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Fiat Lux Freshman Seminars

by: Chelsea Cremin III

Fiat Lux Freshman Seminars PHYSIOL 19

Chelsea Cremin III
GPA 3.89


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Class Notes
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This 38 page Class Notes was uploaded by Chelsea Cremin III on Friday September 4, 2015. The Class Notes belongs to PHYSIOL 19 at University of California - Los Angeles taught by Staff in Fall. Since its upload, it has received 107 views. For similar materials see /class/177848/physiol-19-university-of-california-los-angeles in Physiology at University of California - Los Angeles.


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Date Created: 09/04/15
n Acefylserinefhiollyase Gene Family Demonstrates Compartment Specific Differences in the Re ula rion of C sfeine S n rhesis Heeg C Kruse C Josl k Gulensolm NL kupperl T Wirlz NL and Hell I 2008 Plant Cell 20 168185 netyICnA D acelylserme Presen led by Stephanie DLI Tao Wang Jingwei thmg CHEM 161A Spring 2008 vs rei e Cvs gt Pr o reinogenic amino acid Indispensable for39 any living cell gt Animal cells I Tnkp Ilh Cvlt Fr39nm rlia39l39 Syn rhesize from Me r via rr39anssulfur39yla rion of cys ra rhione in cy rosol hesize Cys in cy rosol from sulfide amp 0 ace rylhomoser39ine by rr39anssulfur39yla rion vs Biosvn39rhe is in Plants gt Independen r pr o rein biosyn39rhesis In 3 compar rmen rs D Cy rosol u Plas rids u Mi rochondr39ia gt Cys can be syn rhesized in each compar rmen r gt Tr ipar fi re process Salute 39 Buf 97 4797495 quotosyn l39hesis pa rhway enzymes 1 HscoA I w M 5 H 6 0 sul de cysmne natal amch o acemsume vs Biosvn rhejs T ipar ri 6 Process Con r39d A cytosol 5 05 OSSImIIg ror y sulfa re seAcCDAOAS 3339 Cys Y CdLICTIOn 6 quotOASTI 9 s l 39cys OAS Diffusion of H25 Through membrane JacqueA5 1936 Thekmencsafpenehmmn xn Hydragermu de J Gen mm 19 39pm sz mm H2 x 12mm Dammmmmw mame reveak he ragmmmyfunc mnaf hemummenccy eme mm pra zm camplg m han92mc7gb9ma mm 52H 197 5257639 vme Dis rr39ibu rion from Previous S rudies uske CR Hill KK Guzman E and Jackson PJ 1996 Subcellular location of Uacefylser39ine sulfhydr ylase isoenzymes in Compartmen rcdion of Cys Synthesis 183 cell cultures and plant Tissues of Dq l ur a innoxia Mill qu nf Physiol 112 659667 Ruf l erl39LIVLLw Lebrun M Droux Mw and Douc I 1995 Subcellular disfr39ibu riien o f Serine ce ryl rr nsfer39aSe fnom Pisum sa rivum and Ehur aei39er quoti on quot quot 39 39 39 39 quot39 quot 39 r quot isofxar39m Eur Bi a ohem quot39 500 u 239 J I A TL ike and 0A TL Pr39o reins gt OASTLIike pr39o reins 5ubs ri ru red Ala syn rhases and belong To The Super39family of pyr39idoxalphospha recon raining enzyme gt OASTL and SAT pr39o reins gt MOST s rr39ongly Transcribed genes n OASTLAJ OASTLA cy rosol n 0A5TLBplas rids OASTLCQmITocnondr39Ia U CYSCJ Hafzfeld Y Maruyama A Schmid r A Noji M Ishizawa K and Sai ro K 2000 b Cyanoalanine synfhase is a mi rochondr ial cys reine svnfhaseIike pr o rein in spinach and Arabidopsis Plan r Physiol 123 W 1172 Hell R Bork C Bogdanova N Frolov I and Hauschild R 1994 Isolafion and characfer izafion of Two cDNAs encoding for comparfmen r Specific isofor39ms of Oace rylser ine Thiol Iyase from Arabidopsis Thaliana FEBS Le 39 351 257 262 Wir39139z M Dr oux M and Hell R 2004 O Ace139yser39inehioyase An enigmafic enzyme of plan r cysfeine biosynfhesis revisi l ed in Arabiidopsis rhali ana J Exp Bot 55 1785 1798 q sulfur s ra rus u Regula ring SAT ac rivi ry gt Regula rion of CSC SAT 0A5 s inactive inactin uc w OASTL L serine AceWICaA aTionale and Siani ican e of The STud gt Few sTudies on cellular coor39dinaTion of Cys hesis among The Three comparTmenTs gt Why Cys biosynThesis is organized in This way or39 JI J2 IJ JI JI III locaTions is unexplained gt TDNA inser39Tion lines eyme acTiviTies and S conTaining meTaboliTes A ma Ajax oAsrLA w 1 3 397 D I I I L55 35m agev 7 H A OASTLB an O J igt v1 9 439L575ALK may QM mi OASTLC 1 T DNA in each MSTgene in appropria re muTan r k T DNA allele was homozygous gt Lines aas39TA amp aas39rB crossed To give homozygous double MB inserTion line Termed as 0057 Loss of mRNA of The Three oasTI genes 7 WA WE w k Confirma rion of compleTe gene ththwt knockouT gt AcTin as posiTive confrol T DNA TDNA i nser39Tions in oasf genes pr39even r de rec rable syn rhesis of encoded pr39o reins gt Cor39r39ela rions of band in rensi ries wi rh r39ela rive Immunoblo r In immunoblots the polyclonal antibody raised against OAS TL C was able to detect OAS TL isoforms A and B because A all ofthese isoforms are encoded by one gene B the antibody cross reacts with OASTL A and B C OASTLAand Bcarry substitutions that make them identical to OASTL C Fresh Weight g E z E a a v 2 u Tim 9 weeks We of oasfInser rion Lines Aerial par rs gt Roo r sys rems n Dnn39l39 Iann l h Dr39y Weigh r mg 395 5 a D m D n Branching pa r rer n gt Dr39 weivh r m D h D E E E E Equot r N a a Mutants and Phenotype Ready quantitative evaluation of contribution from different OASTL proteins in the