FUNGAL
TRANSPORTOSOMICS
Andre Goffeau
Universite de Louvain
Bioinformatic and Comparative Genome Analysis Course
HKU-Pasteur Research Centre - Hong Kong, China
August 17 - August 29, 2009
Genomology
Genome
sequencing
Databases
Deletomics
Interactomics
Genome
comparison
Transcriptomics
Proteomics
post-genomics, functional genomics,
proteomics, transcriptomics,
pharmacogenomics, structural genomics,
metagenomics,epigenomics,
patagenomics!!!
FUNGAL
TRANSPORTOSOMICS
Nomenclature Committee
of the International Union
of Biochemistry and Molecular Biology
(NC-IUBMB)
In consultation with the IUPAC-IUBMB Joint Commission
on Biochemical Nomenclature (JCBN)
Enzyme Nomenclature
http://www.chem.qmul.ac.uk/iubmb/enzyme/
Enzyme Nomenclature
EC 1 Oxidoreductases
EC 1.1 Acting on the CH-OH group of donors
EC 1.1.1 With NAD or NADP as acceptor
EC
EC
EC
EC
…
EC
1.1.1.1
1.1.1.2
1.1.1.3
1.1.1.4
alcohol dehydrogenase
alcohol dehydrogenase (NADP)
homoserine dehydrogenase
(R,R)-butanediol dehydrogenase
1.1.1.37 malate dehydrogenase
EC NOMENCLATURE

ONLY BASED ON CATALYTIC REACTIONS OF
SOLUBLE ENZYMES WHICH ARE
EXPERIMENTALY DEMONSTRATED

NO TRANSPORT REACTIONS

NO SEQUENCE /PHYLOGENY INFORMATION
A membrane protein
Proton-transporting P-type ATPases
TMHMM
membrane span and topology prediction
M.jannaschii
T. acidophilum
M.thermoautotrophicum
M.thermoautotrophicum
Transporter Classification
TCDB
Milton Saier
(validated by IUBMB)
1. A. 1. 1.
First
(number
Second
)
:
(letter)
:
Third
Fourth
Fifth
1
Mechanism class
Mechanism subclass
Phylogenetic superfamily/family
Phylogenetic subfamily
Substrate(s)
Mechanisms
1. Channels/Pores
2. Secondary Transporters
3. Primary Active Transporters
9. Incompletely Characterized Transport Systems
Superfamilies/Families
2.A. uniporters, symporters, antiporters
2.A.1 The
2.A.2 The
Family
2.A.3 The
2.A.4 The
2.A.5 The
2.A.6 The
Major Facilitator Superfamily (MFS)
Glycoside/Pentoside/Hexuronide (GPH): Cation Symporter
Amino Acid-Polyamine-Organocation (APC) Superfamily
Cation Diffusion Facilitator (CDF) Family
Zinc (Zn2+)-Iron (Fe2+) Permease (ZIP) Family
Resistance-Nodulation-Cell Division (RND) Superfamily
…
2.A.73
2.A.74
2.A.75
2.A.76
The
The
The
The
Inorganic Carbon (HCO3-) Transporter (ICT) Family
4 TMS Multidrug Endosomal Transporter (MET) Family
L-Lysine Exporter (LysE) Family
Resistance to Homoserine/Threonine (RhtB) Family
2.A.77 The Cadmium Resistance (CadD) Family
Subfamilies
2.A. uniporters, symporters, antiporters
