFP as well. Whole cell protein extraction for western blotting The whole cell protein extract was prepared as described previously.24 Yeast cells were lysed by glass beads in 20% TCA. The TCA precipitate was washed with ethanol and re-suspended in Tris-Cl (0.5M, pH 7.5) with 2X SDS loading buffer. The sample was boiled at 95oC for 5 minutes. We used anti-phosphop44/42 (Cell Signaling, Catalog 4370) for Slt2 phosphorylation, anti-Mpk1 (Santa Cruz Biotechnology Inc., Catalog SC-6803) for total Slt2, anti-N-terminal GFP (Sigma, Catalog G1544) for Gas1-GFP detections. Primary antibodies for Rap1, Tbp1, and Crg1 are the polyclonal antisera, raised in the rabbit. Total RNA extraction and cDNA synthesis Total RNAs were isolated by heat/freeze RNA isolation protocol.25 Cells were lysed with 1% of SDS in AE buffer (50mM Sodium acetate, 10mM EDTA, pH 5.3). Phase separation was done first with acidic phenol pH 4.2 and then with phenol:chloroform:isoamyl alcohol (PCI; 25:24:1) mixture. The total RNAs were precipitated by sodium acetate (0.3M) and absolute ethanol. The PCR for cDNA synthesis was done using the iScript™ cDNA Synthesis Kit (BioRad, Catalog 1708891). Fluorescence microscopy GPI-anchored protein sorting was analyzed with the help of ZEISS-Apotome.2 fluorescence microscope using Gas1-GFP as a model protein as described previously.23, 26 Purification of Crg1 from yeast Crg1 (UniProt Accession ID: P38892; NCBI Accession ID: KZV10954) purification was done as described previously.4, 27 The crg1 deletion strain was transformed with pYES260-GAL1-CRG1-6xHis, pYES260-GAL1-CRG1-D44A-6xHis,

pYES260-GAL1-D67A-CRG1-6xHis,

and

pYES260-GAL1-

CRG1ΔN-6xHis. Cells were grown at 24oC in SC-URA (2% raffinose) medium (900mL) till midexponential phase, and then induced with 2% galactose (20%, 100mL) for 6h. Cells were harvested, washed with 1xPBS buffer, re-suspended in 7mL of resuspension buffer (20mM HEPES pH 7.5, 1.5M NaCl, 5% glycerol), and lysed with glass beads in the presence of protease inhibitors cocktail (PIC, Sigma). The whole cell lysate was centrifuged at 20,000 rpm for 45 min and diluted four fold with binding buffer (20mM HEPES pH 7.5, 40mM imidazole, 5% glycerol). The 900μL of Ni Sepharose 6 Fast Flow beads (50% slurry in 20% ethanol) were washed once with binding buffer and added to the sample and rotated for 90min, followed by three washes with 40mL of wash buffer (20mM HEPES pH 7.5, 40mM imidazole, 5% glycerol, 0.5M NaCl). For elution of Crg1 protein, the Ni beads were re5 ACS Paragon Plus Environment