three compartments in different strains Protein activity Macroview Mutant loss of protein activities Metabolite concentration in different Microview compartment Reconstruct different 1 contributions His tagged SAT immobilized Affinity for OASTL Elution with OAS The identitiyofOASTL A B and C were confirmed by identification of MALDI TOF sequencing of trypsine digested fragments 7 Didn39t discover post translationalmodification MALDITOF a irr l T dta utnr HEM free It giutn vnllag Q 3 matrix and nmml ym Protein activity Macro view ty 3 o 6 nmol m 1 protein 9 m l T 392 O s A cyt 5 quot 5 39ed t39 quot Normal Size loading crude extract B Plastids 54 reduct39 n C Mitochondria No signi cant reduction AB double mutant 95 reduction Raduced Size Protein activity Microview Total activity of cystein synthesis OASTL related chemical transformation specific activity of OASTLA specific activity of OASTLB specific activity of OASTLC other protein activities mm ASTLB conc 39w In immunoblots the polyclonal antibody raised against OAS TL C was able to detect OAS TL isoforms A and B because A all ofthese isoforms are encoded by one gene B the antibody cross reacts with OASTL A and B C OASTLAand Bcarry substitutions that make them identical to OASTL C Abundance of Individual I solor ms and POStt r anslational pH 5 pH 3 Why 2D gel MALDlTOF data u Contaminants g c c c m B E 39 m a I l I I a I If H c H a Expectation 039 a El Bi 3 i i Within one gel DEA A l 39 1 of Peaks 1 J i 2 Absolute and Relative quota DEA A intensity of Peaks E 2 c c D 3 Identity ofthe peaks LLl l g OAS TL Isoforms or other 3 I I I Til contaminant proteins 5i m 3931 B E El E D I32ll 4quot Between different mutants Change in relative protein expresSIon level H Observation of 2D gel Posttranslational modification MS No compensate effect of protein expression for different isoforms Very low amount of contaminants gtjustify the omission of the other protein activities Correlate with isoform mRNA level Other OASTL like protein mRNA Ievel FT compensation effect Contaminants within elution Microview reconstruction successful TLlike genes Cy526 CysCl CSlike L515 cysm CSJVKE use between Expresswn plum ytic and Interaction Domains of OASTLLike Proteins l Amino acid sequence alignment with Ecoli CysK CysM proteins B8a39a loop necessary for interaction with SAT absent in C526 plastid substantial changes in CysC1 mitochondria and CSIike cytosolic BSaBga loop Posttranslational modification has little effect on protein activity in vitrogt structural stability Further Mass Spec No matching post translational modification Coomassie blue stained gt sensitivity Posttranslational modification probably compensate within each isoform needs further in vitro characterization In vivo different PTM have different interaction with SAT Role of interaction with SAT Scontaining Metabolites steady state concentrations WT mutant differences wildtype sulfate gtgt GSH gtgt Cys OAS gt gamaglutamylcysteine gamaEC cysteinylglycine Significant Changes in Steady State Oacetylserine nmol g FW O U I11 3911 GdNUhmtnathmD oasth oastlAB Same phenotype Different micro level details E 0H Ev 239s 3 QE 5 yGlutamylcysteine nmol 9391 FW Glulalhlone nmol 9391 FW Cysteinylglycine nmol g FW Could it be limitation by mu No 4 O H O N O A C 97 d 2 gt 39o 72 o 10 u o E d U 0 n 0 Carbon Nitrogen Sulfur Back to Big Picture Upstr eam I Cys convergence ofC N S I Titration against SAT protein concentration Nww UIDU39I OOD SAT activity 5 2 o x n is E 3 E 5 0ASTLSAT ratio xfo Id Titration against Cys conc E I 2 I E 80 H Against the Dogma a 3 5 Mitochondria has 3 4 higherCys conc 5 20 o OASTL Leaves Mitochondria Due to the importance of OAS TL s interaction with SAT in determining its activity the Cys synthesis is not monoplied by OAS TL co centration alone but rather an interplay between V 2 conflicts within Complex Story I Phenotype Vs OASTL activity Metabolite conc Vs OASTL protein level Cannot be weaved smooth by taking OASTL alone into account gt a grander picture of regulatory CirCUItH I Previously few studies regarding Cys transport between cytosol and cellular compartments plastas and mitochondria I Arabidopsis mutants obtained byT DNA insertion in the Oastl genes demonstrated the significance ofthe mitochondrial OASTL enzyme for proper growth Implications of Results The CSC regulatory model sulfate limitation p sulfate availability H2f OAS ll H25 5 OAS 391 x t O OASTL IA X r 39 l gang 0 SAT gm sulfate lransporl EgtSulfate transport and reduction and reduction wiru M and Hell R 2006 39 39 39 39 39 hinrhnmiral and regulatory properties Journal of Plant Physiology 163 273286 Efficient transport to explain the viability of Mitochondria m I n2 nc st Cys OAS 4 oastlB 2 oasth 5 oastlAB HS CIT a H S39 Growth impairment caused by sulfide inhibiting mitochondrial respiration Mitochondria t likely in the case st Cys OAS Transport of other reduced sulfur compounds GSH not as likely as Cys transport H25 transport requires further research HS CIT a H S39


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