Molecular Genetics & Genomics

Molecular Genetics & Genomics

A genome-wide screen identifies yeast genes required for protection against or enhanced cytotoxicity of the antimalarial drug quinine

Sandra C. dos Santos1, Isabel Sá-Correia1,2*

1IBB – Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisboa, Portugal.

2Department of Bioengineering, Instituto Superior Técnico, Technical University of Lisbon, 1049-001 Lisboa, Portugal.

*Corresponding author: Prof. Isabel Sá-Correia

Mailing address: IBB - Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

Fax number: +351-218419199.

e-mail:

Running title:Yeast genetic screen for susceptibility to quinine

Key words:Saccharomyces cerevisiae, quinine targets, antimalarial drugs, chemogenomics, drug resistance

Table S1. Yeast genes whose deletion confers hyper-susceptibility phenotypes to quinine.

Gene / Description
cellular metabolic processes
AAC3 / Mitochondrial inner membrane ADP/ATP translocator, exchanges cytosolic ADP for mitochondrially synthesized ATP; expressed under anaerobic conditions; has roles in maintenance of viability and in respiration
CAX4 / Dolichyl pyrophosphate (Dol-P-P) phosphatase with a luminally oriented active site in the ER
CYT2 / Cytochrome c1 heme lyase, involved in maturation of cytochrome c1
DEG1 / Non-essential tRNA:pseudouridine synthase, introduces pseudouridines at position 38 or 39 in tRNA, important for maintenance of translation efficiency and normal cell growth, localizes to both the nucleus and cytoplasm
ELP4 / Subunit of Elongator complex
ERG6 / Delta(24)-sterol C-methyltransferase, converts zymosterol to fecosterol in the ergosterol biosynthetic pathway by methylating position C-24
GCV3 / H subunit of the mitochondrial glycine decarboxylase complex, required for the catabolism of glycine to 5,10-methylene-THF
HTD2 / Mitochondrial 3-hydroxyacyl-thioester dehydratase involved in fatty acid biosynthesis, required for respiratory growth and for normal mitochondrial morphology
KEX2 / Subtilisin-like protease (proprotein convertase), a calcium-dependent serine protease involved in the activation of proproteins of the secretory pathway
LPD1 / Dihydrolipoamide dehydrogenase, the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multi-enzyme complexes
MDJ1 / Protein involved in folding of mitochondrially synthesized proteins in the mitochondrial matrix; localizes to the mitochondrial inner membrane; member of the DnaJ family of molecular chaperones
MRP17 / Mitochondrial ribosomal protein of the small subunit
MRPS16 / Mitochondrial ribosomal protein of the small subunit
NDE1 / Mitochondrial external NADH dehydrogenase, catalyzes the oxidation of cytosolic NADH
PKR1 / V-ATPase assembly factor, functions with other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector (V0)
REF2 / RNA-binding protein involved in the cleavage step of mRNA 3'-end formation prior to polyadenylation, and in snoRNA maturation; part of holo-CPF subcomplex APT
RPL14a / N-terminally acetylated protein component of the large (60S) ribosomal subunit
RPS21b / Protein component of the small (40S) ribosomal subunit
RSM22 / Mitochondrial ribosomal protein of the small subunit
TCM62 / Protein involved in the assembly of the mitochondrial succinate dehydrogenase complex; putative chaperone
TEF4 / Translation elongation factor EF-1 gamma
URM1 / Ubiquitin-like protein with only weak sequence similarity to ubiquitin; is required for normal growth, particularly at high temperature
cell organization and biogenesis
AKR1 / Palmitoyl transferase involved in protein palmitoylation; acts as a negative regulator of pheromone response pathway; involved in cell shape control
ARC18 / Subunit of the ARP2/3 complex, which is required for the motility and integrity of cortical actin patches
BEM2 / Rho GTPase activating protein (RhoGAP) involved in the control of cytoskeleton organization and cellular morphogenesis
BEM4 / Protein involved in establishment of cell polarity and bud emergence
BUD25 / Protein involved in bud-site selection
BUD32 / Protein kinase proposed to be involved in bud-site selection, telomere uncapping and elongation, and transcription; component of the EKC/KEOPS protein complex with Kae1p, Cgi121p, Pcc1p, and Gon7p
DRS2 / Aminophospholipid translocase (flippase) that maintains membrane lipid asymmetry in post-Golgi secretory vessicles
GTR2 / Cytoplasmic GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; subunit of EGO of GSE complexes
LDB7 / Component of the RSC chromatin remodeling complex
MNN10 / Subunit of a Golgi mannosyltransferase complex also containing Anp1p, Mnn9p, Mnn11p, and Hoc1p that mediates elongation of the polysaccharide mannan backbone
NCS2 / Protein with a role in urmylation and in invasive and pseudohyphal growth
NCS6 / Protein with a role in urmylation and in invasive and pseudohyphal growth
