EXPRESSION OF A PARAMECIUM
PROTEIN IN TETRAHYMENA
THERMOPHILA :
ND6P INVOLVED IN EXOCYTOSIS
Delphine Gogendeau, Rachel Lescasse, Anne-Marie Keller
Jean Cohen and France Koll
Centre de Génétique moléculaire,
Gif sur Yvette
THE REGULATED SECRETORY PATHWAY OF
TRICHOCYSTS IN PARAMECIUM
Nd6
WT
Nd6
THE ND6p PROTEIN
1
1925
955
ND6p
ND6p
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
15 82 -P
188 239 294 347 404 522 598 651 701 775 846 909 -
FARLAAG-VNHCLAIQ SE--------GD(9) L WGW GSNEF QALGKPESIDAEIQ--------------------PSPV
LQVACG-NQHS M VLACS Q D--(31aa) -I --- FSSG-SDEFGQ QGR MT DKE QSQ ------------------------ 153
VC QISC G-ANYTLALDIY--------GSV--- YSW G-EGATGCLGLP---------------------MQVD Q Q R
PTRI
M KFISAGPSHAASISEG Q--------GSL--- YTW G-VGTYGQLGH G---------------------INKSSNV
PM SV
VEYVSC G-AMHTACTTTD--------GYA---H TFG-FNKNGQLGTG---------------------DFND R W E
PVKV
YVYAKC G-GNTTFITTLER---------LT--FAFG-SNSKGRLGIK-------------------DEFGS N Y
PEPQ RI
IY QLALS-YHY GLALLQS--------GSI---I SFG-LPFK GILGR D----------------------LQAELL
PKNY
IVDVKC G-AF HTVALTDA--------GEV--- WVW G-SNKC M Q HGLKDE QIIEQVKTRTKFIGPFIQETIAETSYPS QV
VTYLATG-LEY VVVVEN R R---------N
I--- YSW G-KNDKGQLGLG--------------------FVSDFVET
PS ML
VK QVSC G-EDHTLI LDE G--------GSL--- YAVG-SPVH GKLGLG----------------PKNSVVLQT Q KV
PFIF
VAKVAVG-PHHS M ALVIDK-(23aa)--V
I--- YTW G-NGY GGKLGH G--------------------NLDN QIYY
PKLL
FKDVSAG-TN HS GALTIDD---------Q
I---VV W G-VGS -YLGIAKPED DTDDK Q HLKSDKD QEAE------- PYYI
YKSLCLG-DRYN M AISK Q N---------KA--FVW G-LFDYDF QEKLKN Q CA NAA Q REKYVEVLMT----NIVNTPLCD
FTTVSCS-FN HAVALTEN Q---------D
VKELFSW GYD G M TGRLGLG-----------------------YEFIN
PDKE
G ----
RCC1
I
II
III
IV
V
VI
VII
VIII
G ----
F G------
LG
--IVLTL
G-QGDVGQLG-------------------------MERKE
VV QVEAG-GMHTVCLSVS-----------GQ VYSEG-C N DEGALG--------------------------SE MV
VV QVSAG-DSHTAALTDD-----------GRVFLW G----------------------------------SKKS M
VVKVASG-NDHLV MLTAD D-------------LYT
LG-CGE QGQLGLE------------------------RLLV
FQ D AEC G-AYFTFALSHE-----------GHVYGFG-LSNYH QLGES-------------------------CFI
W V G FS
C G-QHHTVC M DSE-----------GKAYSLG-RAEYGPLGEE-------------------------KSI
VSSVAC G-ASVGYAVTKD-----------GRVFAW G-MGTNY QLGED-------------------------A WS
VLSVSSG-GQHT MLLVKD
P-F
PALV
PG KV
PV QV
PKCV
PQ NL
PLI S
PVE M
69
132
184
338
193
448
587
643
700
769
831
906
955
THE ND6p PROTEIN CONTAINS TWO
RCC1-LIKE DOMAINS
955
1
1925
ND6p
1
421
RCC1
The RCC1 protein:
-
Structure in -propeller
RCC1
Conserved from yeast to human
Involved in nucleo-cytoplasmic transport
GTP exchange factor for the small GTPase ran
The RCC1 domains are known to be domains of protein-protein
interactions
THE MUTATIONS IN ND6p ARE IN MAJORITY
FOUND IN THE RCC1 DOMAINS
-
Sequencing of the 12 alleles of ND6
1
1925
THE MUTATIONS IN ND6p ARE IN MAJORITY
FOUND IN THE RCC1 DOMAINS
-
Sequencing of the 12 alleles of ND6
1
-
Nine mutants are touched in the RCC1 domains
1925
THE MUTATIONS IN ND6p ARE MAJORITARY
FOUND IN THE RCC1 DOMAINS
-
Sequencing of the 12 alleles of ND6
1925
1
-
Nine mutants are touched in the RCC1 domains
We tried to express this part of the ND6 protein
INTEREST OF PRODUCING THE RCC1 DOMAINS
OF ND6p
-
Raise antibodies.