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suspended in 1 mL of elution buffer (20mM HEPES pH 7.5, 250mM imidazole pH 7.7, 5% glycerol, 0.25M NaCl) and mixture was rotated for 15 min at 4oC. The eluted Crg1 protein was further concentrated to 250μL using the Amicon 4Kda MWCO filter (Millipore). Mass spectrometric analysis for methyltransferase activity The in vitro enzymatic reaction was performed as described previously.4 0.35μg of pure Crg1 protein was incubated with 200μM of cantharidin and 1.2 mM of SAM at 30oC for two h. The reaction mixture was run through the micrOTOF-Q II 10330 (HR-LCMS) immediately after the end of incubation. The instrument parameters used for samples were; Source type: ESI, Ion Polarity: Positive, Set nebulizer: 0.4 Bar, Focus: Active, Set capillary: 4500 V, Set dry heater: 200 °C, Scan begins: 50 m/z, Scan end: 3000 m/z, Set end plate offset: -500 V, Set dry gas: 4.0 l/min, Set collision cell RF: 130.0 Vpp.3, 4 Prediction of tertiary protein structures using I-TASSER server The primary sequence of Crg1 was taken from the Saccharomyces Genome Database (SGD) and uploaded in the I-TASSER28 or SWISS MODEL29 server in FASTA format. Crg1 has maximum homology and identity with Tmt1; the crystal structure of which is known. So, Tmt1 was taken as a template to model the tertiary structures of WT and mutant Crg1. From the SWISS-MODEL the PDB format files were generated which were used for comparing the wild type and mutant Crg1 structures via PyMOL. The above kinds of software were also used for determining the net charge, buried and exposed residues. RESULTS AND DISCUSSION N-terminal residues of Crg1 are essential for cantharidin resistance in yeast Crg1 methyltransferase was previously characterized by the presence of the methyltransferase domain in two closely related yeast genera, Saccharomyces cerevisiae4, and Candida albicans.3 Though the conserved catalytic sites of Crg1-methyltransferase domain are well known; its upstream and downstream residues function are still unexplored. To address this problem, we characterized the Crg1 residues upstream of the methyltransferase domain. We created the Crg1N mutant by deletion of 41 amino acids from the Crg1 N-terminus (Figure 1A). Additionally, the methyltransferase domain point mutants, D44A, D67A, and E105A-D108A were also created as reported previously (Figure 1A and B).4 We measured their growth sensitivity on cantharidin containing medium where crg1ΔN mutant was found hypersensitive to cantharidin as same as the null mutant, crg1 (Figure 1B and S1A). That suggests the essential role of the Crg1 N-terminal residues in cantharidin resistance. To identify the specific residues essential for cantharidin resistance, sequential truncation of ten amino acids from the 1st to 30th residue: crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30) and an eleven residues truncation crg1Δ(31-41) mutant were created (Figure 1C). Since the 41st residue, serine is a predicted phosphorylation site,30 we substituted it 6 ACS Paragon Plus Environment

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Biochemistry

with alanine (S41A) (Figure 1C). All the crg1 mutants [S41A, D44A, D67A, E105A-D108A, crg1Δ(110), crg1Δ(11-20), crg1Δ(21-30), crg1Δ(31-41) and crg1ΔN] were spotted on cantharidin containing medium to measure the sensitivity. All the truncation mutants showed sensitive growth with cantharidin treatment (Figure 1D). Among the truncation mutants, crg1Δ(1-10) grew better than the rests, indicating lesser essentiality of the first ten residues for cantharidin resistance compared to the remaining 31 residues (Figure 1D). The data also showed no effect of S41A mutation although the crg1Δ(31-41) and crg1ΔN were hypersensitive to cantharidin implying a dispensable role of the residue (Figure 1D and S1B). Altogether, we conclude that the Crg1 N-terminal residues are essential for cantharidin resistance.

Figure 1: Deletion of N-terminal residues of Crg1 causes loss of cantharidin resistance. (A) Schematic diagram, representing yeast Crg1 protein. Crg1 contains a methyltransferase domain composed of a SAM Binding Motif and a Substrate Binding Motif. The model illustrates 41 amino acids truncation in the N-terminus (ΔN) and point mutations created in the methyltransferase domain (D44A, D67A, and E105A-D108A). (B) The 10 fold serial dilutions of 1OD600 cells of WT and crg1 mutants were spotted on SC-URA media containing cantharidin. (C) Schematic diagram illustrating sequential truncations in Crg1 N-terminus. (D) The 10 fold dilutions of 1OD600 cells of WT and crg1 mutants were spotted on SC-URA media containing cantharidin.

