Terecskei, Kata

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Terecskei Kata

University of Szeged

V o l u m e 5 3 ( 1 ) : 5 5 - 7 6 , 2 0 0 9 A c t a B i o l o g i c a S z e g e d i e n s i s http://www.sci.u-szeged.hu/ABS We tried to ident i fy the sumoyla t ion site(s) in Prod by disrupting each potential site one by one, and expressing the HA-tagged mutant pro te ins in t ransfec ted S 2 cells. Next w e immunoprec ip i ta ted the mutant proteins with ant i -HA antibody, and tested their molecular weight and s u m o y l a t i o n o n Western blots. In o n e of the mutan t proteins the h igh molecular weight sumoyla ted band seemd to disappear , and the S 2 ce l l s express ing this protein showed an altered Prod ch romosomal immunosta ining pattern. Th is indicates that the Prod protein is sumoyla ted at the 123rd lysine amirioacid. I F ^ O 1 - 1 T I 'M« ' I V J •> I 'L J 1 " Török T, Benitez C. Takács S. Biessmann H (2007) The protein encoded by the gene proliferation disrupter (prod) is associated with the telomeric retrotransposon array in Drosophila melanogaster. Chromosoma 116(2): 185-195. Takács S. Szülik Á. Török T. The role of sumoylation on Drosophila telomeres, (manuscript in preparation). Supervisor: Tibor Török E-mail: takacssanyi@gmail.com . ': I . i • ' , . ) • ( , . » I « . " , : . ; I . . J . ' -S . . I t l l j f J * . The role of the small GTPase LIP1 in the function of the plant circadian network Kata Terecskei Institute of Plant Biology, Biological Research Center, Szeged, Hungary , \ . ret • * •• i ' I The c i rcad ian clock is a biological t iming mechanism that provides rhythmicity to gene expression, metabol ism, and physiology with ~24h periodici ty. T h e central osci l la tor of eukaryot ic c locks is based on the network of c lock genes and proteins, which are interconnected by t ranscr ipt ional / t ransla t ional negat ive feed-back loops. Cur ren t mode l s of the plant c i rcadian clock postulate three inter locked feedback loops. A pair of single Myb-domain t ranscript ion factors . CIRCADIAN CLOCK ASSOCIATED I (CCAI) and LATE ELONGATED HYPOCOTYL (LHY). p lays central roles in two loops. In o n e loop. CCAI and LHY repress the express ion of the Pseudo-Response Regulator gene TIMING OF CAB EXPRESSION I (TOCI). TOCI c lo ses the first loop by induc ing CCAI and LHY t ranscription for the next cycle. In a second loop. PRR7 and PRR9. are induced by CCAI and LHY. CCAI and LHY are subsequent ly repressed by PRR7 and PRR9. In a third loop, GIGANTEA (Gl) and. possibly. PRR5 are posit ive regula tors of TOCI. Gl is negatively regulated by both CCAI/LHY and TOCI (McClung 2008). T h e lipl-l (light insensit ive period l ) mutant isolated f rom Ambidopsis thaliana d isplays novel circadian phenotypes . lipl-l w a s iso-lated as an ear ly-phase mutan t based on the expression pattern of CAB2:LUC c i rcadian output marker in constant darkness. In wi ld- type plants , per iod length shor tens with increasing light fluence rates and the phase of rhy thms can be shifted by light pulses administered to da rkadap ted plants. In lipl-l. per iod length is nearly insensitive to light intensity and larger phase shi f ts can be induced dur ing the subjec-tive night (Kevei et al 2007) . T h e first a im of o u r work was to de termine the molecular mechan i sm by which LIPl a f fec ts the plant circadian clock. Transcript levels of c lock genes were de te rmined by quanti tat ive real- t ime PCR in lip I mutants. Our data show that LIPl affects the expression of Gl. PRR9 and TOCI. T h e ef fec t on Gl express ion was supported by the analysis of gi-lipl double mutant plants. We genera ted p r o m o t e r : L U C + repor ter gene const ructs for each core clock genes in lipl mutant background and we could prove that the t ranscr ipt ion of all core c lock c o m p o n e n t s is a f fec ted by the mutat ion. Our second aim was to ident i fy how the funct ion of L I P l is controlled. LIPl is a plant-specif ic atypical small GTPase . Small GTPases are molecu la r swi tches shutt l ing be tween the G D P - b o u n d inactive and the GTP-bound act ive states. For this process they require down-s t ream s ignal ing e l emen t s (effectors) ^nd ups t ream signal ing e lements (e.g. GEFs) (Berken et al 2005). We found that LIPl interacts with a m e m b e r of the plant specif ic fami ly of R o p G E F s . R o p G E F 7 in yeast two-hybrid system. However, the insertion mutant allele of RopGEF7 showed no c i rcadian pheno type in planta . T h e fami ly of R o p G E F s consis ts of 14 members . We tested the circadian phenotype of insertion mu tan t s fo r all of them and found that a mutant allele of RopGEF2 has a ///»/-like circadian phenotype . lipl mutant plants show stress pheno type also, they are sensi t ive to salt. RopGEF2 mutant plants display a lipl-like salt phenotype . R o p G E F 2 might be the m e m b e r of the R o p G E F family which p romotes L I P l funct ion. Previous data showed that L I P l is localized in the cytosol . nucleus and in cell compar tments as well. We tested the funct ion of nuclear export signal ( N E S ) o r nuclear ¡ocalizqi'ibn signal (NLS) tagged Y F P - L I P l fusion proteins in lipl mutant background to see if any of the lipl p h e n o t y p e s cou ld be c o m p l e m e n t e d . Y F P - L I P l - N L S fus ion proteins could restore the circadian phenotype. Neither of the const ructs cou ld res tore the salt sensitivity phenotype . These data indicate that L I P l affects the circadian clock in the nucleus, but nucleo-cytosol ic shutt l ing is required to fulfill its role in tolerating salt stress. Berken A. Thomas C. Wittinghofer A (2005VA new family of RhoGEFs activates the Rop molecular switch in plants Nature 436:1176-1179. Kevei É. Gyula P. Fehér B. Tóth R. Vicziin A. Kircher S. Rea D. Doijgotov D. Schafer E, Millar A.J. Kozma-Bognár L. Nagy F (2007) Arabidopsis thaliana .Circadian Clock Is Regulated by the Small GTPase LIPl Current Biology 17:1456-1464. McClung CR (2008) Comes a time. Current Opinion in Plant Biology 11:514-520. Supervisors: László Kozma-Bognár and Ferenc Nagy E-mail: terecs@brc.hu 74 

The role of the small GTPase LIP1 in the function of the plant circadian network

http://acta.bibl.u-szeged.hu/23035/1/biologica_053_numb_001_074.pdf

The role of the small GTPase LIP1 in the function of the plant circadian network

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