2.A.1 Major Facilitator Superfamily (MFS)
2.A.1.1 The Sugar Porter (SP) Family
2.A.1.2 The Drug:H+ Antiporter-1 (12 Spanner) (DHA1) Family
2.A.1.3 The Drug:H+ Antiporter-2 (14 Spanner) (DHA2) Family
2.A.1.4 The Organophosphate:Pi Antiporter (OPA) Family
2.A.1.5 The Oligosaccharide:H+ Symporter (OHS) Family
2.A.1.6 The Metabolite:H+ Symporter (MHS) Family
2.A.1.7 The Fucose:H+ Symporter (FHS) Family
2.A.1.8 The Nitrate/Nitrite Porter (NNP) Family
2.A.1.9 The Phosphate:H+ Symporter (PHS) Family
2.A.1.10 The Nucleoside:H+ Symporter (NHS) Family
2.A.1.11 The Oxalate:Formate Antiporter (OFA) Family
2.A.1.12 The Sialate:H+ Symporter (SHS) Family
2.A.1.13 The Monocarboxylate Porter (MCP) Family
2.A.1.14 The Anion:Cation Symporter (ACS) Family
2.A.1.15 The Aromatic Acid:H+ Symporter (AAHS) Family
2.A.1.16 The Siderophore-Iron Transporter (SIT) Family
…
2.A.1.30 The Putative Abietane Diterpenoid Transporter (ADT) Family
Phylogenetic criteria
-Identify S.cerevisiae ORFs of TMS > 2
-Identify TC # using BLAST
-Apply the following general criteria:
-for 3th digit (Super)Families :
10% identity (E < 10
–20)
-for 4th digit Subfamilies:
20% identity (E < 10
–35)
-for 5th digit Clusters:
35% identity (E < 10-65)
• Explore Transport Protein Diseases.doc
YETI
YEAST TRANSPORTOSOME INFORMATION
BLAST QUERY OF P39980 S.cerevisiae UMP
FASTA
BLAST
P39980
YEH5_YEAST (YEL065W)HYPOTHETICAL 70.6 KDA PROTEIN
1195
0.0
P38731
YHE0_YEAST (YHL040C)HYPOTHETICAL 70.9 KDA PROTEIN
530
e-149
P38724
YHE7_YEAST (YHL047C)HYPOTHETICAL 71.6 KDA PROTEIN
431
e-119
P36173
YK86_YEAST (YKR106W)HYPOTHETICAL 69.0 KDA PROTEIN
393
e-108
P25596
YCH0_YEAST (YCL073C..)HYPOTHETICAL 68.9 KDA PROTEIN
392
e-108
Q08299
ENB1_CHROMOSOME XV READING FRAME ORF YOL158C
280
4e-74
Subfamily 2.A.1.16
The Siderophore-Iron Transporters (SIT)
Clusters
2.A.1.16.1 The ferrioxamine:
H+ symporter
Sit1/Arn3
(YEL065w)
S.cerevisiae
(spP39980)
2.A.1.16.2 The ferric
enterobactin:
H+ symporter
Enb1
(YOL158c)
S.cerevisiae
(pirS66857)
2.A.1.16.3 The ferric
triacetylfusarinine:
H+ symporter
Taf1
(YHL047c)
S.cerevisiae
(spP38724)
ABC TRANSPORTERS in HUMAN DISEASES
DEFECTIVE FUNCTION
CFTR
human
cMOAT
"
SUR
"
TAP1/2
"
ALD
"
PMP70,ALDR
"
Cystic Fibrosis
Dubin-Johnson Syndrome
Hyperinsulinemic glycemia of infancy
Multiple Sclerosis, Behcet's Syndrome
Adrenoleukodistrophy
Zellweger Syndrome
YCF1
YCF1
YCF1
MDL1/2
PXA1
PXA1
AMPLIFICATION
MDR1
MRP
pfMDR2
CaCDR1
EhPgp1
ldMDR
SMDR2
human
"
Plasmodium f.
Candida albicans
Entamoeba h.
Leishmania d.
Schistosoma m.