Deletion of N-terminal residues (crg1ΔN) or point mutations at methyltransferase domain (D44A, D67A, and E105A-D108A) alter the native folding of Crg1 Because the N-terminal truncation mutants show sensitive growth in cantharidin treated medium, we proposed a loss of methyltransferase activity in Crg1ΔN. Crg1 deactivates cantharidin by transferring a methyl group from SAM; this function might be compromised with Crg1ΔN (Figure 2A). To support this hypothesis, we performed mass spectrometry-based enzyme assay to measure the methyltransferase 7 ACS Paragon Plus Environment

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activity of the Crg1ΔN protein. First, we purified the wild-type Crg1 and truncated Crg1ΔN proteins expressed under the GAL1 promoter in crg1 strain (Figure 2B). The purified proteins were confirmed through western blotting using the Crg1 specific antibody (Figure 2C and S8D). The enzyme reaction was set as described previously.4 The mass spectrometry analysis showed loss of methyltransferase activity in Crg1ΔN.

The

mass

spectrometry

chromatogram

of

Crg1ΔN

catalyzed

reaction

(Crg1ΔN+Cantharidin+SAM) didn’t show any peak of methyl cantharidin (m/z = 211), it only showed the unmethylated cantharidin peak (m/z = 197) (Figure 2E) which imply the loss of methyltransferase activity in Crg1ΔN. On the other hand, wild-type Crg1 methylated cantharidin, evident in the mass spectrometry chromatogram of the Crg1 catalyzed reaction (Crg1+cantharidin+SAM) with the appearance of methyl cantharidin peak (m/z = 211) (Figure 2D).

Figure 2: N-terminal truncation of Crg1 causes loss of methyltransferase activity. (A) The biochemical reactions catalyzed by Crg1 or Crg1ΔN using cantharidin and SAM as substrate and co-factor, respectively. (B) CBBR staining of the purified wild-type Crg1-6xHis and mutant Crg1ΔN-6xHis expressed in yeast, separated by SDS-PAGE. (C) Western blot validation of the purified Crg1-6xHis and Crg1ΔN-6xHis with Crg1 specific antibody. The purified proteins (B) were probed against the anti-Crg1 antibody. (D-H) LC-ESI-MS chromatogram of wild-type Crg1 and Crg1ΔN catalyzed reactions. The chromatogram shows distinct peaks of cantharidin (m/z = 197) and methyl cantharidin (m/z = 211). (D) MS chromatogram of wild-type Crg1 catalyzed reaction


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Biochemistry

(Crg1+SAM+cantharidin). (E) MS chromatogram of the Crg1ΔN catalyzed reaction (Crg1ΔN+SAM+cantharidin). (F) The reaction mixture without enzyme (SAM+cantharidin). (G) A control sample containing only cantharidin and protein elution buffer (cantharidin+Elution Buffer). (H) A control sample containing only co-factor SAM and protein elution buffer (SAM+Elution Buffer).

