Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 1983 Mitochondrial transport and processing of methylmalonyl coenzyme A mutase : in cultured buffalo rat liver cells David C. Helfgott Yale University Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Recommended Citation Helfgott, David C., "Mitochondrial transport and processing of methylmalonyl coenzyme A mutase : in cultured buffalo ar t liver cells" (1983). Yale Medicine Thesis Digital Library. 2702. http://elischolar.library.yale.edu/ymtdl/2702 This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. 3 9002 08676 2516 M '1'Cl A M C !) 0 DT ! MIluCHuNlliii/ ■iL (liAiUi Ufil 7 \ID ;B'ROGESSrf t ''■f!;!\/t f'- f\r ivrvi t f 1. m iWilMk luAiL ?lEi MliTASE JN; ' 1 it/CP pc? ! -P '(-•■'.' 1 . i U 11 L .. Il !1U! i' k\ U' I'lMl iJj'l'iS btLU.y Si. ....... -V ‘ •■'5' v IL? L ! 1 W. iv. \ EOt?. Permission for photocopying or microfilming of It (title of thesis) /e Co^n):^^n^ A /A Ci.>Lro>^a> CjOir^ CGlLt^J for the purpose of individual scholarly consultation or refer¬ ence is hereby granted by the author. This permission is not to be interpreted as affecting publication of this work, or otherwise placing it in the public domain, and the author re¬ serves all rights of ownership guaranteed under common law protection of unpublished manuscripts. (Signature of author) PaVI D hiCcf^GOJT (Printed name) (Date) Digitized by the Internet Archive in 2017 with funding from The National Endowment for the Humanities and the Arcadia Fund https://archive.org/details/mitochondrialtraOOhelf ■m m ,ir’ k4 f®' .1^4 ■'>^i '^^-■ i% K-?';n. ■ ■ r.t,T^ f" S M S4t LOi t.KJ 'i. Jr. #1^ 1 ^ T iy ff<*- ijk « <■ ,\ *> ' f- ■ '■ - "A. t- iv,,■;;■•: '■_is ■If 11 MITOCHONDRIAL TRANSPORT AND PROCESSING OF METHYLMALONYL COENZYME A MUTASE IN CULTURED BUFFALO RAT LIVER CELLS David C. Helfgott B.A., University of Pennsylvania, 1978 A thesis Submitted to the Faculty of the Yale University School of Medicine in Partial Fulfillment of the Requirements for the degree of Doctor of Medicine 1983 Med. uk> -TII3 -i'/iO. 5^^ For my parents, with love and appreciation i..v, aiToi iiJJft ,AJa9\Jiq xm no! ; ACKNOWLEDGEMENTS Since I began working on this project in the summer between my first and second years of medical school, many people have contributed to its success and to the enjoyment I have derived from it. I especially want to express my sincere thanks to Dr. Leon Rosenberg; he has been supportive throughout these last three years and was very patient with my slow progress during the time-consuming clinical years. More importantly, he has been a great teacher, an example to follow, and most of all, a friend. I wish him all the best in the future. I am also grateful to Dr. Wayne Fenton, who guided me through this project and made the lab a much more pleasant and interesting place to work. Many others made it fun to keep coming back. Among them are Adele Hack, who also provided essential technical advice and assistance, Fleming, Jan, Frante, Michelle and Alda. This thesis rounds out a great education which I have received at Yale Medical School. I must express my appreciation and thanks to those people who have made these last four years enjoyable and completely worthwhile. In particular, I am deeply grateful to Dr. Vincent T. Andriole, not only for his positive influence and superb teaching, but also for just understanding. He is a great individual and a great clinician who has made an indelible impression on me. Thanks. 1 il r 2-j *, D "{ ' *T * t, ^ C ^ ^ '*** '^ ^ ' • vji!i)<*idw D*u»*J I 9caiZ • N ,.0'-f*'j.- .-''-I 'I a ‘ «J«i Jaiil ta B**wl»d ^^.'.*.7. 