EXPRESSION OF A PARAMECIUM PROTEIN IN TETRAHYMENA THERMOPHILA : ND6P INVOLVED IN EXOCYTOSIS

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1 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

2 THE REGULATED SECRETORY PATHWAY OF TRICHOCYSTS IN PARAMECIUM WT Nd6 Nd6

3 THE ND6p PROTEIN ND6p ND6p I 15 - FARLAAG-VNHCLAIQSE GD(9) L WGW GSNEFQALGKPESIDAEIQ PSPV 69 II 82 -P LQVACG-NQHS MVLACSQD--(31aa) -I --- FSSG-SDEFGQ QGR MTDKEQSQ III VC QISC G-ANYTLALDIY GSV--- YSW G-EGATGCLGLP MQVD Q Q R PTRI 132 IV M KFISAGPSHAASISEG Q GSL--- YTW G-VGTYGQLGH G INKSSNV PM SV 184 V VI VEYVSC G-AMHTACTTTD GYA---H TFG-FNKNGQLGTG DFNDR W E YVYAKC G-GNTTFITTLER LT--- FAFG-SNSKGRLGIK DEFGSNY PVKV PEPQ RI VII IYQLALS-YHY GLALLQS GSI---I SFG-LPFK GILGRD LQAELL PKNY 448 VIII IVDVKC G-AF HTVALTDA GEV--- WVW G-SNKC M Q HGLKDE QIIEQVKTRTKFIGPFIQETIAETSYPS QV 587 IX VTYLATG-LEY VVVVENRR N I--- YSW G-KNDKGQLGLG FVSDFVET PS ML 643 X VKQVSC G-EDHTLI LDE G GSL--- YAVG-SPVHGKLGLG PKNSVVLQTQKV PFIF 700 XI VAKVAVG-PHHS MALVIDK-(23aa)--V I--- YTW G-NGYGGKLGH G NLDNQIYY PKLL 769 XII FKDVSAG-TN HSGALTIDD Q I---VV W G-VGS -YLGIAKPEDDTDDKQHLKSDKDQEAE PYYI 831 XIII YKSLCLG-DRYN M AISK Q N KA--- FVW G-LFDYDF QEKLKN Q CA NAA Q REKYVEVLMT----NIVNTPLCD 906 XIV FTTVSCS-FNHAVALTEN Q D VKELFSW GYD G MTGRLGLG YEFIN PDKE 955 G---- G ---- F G LG P-F RCC1 I --IVLTL G-QGDVGQLG MERKE PALV II VVQVEAG-GMHTVCLSVS GQVYSEG-CNDEGALG SEMV PGKV III VVQVSAG-DSHTAALTDD GRVFLW G SKKS M PVQV IV VVKVASG-NDHLVMLTADD LYT LG-CGEQGQLGLE RLLV PKCV V FQ DAEC G-AYFTFALSHE GHVYGFG-LSNYH QLGES CFI PQ NL VI W V GFSC G-QHHTVC M DSE GKAYSLG-RAEYGPLGEE KSI PLI S VII VSSVAC G-ASVGYAVTKD GRVFAW G-MGTNYQLGED AWS PVE M VIII VLSVSSG-GQHT MLLVKD

4 THE ND6p PROTEIN CONTAINS TWO RCC1-LIKE DOMAINS ND6p RCC1 The RCC1 protein: - Structure in -propeller - Conserved from yeast to human RCC1 - 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

5 THE MUTATIONS IN ND6p ARE IN MAJORITY FOUND IN THE RCC1 DOMAINS - Sequencing of the 12 alleles of ND

6 THE MUTATIONS IN ND6p ARE IN MAJORITY FOUND IN THE RCC1 DOMAINS - Sequencing of the 12 alleles of ND Nine mutants are touched in the RCC1 domains

7 THE MUTATIONS IN ND6p ARE MAJORITARY FOUND IN THE RCC1 DOMAINS - Sequencing of the 12 alleles of ND Nine mutants are touched in the RCC1 domains We tried to express this part of the ND6 protein

8 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.

9 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 ( cells per milliliter) - Stable transformation by biolistic 10 µm - Strong, inducible promotor : pmtt

10 TAXOL SELECTION (Gaertig et al, PNAS, 1994) Cu522 strain : K350M 5 BTU1 BTU1 3 BTU1 BTU2 The K350M mutation in the BTU1 gene hypersensitivity to taxol 5 BTU1 3 BTU1 BTU2 Disruption of the BTU1 gene loss of the taxol sensitivity Introduction of linear DNA at the BTU1 locus

11 QuickTime 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, Cu or Cadmium. The pmtt promotor is thus an inducible promotor, allowing a rapid production of a large among of mrna. et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. QuickTime et un décompresseur TIFF (non compressé) sont requis pour visionner cette image. BICH3 MTT-BICH3 Cadmium 5µg

12 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.

13 DETECTION AND PURIFICATION OF PROTEIN OF INTEREST TAP-tag (localization in C-terminus) Tandem Affinity Purification Tag (Puig et al, Methods, 2001) NH 2 - CBP Tag site TEV protein A -COOH affinity for the calmodulin affinity for IgGs site of cleavage by the TEV protease

14 CONSTRUCTION INTRODUCED IN TETRAHYMENA CBP Protein A 5 BTU1 pmtt RCC1 part of ND6 Tap-Tag 3 BTU Homologous recombination 5 BTU1 3 BTU1 BTU1 Selection of taxol resistant transformants

15 THE EXPRESSION OF ND6p PROTEIN IS INDUCED BY CADMIUM MW 0 15min 30min 45min 1h MW 0 1h 2h 3h 4h One hour of induction with 2,5µg/µl CdCl2 is sufficient to have a maximal expression of our protein

16 THE RCC1 PART OF ND6p IS SOLUBLE MW Total Extract Pellet 5µl Supernatant 5µl MW Total Extract Pellet 20µl Supernatant 5µl

17 FIXATION OF THE RCC1 DOMAIN OF ND6p ON AN IgG COLUMN Total extract MW Total extract Flow through MW Total extract Flow through IgG beads Flow through

18 ELUTION BY THE TEV PROTEASE RCC1 part of ND6 Tap-Tag CBP Protein A IgG bead TEV site A very small quantity of protein is obtained The protein aggregates when we try to concentrate it

19 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