To find the mechanism behind loss of methyltransferase activity in Crg1ΔN, we tracked it's in vivo status. Crg1 proteins (wild-type and mutants) were tagged with daGFP at C-terminus. S41A was taken as a nonspecific mutation that doesn’t affect the Crg1 function. Microscopy analysis of Crg1ΔN-daGFP showed its cytoplasmic aggregates compared to the uniformly distributed wild-type Crg1-daGFP (Figure 3A)4. Additionally, all the cantharidin sensitive point mutants, D44A, D67A, and E105A-D108A also showed the same defect of aggregation (Figure 3B). This observation indicates that either N-terminal truncation or methyltransferase domain point mutation causes the same defect of aggregation. In contrast, S41A didn’t show any defect in its sub-cellular distribution and aggregate formation (Figure 3A). Aggregation of the mutant proteins was further confirmed by western blotting experiment (Figure 3C). Cell lysates of the wild-type and mutant strains were prepared in native condition.4 We found very poor solubility of the Crg1ΔN and D44A; a major fraction of which went into the pellet (Figure 3C). On the other hand, the wild-type Crg1 was found mostly in the soluble fraction (Figure 3C). Moreover, the 3-D structure prediction (I-TASSER/SWISS-MODEL) showed altered confirmation of Crg1ΔN compared to the wild-type Crg1 (Figure 3D) which further supports our conclusions derived from in vivo observations. The deleted first 41 amino acids of Crg1 at the N-terminus forms ‘Coiled-Helix-Coil-Helix-Coil’ structure which seems essential for the functional and stable protein. These N-terminal residues are majorly exposed which and critical for its solubility. On the other hand, the point mutations of D44A, D67A, E105A-D108A alters the net charge which might be the reason for aggregation of these proteins (Figure S9). Altogether, these results describe the actual mechanism of loss of methyltransferase activity upon methyltransferase domain point mutations (D44A, D67A, and E105A-D108A) or N-terminal truncation (Crg1N). These mutations induce structural instability in Crg1 by inappropriate folding and aggregation in the cytoplasm. crg1ΔN mutant exhibits phosphatidylethanolamine (PE) auxotrophy upon cantharidin treatment Our previous study has shown an essential role of PE to tolerate the cantharidin cytotoxicity in the crg1 mutant. The lethal cytotoxic effect of cantharidin can be neutralized by supplementation of ethanolamine (ETA) in the medium.23 ETA supplementation induces PE biosynthesis via the Kennedy Pathway (Figure 4D).31, 32 Similarly, choline (CHO) and inositol (INO) supplementation also lead to phosphatidylcholine (PC) and phosphatidylinositol (PI) biosynthesis, respectively.31-33 We tested the same phenomenon in the crg1ΔN mutant as well. The cantharidin treated medium was supplemented with ETA, INO, and CHO, and the crg1 mutants [S41A, D44A, D67A, E105A-D108A, crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30),


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crg1Δ(31-41) and crg1ΔN] were spotted on it. The ETA supplementation rescued the growth of crg1ΔN in cantharidin treated medium. Similarly, ETA supplementation also rescued the rest of the cantharidin sensitive mutants, D44A, D67A, E105A-D108A, crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30) and crg1Δ(31-41) (Figure 4A, S2). The data suggest that the phenomenon of ETA mediated neutralization of cantharidin cytotoxicity exists for all cantharidin sensitive crg1 mutants [D44A, D67A, E105A-D108A, crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30), crg1Δ(31-41) and crg1ΔN]. Since the lack of PE perturbs the plasma membrane at elevated temperature,34 we checked the growth of the crg1 mutants with a permissible dose of cantharidin (4μM) at a higher temperature (37oC). All the crg1 mutants [D44A, D67A, E105A-D108A, crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30) and crg1Δ(31-41)] except S41A were found sensitive to cantharidin at 37oC (Figure 4B).

Figure 3: Mutations in methyltransferase domain or N-terminus promote misfolding and aggregation of Crg1. (A) CRG1 full length, S41A, and CRG1ΔN tagged with daGFP were observed under ZEISS Apotome fluorescence microscope. (B) CRG1 full length and methyltransferase domain point mutants (D44A, D67A, E105AD108A) tagged with daGFP were analyzed under ZEISS Apotome fluorescence microscope. (C) Whole cell lysates of the WT and D44A strains were prepared in native condition, and the western blotting was performed using antiCrg1 antibody. (D) The 3-D structures of wild-type Crg1 and Crg1ΔN, predicted by SWISS-MODEL and superimposed with the help of PyMOL.


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Biochemistry

The cantharidin sensitivity increased gradually with elevating temperatures (25oC to 37oC) (Figure 4B). When the cells were supplemented with ETA at the higher temperature (37oC), a significant rescue was observed from cantharidin cytotoxicity (Figure 4C). The data demonstrate the essential role of PE in cantharidin tolerance in crg1ΔN cells as well.