-
Determine the precise cellular localization of ND6p.
-
Search for interacting partners.
-
Analyse the composition of proteic complexes
containing ND6p by non-denaturing approaches.
- Test the GEF activity.
-
Obtain the 3D structure of the RCC1 domains.
AVANTAGES OF TETRAHYMENA THERMOPHILA FOR
HETEROLOGOUS EXPRESSION
- Tetrahymena has the same divergent
genetic code as Paramecium (TAA and
TAG encode glutamine)
- Cultures with a high cellular density
(105-106 cells per milliliter)
- Stable transformation by biolistic
10 µm
- Strong, inducible promotor : pMTT
TAXOL SELECTION
(Gaertig et al, PNAS, 1994)
Cu522 strain :
K350M
5 BTU1
BTU1
BTU2
3 BTU1
The K350M mutation in the BTU1 gene
5 BTU1
3 BTU1
Disruption of the BTU1 gene
hypersensitivity to taxol
BTU2
loss of the taxol sensitivity
Introduction of linear DNA at the BTU1 locus
A STRONG AND INDUCIBLE PROMOTOR (Shang et al, PNAS,
2002)
The metallothioneines (MTT)
are small proteins fixing heavy
metals and playing a role in metal
homeostasis and in cadmium
detoxification
QuickTime et un
décompresseur TIFF (non compressé)
sont requis pour visionner cette image.
Their synthesis can be induced
by heavy metals like Zinc, Cu or
Cadmium.
The pMTT promotor is thus an
inducible promotor, allowing a
rapid production of a large among
of mRNA.
QuickTime et un
décompresseur TIFF (non compressé)
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BICH3
MTT-BICH3
Cadmium 5µg + + -
A STRONG AND INDUCIBLE PROMOTOR (Shang et al, PNAS,
2002)
The metallothioneines (MTT) are small proteins fixing heavy
metals and playing a role in metal homeostasis and in cadmium
detoxification
Their synthesis can be induced by heavy metals like Zinc,
Copper or Cadmium.
The pMTT promotor is an inducible promotor, allowing rapid
production of a large among of mRNA.
DETECTION AND PURIFICATION OF PROTEIN OF
INTEREST
TAP-tag (localization in C-terminus) Tandem Affinity Purification Tag
(Puig et al, Methods, 2001)
CBP Tag
protein A
NH2-
-COOH
site TEV
affinity for the
calmodulin
affinity for IgGs
site of cleavage
by the TEV
protease
CONSTRUCTION INTRODUCED IN TETRAHYMENA
1
1928
4875
5430
CBP Protein A
5 BTU1
pMTT
RCC1 part of ND6
1
5 BTU1
Homologous
recombination
1329
BTU1
Tap-Tag
3 BTU1
Selection of taxol resistant transformants
3 BTU1
200
4h
3h
2h
1h
0
MW
1h
45m
in
30m
in
15m
in
0
MW
THE EXPRESSION OF ND6p PROTEIN IS INDUCED BY
CADMIUM
200
116
116
92
92
One hour of induction with 2,5µg/µl CdCl2 is sufficient to have a
maximal expression of our protein
200
116
92
65
45
To
ta
lE
Pe xtr
lle act
t
20
µl
l
M
n
ta
a
n
er
p
Su
µ
t5
W
lE
xt
ra
ct
Pe
lle
t5
µl
To
ta
M
W
THE RCC1 PART OF ND6p IS SOLUBLE
t
na
r
pe
Su
an
µ
t5
l
ug
Flow through
ug
th
ro
ow
le
ct
Fl
W
M
IgG beads
T
a
ot
ra
xt
h
th
ro
x
ow
t
To
e
al
t
Fl
M
W
Total extract
c
tra
h
FIXATION OF THE RCC1 DOMAIN OF ND6p ON AN IgG
COLUMN
ELUTION BY THE TEV PROTEASE
RCC1 part of ND6
Tap-Tag
CBP Protein A
1
983
IgG bead
1168
TEV site
200
116
92
65
A very small quantity of protein is
obtained
45
The protein aggregates when we try
to concentrate it
CONCLUSION
A gene of Paramecium has been introduced in the Tetrahymena
genome, at the BTU1 locus.
The RCC1-part of ND6 is produced by the addition of Cadmium in
the culture, but not overexpressed .
The over-production in a heterologous system of expression may
depend on the protein so Tetrahymena remains a possible way to
produce native proteins