NRP1 / Protein of unknown function, rich in asparagine residues
OSH2 / Member of an oxysterol-binding protein family with seven members in S. cerevisiae; family members have overlapping, redundant functions in sterol metabolism and collectively perform a function essential for viability
PAF1 / RNAP II-associated protein; defines large complex biochemically and functionally distinct from the Srb-Mediator form of Pol II holoenzyme
ROT2 / Glucosidase II catalytic subunit required for normal cell wall synthesis
RTF1 / Subunit of the RNA polymerase II-associated Paf1 complex; directly or indirectly regulates DNA-binding properties of Spt15p and relative activities of different TATA elements
RVS161 / Amphiphysin-like lipid raft protein; subunit of a complex (Rvs161p-Rvs167p) that regulates polarization of the actin cytoskeleton, endocytosis, cell polarity, cell fusion and viability following starvation or osmotic stress
SAC6 / Fimbrin, actin-bundling protein
SEH1 / Nuclear pore protein that is part of the evolutionarily conserved Nup84p complex
SHE4 / Protein containing a UCS (UNC-45/CRO1/SHE4) domain, binds to myosin motor domains to regulate myosin function; involved in endocytosis, polarization of the actin cytoskeleton, and asymmetric mRNA localization
SLA1 / Cytoskeletal protein binding protein required for assembly of the cortical actin cytoskeleton
SLT2 / Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity and progression through the cell cycle; regulated by the PKC1-mediated signaling pathway
SUR4 / Elongase, involved in fatty acid and sphingolipid biosynthesis
TUP1 / General repressor of transcription, forms complex with Cyc8p, involved in the establishment of repressive chromatin structure through interactions with histones H3 and H4
VPS1 / GTPase required for vacuolar protein sorting, required for late Golgi-retention of some proteins including Kex2p
VRP1 / Proline-rich actin-associated protein involved in cytoskeletal organization and cytokinesis
YPK1 / Serine/threonine protein kinase required for receptor-mediated endocytosis; involved in sphingolipid-mediated and cell integrity signaling pathways; localized to the bud neck, cytosol and plasma membrane; homolog of mammalian kinase SGK
regulation of metabolic processes
BRE1 / E3 ubiquitin ligase for Rad6p
NPR1 / Protein kinase that stabilizes several plasma membrane amino acid transporters by antagonizing their ubiquitin-mediated degradation
PDE2 / High-affinity cyclic AMP phosphodiesterase, component of the cAMP-dependent protein kinase signaling system
SCS2 / Integral ER membrane protein that regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1p
SNF4 / Activating gamma subunit of the AMP-activated Snf1p kinase complex (contains Snf1p and a Sip1p/Sip2p/Gal83p family member); activates glucose-repressed genes, represses glucose-induced genes
UBC8 / Ubiquitin-conjugating enzyme that negatively regulates gluconeogenesis by mediating the glucose-induced ubiquitination of fructose-1,6-bisphosphatase
VID24 / Peripheral membrane protein located at Vid (vacuole import and degradation) vesicles; regulates fructose-1,6-bisphosphatase (FBPase) targeting to the vacuole
VID30 / Protein involved in proteasome-dependent catabolite degradation of fructose-1,6-bisphosphatase
ZUO1 / Cytosolic ribosome-associated chaperone that acts, together with Ssz1p and the Ssb proteins, as a chaperone for nascent polypeptide chains
transcription regulation
ADA2 / Transcription coactivator, component of the ADA and SAGA transcriptional adaptor/HAT complexes
AFT1 / Transcription factor involved in iron utilization and homeostasis
MBF1 / Transcriptional coactivator that bridges the DNA-binding region of Gcn4p and TATA-binding protein Spt15p
NGG1 / Transcriptional regulator involved in glucose repression of Gal4p-regulated genes; component of transcriptional adaptor and histone acetyltransferase complexes, the ADA complex, the SAGA complex, and the SLIK complex
STP2 / Transcription factor, activated by proteolytic processing in response to signals from the SPS sensor system for external amino acids
URE2 / Nitrogen catabolite repression transcriptional regulator that acts by inhibition of GLN3 transcription in good nitrogen source
transport
BAP2 / High-affinity leucine permease
FET3 / Ferro-O2-oxidoreductase required for high-affinity iron uptake and involved in mediating resistance to copper ion toxicity
FTR1 / High affinity iron permease involved in the transport of iron across the plasma membrane
ITR1 / Myo-inositol transporter with strong similarity to the minor myo-inositol transporter Itr2p, member of the sugar transporter superfamily
PDR5 / Short-lived membrane ABC (ATP-binding cassette) transporter, actively exports various drugs, expression regulated by Pdr1p
PEX2 / RING-finger peroxin, peroxisomal membrane protein with a C-terminal zinc-binding RING domain
