Veenhuis, Marten

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   University of GroningenWeast microbodies; occurrence, proliferation and metabolic significance.Veenhuis, MartenIMPORTANT 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:1988Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Veenhuis, M. (1988). Weast microbodies; occurrence, proliferation and metabolic significance. 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: 21-06-2022SUUTiARYEukaryotic cells contain a number of distinct subcel-lular compartments (organelles). Each tlpe is involved in anumber of specific netabolic functions and consequentlyharbours its own characteristic group of enzymes and pro-teins. SeveraL of these organelles have been - and sti l l  are-  objects of  extensive research ( i .e .  n i tochondr ia andvacuoles) and much is already knowr about their significancein ceLlular rnetabolism and about their biogenesis. However,the current knowJedge of microbodies, which were only dis-covered recently as a separate class of ubiguitous subcel-lu lar  organel les,  is  s t i l l  f ragrrnentary.  Microbodies arenorphologically simple in construction: they are generallyspher ica l  of  shape,  measure f rorn 0.2-L.5 p* ,  Iack r ibosomesand contain a finely granular proteinaceous matrix sur-rounded by a single membrane of 7-8 nrn. Biochernically theyare defined by the presence of catalase in their matrix. Inaddition a variety of other enzfrme proteins may be presentdepending on the type of organLsm and their developmentalstage and./or environmental condltions. Characteristic fordeveloping microbodies in various eukaryotic cells - inclu-d ing  yeas ts  -  i s  t he i r  i n t ima te  assoc ia t i on  w i t h  t heendoplasrn ic  ret icu lum (ER).  The c lass ical  concept  of  micro-body biogenesis was largely based on these observations andproposed that microbodies develop from the smooth ER bybudding.fn yeasts the development of microbodies can be inducedby a number of organic growth substrates; under these condi-tions the organe).les are thought to be either involved inthe ox idat ive metabol ism or  ass imi lat ion of  these sub-st rates.  This thesis  descr ibes a deta i led u l t rast ructura land biochemical investigation into the mechanisms involvedin microbody biogenesis, substructure and proliferation, aswel l -  as in to thei r  metabol ic  s igni f icance in re lat ion tovar ious condi t ions of  growth.The study was started with the yeast Hansenula polv-morpha in which microbodies are abundantly present duringgrowth on methanol as the sole source of carbon and energy.this c1-compound since they harbour the enzymes ;;;;;;"a t;the init ial oxidation of methanoL, namely alcohol oxidaseand cata lase (chapter  I I ) ,  On the other  hand,  dur ing growthof the cells on glucose activit ies of these enz)rmea $tereabsent or extremely J-ow. At the subcellular level such cell-sgeneral ly  conta ined a s ingle srnal l  rn icrobody (0.1-0.3 pt )local ized in  the v ic in i ty  of  the cel I  membrane and c loselyassociated wi th s t rands of  ER. Biochemical ly  these orga-nel les are character ized by the presence of  very low ac-t iv i t ies of  cata lase,  D-amino acid ox idase,  ur icase and L-c(hydroxy ac id ox idase.  Af ter  a shi f t  o f  g lucose-grown cel lsto media containing methanol as the sole carbon source,alcohol oxidase and catalase are rapidly synthesized. Theresul ts ,  d iscussed in chapter  I I ,  c lear ly  demonstrate thatthese newly synthesized enzymes are imported into the snallmicrobodies originally present in the (glucose-grown) in-oculum cel ls .  Growth of  the organel les,  resul t ing f rom th isprote in import ,  was parale l led by the format ion of  crysta l -l ine cores in the microbody matrix, whose development wascompletely dependent on the presence of aLcohol oxidaseprote in.  