Drug resistant cancers
Drug resistant cancers
Drug resistant malaria
Drug resistant candidosis
Drug resistant amoebiasis
Drug resistant Kala azar
Drug resistant schistosomiasis
STE6
YCF1, YOR1
ATM1
PDR5, PDR10, PDR15
STE6
STE6
STE6
PLEIOTROPIC
DRUG TRANSPORTERS (PDR)
in
YEASTS
also called
Multiple Drug Resistance (MDR)
in
MAMMALIAN CELLS
The The
16 "full
16 "full
- size"
- size"
ABCABC
TRANSPORTERS
transporters
PDR5 - like
Pdr1
Pdr1
Pdr10
2
Pdr10
2
Snq2
Snq2
Pdr15
Ynr07
Pdr15
Ynr07
00
Pdr5
Pdr5
Pdr11
Pdr11
MRP - like
Yor1
Yor1
Ykr103/
Ykr103/
4
4
Yhl035
Yhl035
Bat1
Bat1
Yor011
Yor011
Bpt1
Bpt1
Ycf1
Ycf1
Yol075
Yol075
NEW
(NBF-TM)2
(TM-NBF)2
MDR - like
Ste6
The 16 "full - size" ABC
PDR5 - like
MRP
MRP -- like
like
Pdr12
Pdr1
Pdr10
Pdr10
Snq2
Snq2 2
Yor1
Yor1
Ykr103/4
Ykr103/4
Pdr15
Pdr15
Pdr5
Pdr5
Ynr070
Ynr070
Pdr11
Pdr11
Yhl035
Yhl035
Bat1
Bat1
Yor011
Yor011
Bpt1
Bpt1
Ycf1
Ycf1
Yol075
Yol075
MDR - like
NEW
(NBF-TM)2
(TM-NBF)2
Ste6
MULTIPLE disruptions
SINGLE disruptions
NO disruptions yet
THE YEAST ABC TRANSPORTER PDR5 MEDIATES RESISTANCE
TO MANY DIFFERENT COMPOUNDS
Anticancer drugs Fluorescent dyes
chemosensitizers rhodamine 6G
tamoxifen
daunorubicin
doxorubicin
trifluoperazine
Antibiotics
chloramphenicol
tetracycline
anisomycin
antimycin A1
cycloheximide
lincomycin
oligomycin
tunicamycin
thiolutin
PDR5
rhodamine 123
Detergents
tetradecyltrimethyl-ammonium bromide
hexadecyltrimethyl-ammoniumbromide
zwittergent 3-10
zwittergent 3-12
zwittergent 3-14
brij 58
brij 35
n octylglucoside
n dodecylglucoside
n dodecylmaltoside
 PDR5
Herbicides
sulfomethuron methyl
fluometuron
chloroxuron
difenoxuron
chlorbromuron
diuron
Steroids and their
structural
analogues
5 androstan 3 ol 17 one
5 androstan 3 ol 17 one
5 androstan 17 ol 3 one
5 androstane 3,17 dione
5 androstane 3,17 dione
1,4 androstadiene 3,17 dione
Ionophores
5 pregnan 3 ol 20 one
valinomycin
4 pregnen 20 ol 3 one
nigericin
trans dehydroandrosterone
monensin
4 androstene 3,17 dione
A23187
epiandrosterone acetate
epiandrosterone
Others
estriol 16, 17 diacetate
2,3,5 triphenyltetrazoliumchloride
17 ethynyl estradiol
4 nitroquinoline n oxide
deoxycorticosterone
8 hydroxyquinoline
pregnenolone
diphenylamine
progesterone
p aminodiphenylamine
testosterone
n decylamine
diethylstilbestrol
4 nitrophenol
hexestrol
aniline
4 hydroxytamoxifen
anthron
tetranitrotetrazoliumchloride blue
resazurine
sporidesmin
staurosporine
fluphenazine
cerulenin
compactin
Fungicides
itraconazole
ketoconazole
econazole
miconazole
triadimenol
bitertanol
nuarimol
fenarimol
carbendazim
diclobutrazol
phenapronil
chlorothalonil
cyprodinil
dodine
imazalil
prochloraz
azoxystrobin
flusilazol
tebuconazole
CGA64251
2 mercapto-benzothiazole
krezoxim methyl
nystatin
soraphen A
epoxiconazole
Kolaczkowski et al., 1997
Pdrp are activated
in antifungal-resistant
human and plant pathogens such as
Candida albicans ,
Candida glabrata,
Candida krusei,
Cryptococcus neoformans
and others ??