PHO86 / Endoplasmic reticulum (ER) resident protein required for ER exit of the high-affinity phosphate transporter Pho84p
PHO88 / Probable membrane protein, involved in phosphate transport
TAT1 / Amino acid transport protein for valine, leucine, isoleucine, and tyrosine, low-affinity tryptophan and histidine transporter
establishment of localization
ACB1 / Acyl-CoA-binding protein, transports newly synthesized acyl-CoA esters from fatty acid synthetase (Fas1p-Fas2p) to acyl-CoA-consuming processes
AIR1 / RING finger protein that interacts with the arginine methyltransferase Hmt1p to regulate methylation of Npl3p
ATG11 / Peripheral membrane protein required for delivery of aminopeptidase I (Lap4p) to the vacuole in the cytoplasm-to-vacuole targeting pathway
CHS5 / Component of the exomer complex, which also contains Csh6p, Bch1p, Bch2p, and Bud7p and is involved in export of selected proteins
GOS1 / v-SNARE protein involved in Golgi transport
MSN5 / Karyopherin involved in nuclear import and export
PEP3 / Vacuolar peripheral membrane protein that promotes vesicular docking/fusion reactions in conjunction with SNARE proteins, required for vacuolar biogenesis, forms complex with Pep5p that mediates protein transport to the vacuole
SAC1 / Lipid phosphoinositide phosphatase of the ER and Golgi, involved in protein trafficking and secretion
SPC2 / Subunit of signal peptidase complex (Spc1p, Spc2p, Spc3p, Sec11p), which catalyzes cleavage of N-terminal signal sequences of proteins targeted to the secretory pathway
TIM11 / Subunit e of mitochondrial F1F0-ATPase
TIM21 / Constituent of the mitochondrial inner membrane presequence translocase (TIM23 complex)
VPS52 / Component of the GARP (Golgi-associated retrograde protein) complex, Vps51p-Vps52p-Vps53p-Vps54p, which is required for the recycling of proteins from endosomes to the late Golgi
VPS64 / Cytoplasmic protein required for cytoplasm to vacuole targeting of proteins
cell ion homeostasis
GEF1 / Chloride channel localized to late- or post-Golgi vesicles, involved in iron metabolism
HAL5 / Putative protein kinase; overexpression increases sodium and lithium tolerance, whereas gene disruption increases cation and low pH sensitivity and impairs potassium uptake, suggesting a role in regulation of Trk1p and/or Trk2p transporters
ISU2 / Conserved protein of the mitochondrial matrix, required for synthesis of mitochondrial and cytosolic iron-sulfur proteins
PPA1 / Subunit c'' of the vacuolar ATPase, which functions in acidification of the vacuole
SPF1 / P-type ATPase, ion transporter of the ER membrane involved in ER function and Ca2+ homeostasis
TFP3 / Vacuolar ATPase V0 domain subunit c', involved in proton transport activity
TRK1 / Component of the Trk1p-Trk2p potassium transport system
VMA13 / Subunit H of the eight-subunit V1 peripheral membrane domain of the vacuolar H+-ATPase (V-ATPase)
VMA2 / Subunit B of the eight-subunit V1 peripheral membrane domain of the vacuolar H+-ATPase (V-ATPase)
VMA22 / Integral membrane protein that is required for vacuolar H+-ATPase (V-ATPase) function, although not an actual component of the V-ATPase complex; functions in the assembly of the V-ATPase
response to stress
DBF2 / Ser/Thr kinase involved in transcription and stress response
GSH2 / Glutathione synthetase, catalyzes the ATP-dependent synthesis of glutathione (GSH) from gamma-glutamylcysteine and glycine; induced by oxidative stress and heat shock
HOF1 / Bud neck-localized, SH3 domain-containing protein required for cytokinesis
STE50 / Protein involved in mating response, invasive/filamentous growth, and osmotolerance
TPS2 / Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex; expression is induced by stress conditions and repressed by the Ras-cAMP pathway
YCL033c / Putative protein-methionine-R-oxide reductase; involved in response to oxidative stress
cell cycle
BFR1 / Component of mRNP complexes associated with polyribosomes; implicated in secretion and nuclear segregation
CDC50 / Endosomal protein that regulates cell polarity
CIK1 / Kinesin-associated protein required for both karyogamy and mitotic spindle organization, interacts stably and specifically with Kar3p and may function to target this kinesin to a specific cellular role
GID8 / Protein of unknown function, involved in proteasome-dependent catabolite inactivation of fructose-1,6-bisphosphatase;
KEM1 / Evolutionarily-conserved 5'-3' exonuclease component of cytoplasmic processing (P) bodies involved in mRNA decay
SWI6 / Transcription cofactor, forms complexes with DNA-binding proteins Swi4p and Mbp1p to regulate transcription at the G1/S transition
TOP1 / Topoisomerase I, nuclear enzyme that relieves torsional strain in DNA; functions in replication, transcription, and recombination
UME6 / Key transcriptional regulator of early meiotic genes