Dur ing pro longed incubat ion of  ce l ls  in  methanol ,the number of microbodies gradually increased by the sepa-ration of small organeJ.les from mature ones; the latterorganelles measured up to 1.0 p* and contained large crys-ta l l i ne  i nc lus ions .The mode of nicrobody development described above r.lasconf i rmed in fur ther  s tudies wi th H.polvmorpha dur ingvegetative and generative reproduction of celIs on methanol(Chapter  I I I - Iv) .  S imi lar ly ,  microbodies present  in  deve-loping buds originated fron mature organelles present in themothercel l .  F iss ion general ly  took p lace in  the neck betweenmothercell and bud. These studies also revealed that both inascospores and vegetative cells, apart from the enzymaticcomposi t ion,  a lso the.  u l t imate shape,  sLze,  substructure andvolume fraction of microbodies is largely determined bygrowth conditions. In methanol-Iinited chemostat-grown cellsinvar iably  conplete ly  crysta l l ine organeJ.Les were foundwhich,  under appropiate condi t ions (D=0.03 h- l ) r  nay occupyup to 80 t of the cytoplasmic vo.Lume. Chapter V describesthat  these crystaJ- l -o ids are composed of  a lcohol  ox idaseprote in.  A three-dimensional  model  of  th is  crysta l lo id ispresented,  which a lso expla ins the observed f ree mobi l i ty  ofother  microbody-matr ix  prote ins.Chapter VI describes the turnover of microbodies inmethanol-groe/n H.polvmorpha under conditions where theirnaJor matrix proteins are no longer reguired for growth. Ourresults indicated that the degree to which the microbodypopulat ion,  or ig inal ly  present  in  the cel l -s ,  is  degraded,  isdependent on the extent of repression of alcohol oxidaseslmthesis in  the new envi ronment .  Ful l  repression of  a lcoholoxidase slmthesis by adding excess glucose or ethanol to theincubation media led to the total degradation of the alcoholoxidase-containing rnicrobodies. The organelles were degradedindiv idual ly  by an autophagic process,  resul t ing in  thecomplete turnover of their matrix proteins. This process wasinit iated by the segestration of the organelles frorn thecytosol by a number of mernbranous layers. ?he vacuole playeda cruc ia l  ro le in  the degradat ion process s ince i t  admini -strated the hydrolytic enzymes reguired for proteolysis tothese compartments. The degradation was found to be an ir-revers ib le,  energy dependent  and -  above a l l  -  fast  process;individual organelles could be fully degraded within a timeintervaL of  30 min.  Not  a l - l  the organel les present  in  onecel l  f ina l ly  d isappeared.  Depending on the composi t ion ofthe new environment one or more organelles remained, whichdeveloped from the alcohoL oxidase-containing microbodiesbut whose enzymic composition now $tas a reflection of thenew environment.Chapter  7 and 8 descr ibe examples of  microbodies in-volved in the metabolism of both the carbon and the nitrogensource. The first example was encountered in newly isolatedstrains of Candida famata and Trichosporon cutaneum capableof  ut i l iz ing ur ic  ac id as the combined carbon- and n i t rogensou rce  ( chap te r  V I I ) . .G row th  a t  t he  expense  o f  u r i c  ac id  wasassociated with the development of rnany microbodies in thecel Is  which conta ined h igh levels of  urate ox idase andcatalase. The second example was observed in a newly iso-Iated strain of Trichosporon cutaneum X4 that could grov/ oncatatase,  as was shor .m cytochemi. ; ; ; ; : - ; ;  uotn orsanisms theurate ox idase-  or  amine oxidase-conta in ing microbodies deve-loped f rom organel les present  in  the inoculum cel ls .  I t  mustbe st ressed that  growth of  the above organisms on ur ic  ac idor ethylamine provide unigue examples of the proliferationand physio logical  funct ion of  yeast  rn icrobodi-es,  s inceenzymes other than microbody-borne urate oxidase and amineoxidase which are invofved in the in i t ia l  rnetabol ism ofthese substrates,  were not  detected.  