Figure 4: ETA supplementation rescues crg1ΔN mutant from cantharidin cytotoxicity. (A) The phospholipid precursors; ETA, INO, and CHO were added into SC-URA agar media with or without cantharidin. Ten-fold serial dilutions of WT and crg1 mutants were spotted on the prepared media. (B) The 10 fold serial dilutions of WT and crg1 mutant cells were spotted with the permissible dose of cantharidin (4μM) and incubated at different temperatures (25oC, 30oC, and 37oC). (C) The 10 fold serial dilutions of WT and crg1 mutants were spotted on SCURA media containing a permissive dose of cantharidin (4μM) with or without ETA and incubated at 37oC. (D) Phospholipid biosynthesis pathways in yeast. INO is used as a substrate by Pis1 to synthesize PI in ER. SER is converted into PS by the action of Cho1 in ER. PS is transported to the mitochondria where Psd1 converts it into PE. PE is transported again into the ER where Cho2 and Opi3 convert it into PC in a sequence of reactions. PE and PC biosynthesis is also possible by an alternate pathway, Kennedy Pathway, where ETA or CHO is used as a substrate to synthesis PE or PC, respectively, in two distinct cascades of reactions. Our hypothesis proposes PE as a potential target of cantharidin.


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N-terminal truncation of Crg1 reduces the unfolded protein response Cantharidin induces ER stress by alteration of phospholipid and redox homeostasis in ER.4 crg1 mutant exhibits decreased unfolded protein response upon cantharidin treatment.23 We checked this phenotype in crg1ΔN as well, for which we examined the synergistic effect of cantharidin and unfolded protein response inducers (DTT/TM) on crg1ΔN. The growth sensitivity assay confirmed that the combination of cantharidin with DTT or TM increases cytotoxicity in crg1ΔN, similar to crg1 (Figure 5A and S3).23 Besides, the rest crg1 mutants [D44A, D67A, E105A-D108A, crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30), crg1Δ(31-41)] also showed hypersensitivity to the combination of cantharidin+DTT or cantharidin+TM, implying synergistic toxicity by both the molecules on crg1 mutants. Contrarily, the S41A didn’t show any sensitivity in any conditions, suggesting a dispensable role of this residue in methyltransferase activity. ER homeostasis also depends on the redox balance maintained by GSH buffer.35 We have seen a lethal synergistic effect of cantharidin and GSH on crg1 mutant;23 this effect was also tested on the crg1ΔN mutant. Both the combinations, cantharidin+GSH, and cantharidin+NAC, inhibited the growth of crg1ΔN mutant strongly compared to cantharidin alone (Figure 5B and S4). The synergistic cytotoxic effect of cantharidin+GSH or cantharidin+NAC was also evident on other crg1 point mutants, D44A, D67A, E105A-D108A, and the N-terminal truncation mutants, crg1Δ(11-20), crg1Δ(21-30) and crg1Δ(31-41) (Figure 5B and S4). To identify the underlying mechanism responsible for sensitive growth upon ER stress, we checked the unfolded protein response in crg1ΔN by measuring the HAC1 mRNA splicing. The mutants, crg1 and crg1ΔN, showed decreased splicing of HAC1 mRNA compared to WT in an untreated condition which further decreased upon cantharidin treatment (Figure 5C, D).23 Based on these data we conclude that cantharidin induced ER stress in crg1ΔN is as similar as the null mutant crg1, indicating that the lack of N-terminal residues causes complete loss of Crg1 function. Cantharidin perturbs cell wall integrity in crg1ΔN mutant Cantharidin induces the CWI pathway through perturbation of lipid homeostasis, ER stress and GPIanchor missorting.3, 4, 23 As per our previous study, the above phenotypes were more prominent in crg1 strain than wild-type.23 That motivated us to investigate these phenotypes in crg1ΔN mutant as well. Firstly, we checked the synergistic effect of cantharidin and cell wall perturbing molecules, Congo Red (CR) and Calcofluor White (CFW) on crg1ΔN. The crg1ΔN mutant showed hypersensitivity to cantharidin+CFW and cantharidin+CR, identical to crg1 mutant (Fig 6A and S5A).23 This synergistic effect was also evident in crg1 point mutants (D44A, D67A, and E105A-D108A) and N-terminal truncations [crg1Δ(11-20), crg1Δ(21-30), crg1Δ(31-41)] (Figure 6A and S5A). Quite the opposite, S41A didn’t show any sensitivity in the given conditions. Next, the cantharidin containing medium was supplemented with sorbitol (SORBITOL), but didn’t rescue the growth of the sensitive crg1 mutants [D44A, D67A, E105A-D108A, crg1Δ(1-10), crg1Δ(11-20), crg1Δ(21-30), crg1Δ(31-41) and crg1ΔN] 12 ACS Paragon Plus Environment