Table S2. Yeast genes whose deletion confers susceptibility phenotypes to quinine.

Gene / Description
cellular metabolic processes
AAT2 / Cytosolic aspartate aminotransferase, involved in nitrogen metabolism
ADD37 / Protein of unknown function involved in ER-associated protein degradation
ARG82 / Inositol polyphosphate multikinase (IPMK), sequentially phosphorylates Ins(1,4,5)P3 to form Ins(1,3,4,5,6)P5
ARV1 / Protein required for normal intracellular sterol distribution and for sphingolipid metabolism
BLM10 / Proteosome activator subunit; required for resistance to bleomycin, may be involved in protecting against oxidative damage
BNA2 / Tryptophan 2,3-dioxygenase, required for biosynthesis of nicotinic acid from tryptophan
BRE5 / Member of the Chs5p-Arf1p-binding proteins (ChAPs) (Bch1p, Fmp50p, Bud7p, Chs6p) that mediate export of specific cargo proteins, from the Golgi to plasma membrane
COQ3 / O-methyltransferase, catalyzes two different O-methylation steps in ubiquinone (Coenzyme Q) biosynthesis
COX19 / Protein required for cytochrome c oxidase assembly
CSH1 / Probable catalytic subunit of a mannosylinositol phosphorylceramide (MIPC) synthase
CYS4 / Cystathionine beta-synthase, catalyzes the synthesis of cystathionine from serine and homocysteine
DER1 / Endoplasmic reticulum membrane protein, required for ER-associated protein degradation of misfolded or unassembled proteins
DLD3 / D-lactate dehydrogenase, part of the retrograde regulon which consists of genes whose expression is stimulated by damage to mitochondria and reduced in cells grown with glutamate as the sole nitrogen source
ECM40 / Mitochondrial ornithine acetyltransferase, catalyzes the fifth step in arginine biosynthesis
ERG24 / C-14 sterol reductase, acts in ergosterol biosynthesis
ERG3 / C-5 sterol desaturase, catalyzes the introduction of a C-5(6) double bond into episterol, a precursor in ergosterol biosynthesis
FEN1 / Fatty acid elongase, involved in sphingolipid biosynthesis
FES1 / Hsp70 (Ssa1p) nucleotide exchange factor
FMC1 / Mitochondrial matrix protein, required for assembly or stability at high temperature of the F1 sector of mitochondrial F1F0 ATP synthase
HFA1 / Mitochondrial acetyl-coenzyme A carboxylase, catalyzes the production of malonyl-CoA in mitochondrial fatty acid biosynthesis
IMP2 / Catalytic subunit of the mitochondrial inner membrane peptidase complex, required for maturation of mitochondrial proteins of the intermembrane space
KGD1 / Component of the mitochondrial alpha-ketoglutarate dehydrogenase complex, which catalyzes a key step in the tricarboxylic acid (TCA) cycle
LEM3 / Membrane protein of the plasma membrane and ER, involved in translocation of phospholipids and alkylphosphocholine drugs across the plasma membrane
LIP2 / Lipoyl ligase, involved in the modification of mitochondrial enzymes by the attachment of lipoic acid groups
LYS20 / Homocitrate synthase isozyme, catalyzes the condensation of acetyl-CoA and alpha-ketoglutarate to form homocitrate
MDM20 / Non-catalytic subunit of the NatB N-terminal acetyltransferase
MLS1 / Malate synthase, enzyme of the glyoxylate cycle, involved in utilization of non-fermentable carbon sources
MRPL28 / Mitochondrial ribosomal protein of the large subunit
MRPL51 / Mitochondrial ribosomal protein of the large subunit
MSY1 / Mitochondrial tyrosyl-tRNA synthetase