Therefore,  in  theseorganisms all the carbon and nitrogen required for growLhflows via microbodies where enzymes are present that arecrucia l  for  the in i t ia l  metabol ism of  these combined carbonand n i t rogen sources.Ut i l izat ion of  ur ic  ac id or  ethy lamine as carbon sourceinevitably leads to the excretion of amrnoniurn into thecuLtivation medium as a result of the low carbon (C) overni t rogen(N) rat io 's  in  both substrates compared to cefLma te r i a l  (C :N  u r i c  ac id  =  5 :4 ;  C :N  e thy lam ine  =  2 tL  ve rsusce .L l  ma te r i a l  C :N  =  7 :1 ) .  U t i l i za t i on  o f  t hese  subs t ra tes  asC-source could be explained by the finding that the syn-thesis of both amine oxidase and uric acid oxidase wasinsensi t ive to repression by f ree arnmonium ions.  Our resul tsindicated that the synthesis of these enzymes was regulatedby induct ion.The physio logical  s igni f icance of  the microbodies inethylamine-grown T. cutaneum X4 was further investigated byfract ionat ion studies.  In tact  protoplasts were gent ly  hono-genized and subseguent ly  f ract ionated by means of  d i f fer-ent ia l  and sucrose gradient  centr i fugat ion.  The resul tsshowed that  the iso lat ion of  in tact  microbodies was cr i t i -ca l ly  dependent  on the pH, cornposi t ion and osmot ic  s t rengthof  the homogenisat ion medium. Analys is  of  the d i f ferentfractions obtained indicated that microbodies of ethy)-aminegrown T.  cutaneum X4 conta ined,  besides cata lase and amineoxidase, key enzymes of the glyoxylate cycle narnely iso-citrate lyase and nalate slmthase, together with malatedehydrogenase, glutamate:oxaloacetate arninotransferase andan enzyme not encountered before in microbodies, namely(NAD-dependent) gLutamate dehydrogenase. This subcellularIocalization of these enzflmes reguires interlocking of thernetabol.ic activit ies of microbodies and mitochondria forbaLanced growth at the expense of ethylamine. The rnicro-bodies produce aspartate which serves as a maJor cytosolicin termediate for  carbon assimi lat ion.fn chapter 9 the presence and proliferation of micro-bod ies  i n  Saccha romvces  ce rev i s i ae  (bake r ' s  yeas t )  i sdescribed. In this industrially inportant yeast microbodiesare generally present in very small numbers and consequentlylvere only poorly characterized. Attempts to induce theirpro l i ferat ion had so far  fa i led.  This general  behaviour  ofbaker 's  yeast  is  conf i rmed by the resul ts  d iscussed inchapter 9. Microbody proJ-iferation was not observed duringincubation of cells under a variety of growth conditionsthat are known to induce microbody proliferation in otheryeasts with only one exception, namely during growth onoleic acid. Marked proliferation of microbodies was observedaf ter  a shi f t  o f  ce l ls  f rorn g lucose to media conta in ingole ic  ac id.  This was associated wi th induct ion of  act iv i t iesof B-oxidation enzymes and catalase. Ultrastructural inves-tigations carried out at different t ime intervals suggestedthat also in baker's yeast microbodies developed from aI-ready existing organelles by growLh and division.The resu.Its discussed in this thesis support the generalhllpothesis that at least in aerobically-gror^rn yeasts micro-bodies are invariably present inespective of the cultiva-tion conditions. Their function can becorne important for themetabolism of certain carbon and/or nitrogen sources byimport of several- substrate- specific enzymes. During adap-tation of cells to a different environment newly fornedmic robod ies (peroxisomes,  g lyoxisomes or  funct ional lyintermediate forms ) develop frorn organelles already presentin the ceI I  pr ior  to  the t ransi t ion.  Their  u l t imate shape,volume fraction, substructure and enzymatic composition iscornpletely dependent upon environmental conditions.

Weast microbodies; occurrence, proliferation and metabolic significance.

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Weast microbodies; occurrence, proliferation and metabolic significance.

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