MIYAKAMA et al, 2005
ECL3
Balzi et al., JBC (1994)
ECL6
The catalytic Lys from NH2 Walker A is
substituted by Cys in Pdr sensu stricto
Linker region
Walker A
Pdr5-NH2
GRPGSGCTT
Pdr5-COOH
GASGAGKTT
*
* * **
Walker B
Fig. 1
TMD1
TMD2
EL3
NBD1
EL1
1
3
4
IL1
N
Walker A1
(GX4GC(S/T))
Helical
domain
P-loop Q-loop
(E)
EL4
EL2
2
EL6
NBD2
5
6
7
IL2
ABC 1 Walker B1
(VSGGE) (X3WD)
C-loop pro-loop D-loopH-loop
(X)
(D)
(Y)
8
EL5
9
IL3
Walker A2
Helical
(GX2GXGK(S/T)) domain
P-loop
Q-loop
(Q)
10
11
12
IL4
ABC 2 Walker B2
(LNVEQ) (hhhhD)
C-loop pro-loop D-loop H-loop
(P)
(E)
(H)
C
Pdr Transporters
only in fungi, plants and protists
• Sensu stricto Pdr (Pdr5/10/12/15/18, Snq2)





Reverse topology
Cysteine in N-terminal Walker A
Expanded ECL3 and ECL6
Efflux pump
Honorary Pdr (Aus1, Pdr11, YOL075)


Reverse topology
Influx pump?
Snq2
Sensu stricto Pdrp in
SACE
YNR070w (Pdr18)
Pdr12
Pdr10
Pdr5
Pdr15
COMPARATOMICS
Hemiascomycetes
Saccharomyces cerevisiae
Dujon et al.
2.A.5.Y1
Golgi
Mn
YALI-0D19008g
KLLA-0F17886g
DEHA-0E06105g
2.A.5.Y2
no data
no data
KLLA-0A07601g
SACE-YKE4
SACE-ATX2
CAGL-0K05577g
YALI-0F15411g
YALI-0E00748g
YALI-0D00759g
DEHA-0B16335g
DEHA-0E25388g
SACE-ZRT2
SACE-ZRT1
CAGL-0M04301g
YALI-0F21659g
2.A.5.1
KLLA-0D16434g DEHA-0B07337g
CAGL-0E01353g
plasma membrane
Zn
The Yeast ZIP Family (ZINC Iron Porters). TC # 2.A.5.
2.A.5.2
ER
Zn
CONCLUSIONS
REDONDANT TRANSPORTERS ARE LOCATED IN DIFFERENT MEMBRANES
SOME TRANSPORTER SUBFAMILLIES HAVE NO SACE MEMBERS
In practice – the most variable families
YALI
DEHA
KLLA
CAGL
SACE
Mean
Variance
Subfamily
2.A.1.1
Sugar Porter (SP)
27
48
20
17
34
29.2
153.7
2.A.1.14
Anion Cation Symporter (ACS)
39
27
13
6
10
19.0
187.5
2.A.1.2
Drug Proton Antiporter 1 (DHA-1)
33
24
8
10
12
17.4
114.8
9.A.5.1
Peroxisomal Protein Importer (PPI)
27
7
10
11
10
13.0
63.5
2.A.67.1
Oligopeptide Transporter (OPT)
17
4
3
0
2
5.2
45.7
2.A.1.16
Ferrioxamine H+ symporter (SIT)
14
5
4
1
6
6.0
23.5
2.A.1.13
Fructose uniporter (FRU)
5
8
12
3
0
5.6
21.3
9.B.17.1
The Putative Fatty Acid Transporter (FAT-1)
14
3
3
5
5
6.0
21.0
3.D.1.2
NADH Dehydrogenase I (NDH 1)
8
8
0
0
0
3.2
19.2
2.A.3.10
AminoAcid-Polyamine-Organocation Yeast
Transporter( APC-YAT )
14
24
16
14
18
17.2
17.2
1.A.20.5
Yeast Metal Channel ( Cyt B-FRE )
11
7
5
1
7
6.2
13.2
BLAST of Snq2 in SACE and KLLA
ScSnq2
ScYNR070
KlSnq2a
ScPdr12
KlSnq2b
ScPdr5
KlSnq2c
ScPdr15
ScPdr10
KlSnq2d
BLAST E-value is not discriminating
BLAST score distinguishes four Snq2 homologs in KLLA
KlSnq2a clusters with ScSnq2
KlSnq2b clusters with ScPdr12
KlSnq2 c and d cluster with Sc Pdr5/10/15
Neighbors analysis
Pdr5/15 filiation
SACE
YOR150
YOR151
PDR5
YOR155
YOR156
SACE
YDR400
YDR402
YDR403
YDR404
YDR405
PDR15
YDR407
YDR408
YDR409
YDR410
YDR411
CAGL
CAGL0F02585g
CAGL0F02607g
CAGL0F02651g
CAGL0F02673g
CAGL0F02695g
CAGL0F02717g
CAGL0F02739g
CAGL0F2761g
CAGL0F02783g
CAGL0F02805g
CAGL0F02827g
RO5530
RO5529
RO5513
RO5509
RO5508
ZYRO
RO5533
RO5532
RO5523
RO5505
Ustilaginom.
