Komduur, Janet

English

ARTS repository - University of Groningen

   University of GroningenMolecular aspects of peroxisome degradation in Hansenula polymorphaKomduur, JanetIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2004Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Komduur, J. (2004). Molecular aspects of peroxisome degradation in Hansenula polymorpha. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-2023S UMMARYFrom an evoludonary point of vierv, all organisms are crivic-lecl i 'tothree kingcloms: the kir-rgclom of archaea (u'iceilurar organisms), rhekingclonr of prokaryotes (rracteria), ancl the kingcro- ni .uk"ryores.Plants' animals, a'd fungi (a'c'r so yeasr as welr) belong to the rast ofthe three' Ma.y r1'pes of comprex biochemicar reacrions, which together form the lrasis of life, rake place within the cells of all organ_isms' To avoid clisturbances among the interactio's, a'cl to improvethe efficiency of the reactior-rs, eukaryotic cells are cliviclecl i'to com-paÍtments, the so-callecl orga'elles. The work described in the prese'tthesis focuses (xr two of these: the vacuole (or lysosome) ar-rd the per-oxisome.It is i ' rhe vacuole rhat the material taken fron-r outsicle the cell isbeirg degracleci. Ancl the materiar within the celr (incrucli 'g whole or_ganelles) is, w4ren necessarv, also cregracieci there. In this r,vay, the ceilobtains building blocks f.r growth ancl repair. To perform this furc_tion, a mechanism for'acuolar transportation of the enzymes ancr ma_terial to be clegradecl must exist. Such transportation must rre weil-regulated, so that the funcrional compo'ents of the vacu.le-enzymesthat actually perform the clegrac-lation work-arrive at their proper cres-tination, for they rnust be active only lvithin the vacuole, ancr not inother cellular compartments.The peroxisome is defined as an orga'elre in which enzymes rhatproduce ancl process hydrogen peroxicre (H.o.) reside. Due to theirsimultaneous presence within the peroxisome, the proclucecr H.o. isimmecliately used by another enzyme, ancl not spread rhroughout thecytoplasm' n'here it woulcl cause c{amage. Like vacuoles, peroxisomesalso have rransporrarion roures for the clelivery of the opi.op.iot. .,-,-zymes and substratcs.IZTS U M M A R YAlthough simple in builcl-essentially a bag with enzy'mes-, perox-isomes can function in a large variery of metabolic pathlvays. In man,they are invoivecl in, for instance, the B-oxiclation of the so-called verylong chain fatty acids (VLCFAs). The importance of their properfunctioning is probably best seen negatively, in the cliseases induced byrnalfunctioning peroxisomes, such as Zelhveger syrclrome ancl aclreno-leukodystrophy, which lead to physical deformation and mental retar-dation, usualfy resulting in early cleath. In the fungus Penicilliunt chry.so{aenum, peroxisomes are involvecl in the synthesis of BJactam (peni-cill in-related) antibiotics-a fact that makes the research into perox-isomes interesting for the industry. Another motive for the research liesin the funclamentai scientific curiosiry- as to the basis of regulation andtransportation processes within the cell.Besides for the biogenesis, the specific degraclation of peroxisomesis also irxportant for the proper functioning of the cell. In the presentthesis, research into the degraclation of peroxisomes, known as pexo-phagy, is clescribed. Superfluous peroxisomes have to be transportedto the vacuole for degradation. Keeping these organelles in circum-stances under which they are not necessary for growth would cost en-ergv that could otherwise be usecl for more important tasks in the cell.The clegradation process occurs also lvhen perorisomes are dam-aged-yet another important function, since peroxisomes with incom-plete membranes could spread the produced H.O. throughout thecell, causing damage and leading to its rapid ageing.To study peroxisome degradation, lve used Hansenula