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Biochemistry

(Figure 6B, S5A), symptomatic of irreversible cell wall damage. For more evidence, we measured chitin content in crg1ΔN upon cantharidin treatment and found increased deposition of chitin in the cell wall of crg1ΔN similar to the crg1 mutant (Figure 6C). Additionally, we measured Slt2 phosphorylation in crg1ΔN mutant upon cantharidin treatment where the crg1ΔN showed hyperphosphorylation of Slt2 similar to the crg1 mutant (Figure 6D, E and S5B). We also found rescue in Slt2 phosphorylation upon ETA supplementation, consistent with our previous observation.23 Overall, crg1ΔN and crg1 mutants display similar phenotypes related to the cell wall integrity giving evidence for the loss of function in Crg1 after the N-terminal truncation.

Figure 5: cantharidin shows synergistic effect with ER stress inducers (TM/DTT) and antioxidants (GSH/NAC) in crg1ΔN. (A) The normalized 10 fold serial dilutions of WT and crg1 mutants were spotted on cantharidin containing SC-URA media with and without TM/DTT and incubated at 30oC for 72h. (B) The normalized 10 fold serial dilutions of WT and crg1 mutants were spotted on cantharidin containing SC-URA media with and without antioxidant molecules GSH/NAC and incubated at 30oC for 72h. (C) WT and crg1 mutants were grown in SC-URA media at 30oC till mid-exponential phase (0.8 OD600), treated with cantharidin (6μM) for 2h. Cells were harvested, processed for cDNA synthesis and semi-qPCR. HAC1 (u): HAC1 uninduced/unspliced, HAC1 (i): HAC1 induced/spliced (D) Densitometric quantification of HAC1 mRNA splicing by ImageJ software. The data represents mean ± standard deviation of the four individual repeats; the data shows the mean ± standard deviation. The statistical significance was calculated applying students t-test, where p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***).


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Cantharidin alters GPI-anchored protein sorting in crg1ΔN mutant GPI-anchored proteins play a major role in the biosynthesis and maintenance of yeast cell wall, lack of which affects the cell wall integrity.36, 37 Our previous study showed altered GPI anchored protein sorting in crg1 mutant with cantharidin treatment.23 So, we decided to examine this phenotype in crg1ΔN mutant as well. Gas1-GFP was used as a model GPI-anchored protein,26, 38 expressed in the crg1ΔN mutant. We observed missorting and aggregation of GPI-anchored Gas1-GFP in crg1ΔN cells upon cantharidin treatment, similar to the crg1 null mutant (Figure 7A and S6). Western blot analysis of Gas1-GFP also showed its reduced level in crg1ΔN and crg1 cells upon cantharidin treatment (Figure 7B, C and S6), which might be due to targeted degradation of the aggregated proteins.39-41

Figure 6: cantharidin perturbs cell wall integrity in crg1ΔN. (A) The normalized 10 fold serial dilutions of WT and crg1 mutants were spotted on cantharidin containing SC-URA agar media with and without cell wall perturbing molecules CR or CFW. (B) The normalized 10 fold dilutions of WT and crg1 mutants were spotted on cantharidin containing SC-URA media with or without sorbitol. (C) The cantharidin treatment increases chitin content in the cell wall of crg1 mutants. WT and crg1 mutants were grown in SC-URA media at 30oC till mid-exponential phase (0.8 OD600) and treated with cantharidin (6μM) for 3h. Cells were stained with CFW (5μg/mL) in growth condition for 15 minutes. Fluorescence intensity was measured using Biotek Eon 96 well fluorescent plate reader (FLx800). The data shows mean ± standard deviation. The statistical significance was calculated applying students t-test, where p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). (D) WT and crg1 mutant cells were grown in SC-URA media at 24oC till mid-exponential phase and then treated with cantharidin (6μM) for 2h. Western blotting was performed with antibodies specific for phospho-Slt2 (pSlt2), Slt2, and Tbp1. (E) Densitometry quantification of the three independently performed experiments for western blotting shown in (D), the data shows mean ± standard deviation.