MTF1 / Mitochondrial RNA polymerase specificity factor with structural similarity to S-adenosylmethionine-dependent methyltransferases and functional similarity to bacterial sigma-factors
NCS6 / Protein with a role in urmylation and in invasive and pseudohyphal growth
PDB1 / E1 beta subunit of the pyruvate dehydrogenase (PDH) complex
POT1 / 3-ketoacyl-CoA thiolase with broad chain length specificity
PRE9 / 20S proteasome beta-type subunit
RML2 / Mitochondrial ribosomal protein of the large subunit
RPS24b / Protein component of the small (40S) ribosomal subunit
RSM25 / Mitochondrial ribosomal protein of the small subunit
RSM27 / Mitochondrial ribosomal protein of the small subunit
SCS3 / Protein required for inositol prototrophy, appears to be involved in the synthesis of inositol phospholipids from inositol
SER3 / 3-phosphoglycerate dehydrogenase, catalyzes the first step in serine and glycine biosynthesis
SHM2 / Cytosolic serine hydroxymethyltransferase, involved in one-carbon metabolism
SLM5 / Mitochondrial asparaginyl-tRNA synthetase
TUF1 / Mitochondrial translation elongation factor Tu
UBP15 / Ubiquitin-specific protease that may play a role in ubiquitin precursor processing
VID22 / Glycosylated integral membrane protein localized to the plasma membrane; plays a role in fructose-1,6-bisphosphatase (FBPase) degradation
YND1 / Apyrase with wide substrate specificity, involved in preventing the inhibition of glycosylation by hydrolyzing nucleoside tri- and diphosphates
cell organization and biogenesis
ARD1 / Subunit of the N-terminal acetyltransferase NatA (Nat1p, Ard1p, Nat5p); N-terminally acetylates many proteins, which influences multiple processes such as the cell cycle, heat-shock resistance, mating, sporulation, and telomeric silencing
ATG15 / Lipase, required for intravacuolar lysis of autophagic bodies
BRE2 / Subunit of the COMPASS (Set1C) complex, which methylates histone H3 on lysine 4 and is required in transcriptional silencing near telomeres
BUD7 / Member of the Chs5p-Arf1p-binding proteins (ChAPs) (Bch1p, Fmp50p, Bud7p, Chs6p) that mediate export of specific cargo proteins from the Golgi to plasma membrane
CDC73 / Constituent of Paf1 complex with RNA polymerase II, Paf1p, Hpr1p, Ctr9, Leo1, Rtf1 and Ccr4p, distinct from Srb-containing Pol II complexes; required for expression of certain genes, modification of some histones, and telomere maintenance
CGI121 / Protein involved in telomere uncapping and elongation as component of the KEOPS protein complex with Bud32p, Kae1p, Pcc1p, and Gon7p
CHS1 / Chitin synthase I, requires activation from zymogenic form in order to catalyze the transfer of N-acetylglucosamine (GlcNAc) to chitin
CMS1 / Putative protein of unknown function that may participate in DNA replication
CTK1 / Catalytic (alpha) subunit of C-terminal domain kinase I (CTDK-I), which phosphorylates the C-terminal repeated domain of the RNA polymerase II large subunit (Rpo21p) to affect both transcription and pre-mRNA 3' end processing
CTK3 / Gamma subunit of C-terminal domain kinase I (CTDK-I), which phosphorylates the C-terminal repeated domain of the RNA polymerase II large subunit (Rpo21p) to affect both transcription and pre-mRNA 3' end processing
CWH41 / Processing alpha glucosidase I, ER type II integral membrane N-glycoprotein involved in assembly of cell wall beta 1,6 glucan and asparagine-linked protein glycosylation