polvmor-plt*-a yeast species isolated from the flow fields of a cane sugar dis-tillery ir.r Brazil-as model organism. H. polymorpha is a ubicluitousyeast species, occurring naturally in spoiled orange juice, in the gut ofcertain insects, and in the soil. Its large peroxisomes can be inducedand degraded on demand, by varying the grou,.th circumstances of tlrecells, which makes these processes easy to observe. In H. Ttolvmorphe,IIIct:hoI l tbrrhl teofsl"lofpoatethedarfror\\rerlvrtlrtureotheterln(t ionturbet ion' tv',fryc-tr-r-ni-)x-iesndnesent)xo-:tedum-: en-cell.lam-:om-themoÍ'' d i s -,tousut ofucedf therpha,S U M M A R Yperoxisomes are inducecl when the cells grow ()n methanol-containingmeclia, under which conciitions the organelles contain the kcy cnz)'mcsfor methanol metabolism (alcohol oxiclase [AO], for instance). When,next, these cells are transferred to glucose- or ethanol-conteining me-dia, the peroxisomes, being no longer necessary for grou.th, are de-gracled. The process of pexophagy takes place as follows: first, therehas to occur a signal that starts peroxisome degradation; next, a layerof membranes is formecl tighdy arouncl a single peroxisome; ancl, fi-nally, the outer membrane of the layer fuses r,vith the vacuolar mem-brane. In this way, the peroxisome is delivered to the enzymes rvithinthe vacuole ancl degradecl.For ali these processes to take place, many specitrlizecl proteins arenecessary. Which proteins these are and how they function, was oneof the questions of the present research. Each protein deper-rds for itssynthesis on a gene which is, in eukaryotes, present \\.ithin the nucleusof the cell. To isolate genes that are ir-rvolved in pexophagy, a ranclompool of mutants (organisms with a random gene clamagecl) was cre-ated. Next, cells that were disturbecl in pexophapy were taken fromthe pool, anc-l it was determined rvhich gene in these mutants \À'asdamagecl. Finally, the function of the protein that was transcribec'lfrom this gene was analyzecl by finding out (r) which other processeswere disturbecl ancl (z) its localization in the cell.In Chapter One, an overview is given of the current state of affairsr,vithin the research into pexophagy, including ideas about possible fu-ture steps. We emphasize the specificiry of the process compared toother, non-selective processes, ancl the most likely origin of the seclues-ter ing membranes.Chapter Two clescribes a technique that can be usecl for the isola-tion of those clones from a ranclom pool of mutants which are clis-turbecl in peroxisome degradation. It can also be used for the selec-tior-r of complementecl mutants; these are mutants that have beenS I I M M A R Ygiven a treu', unclAmagecl coprr of the rlam,rgccl gene br the researcher.The technique is based on the cletection of tl-re prescnce of peroxis-omal AO in cel ls,  in circumsti lnccs uncler lvhich peror isornes are usu-ally c'legraclecl. When cclls that stil l contain AO are subjectecl to alll ' lalcohol, thcy' c'lie, because AO convcrts aliy.l alcohol into acroleirr, asubst:urce xtrenrelv tor ic to the cel l .  \VT or conrplenrentcd cel ls,  onthc other l iand, survive such treatmerrt ,  s ince al l  AO is,  in their  case,degracled. A pitfall of this techniclue, r,vhen uscc-l for complcmcrltetionstuclies, is that otten a group of gcncs (denontin:Ltecl HYEr, -: and -i)thr-rt gives a false positive result gets isolatecl. A probatrle cxplrrnationof this phenonrcnon is that the }{.ve proteins detoxifv thc formecl ac-rolein into less l-rarnrful substances. We suggest a way to overcome theprroblerrt-namell', by pcrforrning i,u-r extrrl assil)' ol.] the sclectecl colo-r-rics. This is an acccptecl, lvicleh' usecl plate assa\', basecl on a color re-act ion for AO. Alt l iough a laborious prcceclure, in this case ir  is notnecessary to apply it to tl-re whole group, as the ar-ralysis of only' a snralinurnlrcr of mutants rvill sultfice. In Chapters