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Biochemistry

The statistical significance was calculated applying students t-test, where p ≤ 0.05 (*), (***).

p ≤ 0.01 (**), p ≤ 0.001

We also checked genetic interaction between CDC1 and CRG1 taking two different alleles of CRG1, crg1, and crg1ΔN, where both the alleles showed synthetic lethality with cdc1-314 evident in the Slt2 phosphorylation pattern (Figure 7D, E).42 The data shows a similar effect of cantharidin on GPI-anchored protein sorting in crg1 and crg1ΔN mutants implying a complete loss of function in crg1ΔN.

Figure 7: Cantharidin alters GPI-anchored protein sorting in crg1ΔN. (A) WT and crg1 mutants expressing Gas1-GFP were grown in YPD till mid-exponential phase, treated with cantharidin (25μM) for 4h and visualized under ZEISS Apotome fluorescence microscope. (B) Whole cell lysate prepared from cells in (A) were probed with anti-GFP and anti-Tbp1 antibody. (C) Densitometry quantification of the four independently performed experiments for western blotting shown in (B), the data show mean ± standard deviation. The statistical significance was calculated applying students t-test, where p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***). (D) WT, cdc1, crg1, crg1ΔN, single and double mutants were grown till the mid-exponential phase and then treated with cantharidin for 2h. Western blotting was done with anti-phospho-Slt2, anti-Slt2, and anti-Tbp1 antibodies. (E) Densitometry quantification of the three independently performed experiments for western blotting shown in (D), the data shows mean ± standard deviation. The statistical significance was calculated applying students t-test, where p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***).


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CONCLUSION In this study, we characterized the catalytic role of the Crg1 N-terminus for methyltransferase activity. Previous studies identified Crg1 as a methyltransferase enzyme by the presence of a conserved methyltransferase domain positioned centrally in the primary structure3, 4. Our study affixes new information on the existing knowledge of Crg1 methyltransferase. We identified an indispensable role of the Crg1 N-terminal residues in methyltransferase activity. The inability of the N-terminally truncated mutants to grow in cantharidin treated medium gave first evidence supporting our hypothesis. Biochemical enzyme assay further proved the essentiality of the Crg1 N-terminus, since the mutant Crg1ΔN was unable to methylate cantharidin in vitro. It helped us to conclude that the deletion of the Crg1 N-terminal residues leads to loss of methyltransferase activity. Microscopic visualization of the fluorescent tagged Crg1ΔN revealed defective folding that leads to the cytoplasmic aggregation of the mutant protein (Figure 8). Additionally, we observed the previously characterized Crg1 methyltransferase domain point mutants (D44A, D67A, and E105A-D108A) also form cytoplasmic aggregates, indicating the same folding defect due to methyltransferase domain point mutations (Figure 8). This is the actual mechanism behind the loss of enzyme activity or cantharidin resistance in methyltransferase domain point mutants (Figure 8).3, 4 Phenotypic characterizations of crg1ΔN also showed identical phenotypes as crg1 null mutant. Both the mutants exhibit PE auxotrophy, reduced unfolded protein response, cell wall damage, and GPI anchor missorting upon cantharidin treatment suggesting a complete loss of function in crg1ΔN. Altogether, our study concludes that the Crg1 N-terminal residues are essential for its methyltransferase enzyme activity, and so the intact protein is required to methylate cantharidin and develop resistance.

Figure 8. N-terminal truncations methyltransferase domain point mutations impair the native folding of Crg1. The schematic diagram illustrates the substitution mutations in the methyltransferase domain and deletion of


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Biochemistry

N-terminal residues of Crg1. The indicated mutations affect the native conformation of Crg1 by impairing the appropriate folding, resulting in the formation of aggregates.