7'y '■ : « seairiiio Ji oi t><>^udliJiiOO tvad «Xqoiiq r.., -..V I ii •0‘tl b»vl-i*»ft •v*il I , H ^ .1 = i ,, ; y 7 •■ O .J ? ?. u li no?*.; . 1 0 0 ^ 8 Jl n »d .1 • n ir 0 9 i 8 ,, , . ., . •. .. , ->u7';. ^enl 7a<511 iMonjudtdi avJtinoqqoe ■ r' . a . = ^i..' ■’ ^ .;i T 1 y b j •> . t n 0 -j q w 0 i <1 t * d i tn • X 4 8 q . ^ ^ ^ 3 0 • . *'.■ • 2 ? if . V ! ‘ ^( ii j .] fl* 1 a '1. I n 1M • X i 81/ X n X X 0 [ r ..'7' ,'. i 1 lo i''■ c n i'ij* K .’iro' Aiqvsna •* ,*i<krt08*4 •di >1 4a«« tdi IXi Bid ii«X» ?r. r, :. , • .-V,.,'. if. oi 06 !• BA I JII .-'rr- .’.-u® . ■ •••.BC *5.7 8 JoatoT*? «l«i4 dgUO'idX v' iOv,' fti jfll48l>'i»4flX Mfcd .<n.a.n .■ )vy j» tbi'-r •'1»dJ<‘ xn*M -).'> '-'j's J.a I X« i 4.% r-i-f ?? *»*>£> ^v-.^nq aaia t ft Iff ,dogf^.0liftA •*ia- .roi . .-H-cji-i ,.-.>X .artiaail ,«os»i«Xiaa ban r 3 <*o jt.-j ^ ts'Mwoi nldT va 'jatn;: .Te>4? I looriaR a 6 alal 4f btvXBOBl :?^.»<i.» •7bi' >“w ii'io'>>y o'jgilJ q4 e>ln»dJ bn8 aoX^nXoBT'iq• nl .» £ iff'rf'i7-.^ •» ’V 4 * in *’00 tjft*- aXdayot^f* Biit-s: luol JmaI ,.*5 io •■•ioA .t j 171‘< jai,»' .,7<£ fjj Xy*i« .’8 >j xX^>*<»b ora 1 ,'i.*XwolX iufl (f’Jaaui boA »oasi.noJL avjt.flee'y lid tXoo Jon 8 bam l8«jblvlb«i Jntn^ a cX a'H .g/iAODuif48«t oaXfl .98 no noX»B9’iqat oidXXabnX a* abi;® afci ortif naXaXdllo .•inadl TABLE OF CONTENTS Acknowledgements.i Table of Contents.ii Abstract.1 Introduction.2 The Methylmalonic Acidemias.8 Transport Characteristics of Mitochondrial Proteins Synthesized in the Cytoplasm.25 Materials and Methods.45 Resul ts. 51 Discussion.66 Bibliography.75 ±L ^ • uetif - • • * .noiioMtoiJal Ssn« .- ♦» . ... .5iiJlttll 1^.- . ... ... , * . , .AO Jteduoititi 36 ... • • • • ■• ' t - % 1' , , ► . ,t<<l«'^goUd±tt ^ r. , . j ■ ♦ ► * » V <* r ., , . »■ ‘ 4 ‘ ' '' ■■!'?.•■•■ ABSTRACT Methy1malonyl coenzyme A mutase, a cytopi asm1ca 1ly synthesized mitochondrial enzyme, catalyzes the conversion of L-methy1ma 1ony1 coenzyme A (CoA) to succinyl CoA. Methylmalonic acidemia is an inborn error of metabolism which is characterized by derangements in the activity of this enzyme, secondary either to defects in the synthesis of its cofactor, adenosy1coba1 amin, or to defects in the structural integrity of the enzyme itself. The work presented here shows that, like many other previously studied cytoplasmically synthesized mitochondrial proteins, methylmalonyl CoA mutase is synthesized first as a precursor which is subsequently processed by the mitochondrion to the mature form. This maturation can be blocked by 2,4- dinitrophenol, an uncoupler of oxidative phosphorylation which deprives the mitochondrial inner membrane of energy. The conversion of precursor to mature methy1ma 1ony1 CoA mutase subunit takes place with a 6-9 minutes. In contrast to the few reports published for other proteins, the precursor form of mutase is not rapidly degraded if maturation is inhibited. The implications of these findings are discussed with respect to the biosynthesis of mitochondrial proteins made in the cytoplasm and with respect to the possible molecular defect in the mut° mutant group of methylmalonic acidemia. 1 1 'i o 5. ^ A iv^ssttoo i xoo X»*i Yrf4#M r o ^ ' ?>il '■ f .'jj.'TC; .9517?.^^ i n llfenO^OO j X fi b * »1 C 8»dy U’i# . .\ -j !' J. V n 1 J j J V -• ^' ^ d E 'C . 0 ^ i t ^ ^ 'i ^ ® ® “ ® nu.rlo''' n To*:‘!e nnv^J.t: fis if.laetioa ■\.;. '{3s'^:fr: J ^ J ft f' ’ .■ a t 3 b \icl b • J A i « - o • O if< W ,,_ . _ I ^:J.■^l.■2-L cJ ^mQJ ii ^ j j j ' I 3 ’. J iO , n .i 0 J a d 0 '- I ^ fi d u • u i 7 §^oo ti X r^,ii ' J 10 t#nti30UTJ« rijL' •<aori« ai»d l5D^fl#*»nq lai Iffl t'> . i • fcxM n x* x f ‘ ^ ^ r -i 1 I o-J Ti; tf ^.*<fTtI bor f«» n^aJuM hcO itno ^.nJ .1'-’1 a. 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