Three ancl Four, nvoge nes-isolatecl  bv the clcscr ibecl  techniques*are ic lent iFiecl  as bcing in-r ' , , l vc . l  in  pcrophegr .I t  is becoming increasingly clear that there is a big overlap in thegenes irrvolvecl  in pexoprhirgy ancl other transportet i ()u r()utes to thel'rrcuole, e.g. autophagl' ancl the Cvt (c1'toplasrn-to-r'acuole trrrgcting)route. Autoph:11ry' is a proccss u'l-rich occurs u'hen cells are expericnc-ing a shortage of nitrogen. Thel' react to the situatior"r by clelir.eringparts of the cytoplasm ancl organelles to the vacuolc for clegrirdationancl recl'cling. Irr this rvay', the cell obtains ener€ï' ancl br.rilcling blocksnceclecl to sun'ive the periocl of famine. The Cr.'t route is a prccess forthe transportat ior-r  of  aminopeprt ic laser ancl c[-nrannosiclaser to thevacuole. These are two of the enzymes involvecl in the clegraclationprocesses that take place rvi thin this colnpartment.  ' \X/e havc, so far,not cleterminecl u'hether H. po/.rntorp/ta cloes possess rr Crt rollte,I ) l. - ïb u t  ie r s _ v^/ l!{eÍteproterlre grkrguephap.111.1in ChrrI t  i s  l t ( lthe i r  cproccs:sible Í,rg lucoscAtgrr l rpr()ce.ssappear Ist i tuel t tsChalturc in ccific forpattern \reirson [-rtaken inttentperatSuperf ic iathe intcr i r:rer.)XiS-usu-allyl[ r ài ,  c)nlase,ltion,d -,;)adon:d ac-re thecolo-or re-is notsmall:, NVOing in-in theto theieting):rienc-veringiationrlocks:ss foro thelationo far,:oute,S U M M A R Ybut it has been studiecl extensively in Saccharont),ces cereuisiae (baker 's yeasr).An example of overlappirg vacuolar transp.rtation processes is thegene PDD7, describecl in Chapter Three. This gene codes for Pdc{7pprotein, which is involved in autophagy as well as in pexophagy. Whenthe gene is damaged, both processes are clisturbecl. Ancl the sc homo-logue of this gene, ATGt, is involvecl not only in autophag,v and pexo-phagy but also in the Cvt pathlvay.Another exanrple of partial overlap is ATGrr, rhe ger-re clescribec{in chapter Four. ATGrr is imporrart for peroxisome c{egradation, butit is not involvec'l in autophagy. cells lacking nirrogen degrade parts oftheir cytosol, but surprisingly, peroxisomes are excluded from thisprocess. It appears that a special mechanism involving Atgrrp, respon-sible for specific peroxisome degradation, nor only after adc{ition ofglucose, but also during autophagy, exisrs.  The Sc homologue ofAtgrrp has been describecl as being involvecl in the cvr roure. All theseprocesses (peroxisome degraclation, autophagy and the Cvt pathway)appear to partly make use of the same mechanism, though unique con-stituents for each pathway also exist.Chapter Five presents the observatiorr that a lowering of tempera-ture in cells ir-rduces peroxisome c'legracladon. The degradation is spe-cific for peroxisomes, since microscopic analysis shows the knownpattern with inclividual sequestrarion and fusior-r with the vacuole. Tl-rereason behincl the phenomenon is unclear. The observation has to betaken into account cluring experiments performed, as is usual, at roomtemperature, such as microscopy of living cells.*Superficially regarded, H. ltolymo4tha is nor reminiscent of man. Yer,the interiors of all eukaryotic cells have much in common, and thisrz5SUMMARYprinciple, known as the 'unity of biochemistry', suggests thar under-standing the biochemical processes occurring in H. polymorpha c nteach us much about similar processes occurring in other eukaryoticcells, and, thus, help us understand human diseases, as well as con-tribute to the research towards the production of antibiotics. In thepresent thesis, small steps toward such understanding have been made,yet steps that could eventually lead to full elucidation of one of themany processes which together form the basis of life.LpdnStbrIe,dereíende:ideschstaÍbehnaa-IChapter  11Growth factor- induced phenotypic modulat ionlsolated smooth muscle cel ls in cul ture do not immediately start  div iding, even in amedium containrng al l  nutr ients required Before enter ing the cel l  cycle. the cel ls í , rstaccommodate their  phenotype to their  new environment.  