CRG1 doesn’t show any notable phenotype in standard growth conditions, but, it has been shown to be essentially required in the pathogenesis of Candida albicans.3 Two different features; (i) a drug-resistant methyltransferase and (ii) a virulence factor, makes Crg1 a potential antifungal drug target.3 Crg1 is also shown to interact with mRNAs which suggests its possible role as mRNA methyltransferase.43 Since Crg1 is conserved in higher eukaryotes; it will be interesting to study its human homologs against the small molecules.4 ACKNOWLEDGMENT We are thankful to Benjamin S. Glick and Won-Ki Huh for providing us the cdc1-314 mutant and pRS413-GAS1-GFP plasmid, respectively. We acknowledge all the lab members for their crucial inputs throughout the study. FUNDING SOURCES DBT-India is acknowledged for providing the JRF/SRF support to PKS (DBT/2014/IISER-B/195). CSIRIndia is conceded for the JRF/SRF support to SC [09/1020(0036)/2012EMR-I]. This work was financially supported by SERB Govt. of India (Grant No. EMR/2015/001797) to RST. SUPPLEMENTARY INFORMATION The supplementary file contains Methods, Tables S1-S3 (list of strains, plasmids, and primers), and Supplementary Figures S1-S9. REFERENCES [1] Katz, J. E., Dlakic, M., and Clarke, S. (2003) Automated identification of putative methyltransferases from genomic open reading frames, Molecular & cellular proteomics : MCP 2, 525-540. [2] Schubert, H. L., Blumenthal, R. M., and Cheng, X. (2003) Many paths to methyltransfer: a chronicle of convergence, Trends in biochemical sciences 28, 329-335. [3] Lissina, E., Weiss, D., Young, B., Rella, A., Cheung-Ong, K., Del Poeta, M., Clarke, S. G., Giaever, G., and Nislow, C. (2013) A novel small molecule methyltransferase is important for virulence in Candida albicans, ACS chemical biology 8, 2785-2793. [4] Lissina, E., Young, B., Urbanus, M. L., Guan, X. L., Lowenson, J., Hoon, S., Baryshnikova, A., Riezman, I., Michaut, M., Riezman, H., Cowen, L. E., Wenk, M. R., Clarke, S. G., Giaever, G., and Nislow, C. (2011) A systems biology approach reveals the role of a novel methyltransferase in response to chemical stress and lipid homeostasis, PLoS genetics 7, e1002332. [5] Dzyubak, E., and Yap, M. N. (2016) The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling, Antimicrobial agents and chemotherapy 60, 7178-7188. 17 ACS Paragon Plus Environment

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[38] Nuoffer, C., Jeno, P., Conzelmann, A., and Riezman, H. (1991) Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane, Molecular and cellular biology 11, 27-37. [39] Sikorska, N., Lemus, L., Aguilera-Romero, A., Manzano-Lopez, J., Riezman, H., Muniz, M., and Goder, V. (2016) Limited ER quality control for GPI-anchored proteins, The Journal of cell biology 213, 693-704. [40] Field, M. C., Moran, P., Li, W., Keller, G. A., and Caras, I. W. (1994) Retention and degradation of proteins containing an uncleaved glycosylphosphatidylinositol signal, The Journal of biological chemistry 269, 10830-10837. [41] Ha, C. W., Kim, K., Chang, Y. J., Kim, B., and Huh, W. K. (2014) The beta-1,3glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae, Nucleic acids research 42, 8486-8499. [42] Losev, E., Papanikou, E., Rossanese, O. W., and Glick, B. S. (2008) Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling, Molecular and cellular biology 28, 3336-3343. [43] Tsvetanova, N. G., Klass, D. M., Salzman, J., and Brown, P. O. (2010) Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae, PloS one 5.

UniProt Accession ID: P38892; NCBI Accession ID: KZV10954


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Biochemistry

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The Crg1 N-Terminus Is Essential for Methyltransferase Activity and

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