Basidiomycetae
Tremellom.
Agaricom.
Taphrinomycotina
P
P
P
Homob.
P
Ascomycetae.
P
Saccharomycotina
P
P
P
P
P
Onygenales
P
P
P
P
Dothideomycetes
Pezizomycotina
P
P
P
P
PP
Eurotiomycetes
P
P
(P)
P
P
P
P
Leotiomycetes
P
Sordariamyces.
P
P
P
P
PHYLOGENETIC ANALYSIS
OF 78 PDR
(SENSU STRICTO AND HONORAY)
FROM 11SPECIES
SACHARO/PEZIZO/BASIDIO
PHYLOGENETIC CLUSTERS OF FUNGAL PDRs
CONCLUSIONS
•
A total of 8 clusters (A to H)
Three clusters contain only SACE members (A,D,E)
Two honorary clusters are conserved (E,F)
Four new clusters not present in SACE(B,C,G,H)
The four new clusters are sensu stricto Pdrp (see Cys in Walker A1)
Are the new clusters drug efflux pumps? Which specificity?
PHYLOGENETIC ANALYSIS of
349 Pdr from 56 species
distributed in 5 subphyla
C
B
Fig. 3
A
PDR5
E
AUS1
H1
F
YOL075
H2
G
-
D
SNQ2
Conclusion
Only one more cluster H1/H2 that
belongs to sensu stricto PDR
STRUCTURE PREDICTOMICS
CONCLUSION
1.Combined to analysis of mutants,the elucidated sequence
motifs and the prediced topology will become important for
interpretation of crystallographic data and for elucidation of the
catalytic mechanism of the efflux pumps including the amazing
pleiotropic specificity for substrates and the asymetry of the
two NBD.
2.This infornation must be taken into account to understand
the molecular evolution of this important protein family (>500
millions years?)
3.This molecular information should facilitate the design of
more sensitive antifungals.
COLLABORATORS
OTAGO
•
•
•
•
MONK brian
CANNON richard
NIMMI kyoko
LAMPING erwin
LLN/GENA
•
•
•
•
BARET philippe
DE HERTOG benoit
DIFFELS julie
SERET marie line
LLN/FYSA
• BALZI elizabetta
• KOLASCZKOWSKI marcin
SAN DIEGO
• SAIER milton
ACKNOWLEDGEMENTS
BIBLIOGRAPHY REVUES PDR
Monk B.and Goffeau A. 2008,
SCIENCE
321:367-369
Richard Cannon et al. 2009,
CLINICAL MICROBIOLOGY REVIEWS
22:291-321
Phylogeny of human fungal pathogens
Coccidioides immitis
Histoplasma capsulatum
Aspergillus nidulans
Aspergillus fumigatus
Aspergillus oryzae
Coccidoides-Histoplasma Cluster
Aspergillus Cluster
Aspergillus terreus
Candida lusitaniae
Candida guillermondii
Candida parapsilosis
Candida tropicalis
Candida dubliniensis
Candida albicans
GTG
Candida glabrata
Saccharomyces cerevisiae
WG
D
Pneumocystis Cluster
Cryptococcus neoformans
Cryptococcus Cluster
Rhizopus oryzae
Homo sapiens
800
600
400
200
Millions of years
0
Saccharomyces Cluster
Pneumocystis carenii
Chytridium comfervae
1000
Candida Cluster
CHYTRIDS
ZYGOMYCETES
Snq2p in Hemiascomycetes
Z. rouxii
RO1382
K. thermotolerans
TH11558
RO1385
TH11559
RO1386
RO1387
RO1392
TH11563
TH11564
TH11568
WA12062
WA12069
K. waltii
K. lactis
WA12916
WA12917
KLLA0D03542g
KLLA0D03520g
KLLA0D03498g
WA12920
WA12921
WA12924
KLLA0D03476g
KLLA0D03454g
KLLA0D03432g
RO1393
RO1395
TH11579
TH11581