They lose their  contract i lepropert ies and modulate to a prol i ferat ive and synthet ic phenotype, character ised bydecreased contract i le protein and M, receptor expression. but enhanced expressionof PKC and synthet ic organel les [1;2].  This process of phenotypic modulat ion hasbeen postulated to occur in vivo during periods of airway inf lammation, inviewof theincreased airway smooth muscle (ASM) mass seen in asthmatics which is part ly dueto an increase in cel l  number [3]  A central  role for phenotypic modulat ion in smooth_ , , ^ ^ r ^  ^ - ^ . . , + L  i ^  ^ r ^ ^  ^ ^ r . ^ ^ . . , r ^ ! ^ ^ r  ; ^  r / 2 s í : l  r l a r  r e m n d c l i n n  l n  a t h e r o s c l e . o t i cl i l U J U l V  9 l U V V L l  I  l J  d l S U  d U n l  I U v V I U U V U U  l l l  v q r u u r u r  r u r r r v u u i l r  r V .lesions, neoint ima formation is accompanied by modulat ion of smooth muscle cel lphenotype to favour condit ions of growth and extracel lular matr ix deposit ion [4] .ASM phenotypic modulat ion can be rnduced by exposing cel ls to high concentrat ionsof fetai  bovine serum (FBS). The main disadvantage of studyrng phenotypic plast icrtyin cel l  cul ture is,  however,  that cel l  to cel l  contacts and cel i  to matr ix contacts aredisrupted. Even at conf luence, cel l  cul ture cannot mimic the three-dimensionaicontext of  intact muscle. The extracel lular matnx in which the muscle cel is areembedded has been found of major importance: vascular smooth muscle cel ls canbe retained in their  contract i le phenotype when cultured in lamrnrn-coated f lasks oron matr igel ,  which contarns solubi l ized basement membrane proteins [5;6] Inaddit ion. human ASM cel ls cul tured on laminin exhibi t  a contract i le phenotypewhereas col lagen I  and f ibronect in favour progression to a prol i ferat ive and lesscontract i le phenotype [7].To study the impact of the natural  mix of extracel lular matr ix components onphenotypic modulat ion, Tao et al .  cul tured ASM cel ls on top of a ethanol- f ixed layerof dead ASM cel ls,  reasoning that the complex mix of extracel lular matr ix proteinssynthesized by smooth muscle cel ls would create a more physiological  environment[B].  They found that cel ls cul tured under such condit ions did not lose contract i leresponsiveness to methachol ine, suggestrng that phenotypic modulat ion is lessprominent in this sett ing Also. these cel ls did not spread to the extent hat smoothmuscle cel ls do when plated on glass and they did not acquire stress f ibres i t  isimportant to note, however,  that the condit ions used to create this matnxenv i ronment  (smooth  musc le  ce l l s  on ly ,  s t imu la t ion  w i th  10  % FBS)  prec lude aproper comparison to intact muscle.Therefore, we used an organ culture approach in our studies, s ince this model hasthe advantage of having al l  endogenous extracel lular matr ix const i tuents and cel l  tocel l  contacts intact.  Using this model,  we showed that growth factor- inducedphenotypic modulat ion can be induced in the intact muscle (Chapter 2).  ln summary,this study demonstrated that t reatment of bovine tracheal smooth muscle (BTSM)str ips with FBS caused a t ime-dependent ( , .  = 2.8 days) decrease in maximalcontract ion compared to serum-deprived control  str ips. Contract i l i ty was "ott r )+ccl nt n{^t óTIC LCCmCCe)ReorrF Er inCOth rm (I to pt n (CCp rCOÍ ^ ,t d rPf,Tt-0 l - lS NvteJ Em rI n is t lmF tpra (prd (fap í

Molecular aspects of peroxisome degradation in Hansenula polymorpha

https://pure.rug.nl/ws/files/2952753/Komduur.PDF

Molecular aspects of peroxisome degradation in Hansenula polymorpha

Abstract

Similar works

Full text

Available Versions

ARTS repository - University of Groningen