Rotenone sensitivity in Candida utilis yeast: Exponential vs stationary phase

UPdate 2003: This is old. Go to the pdf of Katz, Kilpatrick and Chance

Note: 1999 This was the first mention that Candida (=Torulopsis) yeasts have site 1 energy conservation = site 1 oxidative phosphorylation = site 1 of the electron transport chain in stationary phase, but not in exponential phase = log phase of growth.

Rotenone Sensitivity in Torutopsis utilis

Acquisition and Loss of Rotenone Sensitivity in Torulopsis utilis

Richard KATZ, Laurie KILPATRICK, and Britton CHANCE

Johnson Research Foundation, Philadelphia

( cited as Katz R., Kilpatrick L., Chance B. (1971). Acquisition and loss of rotenone

sensitivity in Torulopsis utilis. Eur. J. Biochem. 21, 301-307.)

Received January 16/March 13, 1971)

Torulopsis utilis yeast cells growing exponentially on a synthetic medium with ethanol as carbon and energy source are insensitive to rotenone, acquiring the rotenone-sensitive component of the mitochondrial electron transport chain upon entering an ethanol-depleted stationary phase.

The rotenone-sensitive site is lost when growth is restimulated with ethanol, but not when growth is restimulated with glycerol or acetic acid. Growth limitation by iron or by glucose or glycerol is also characterized by acquisition of the rotenone-sensitive site, upon entering stationary phase.

The transition from rotenone-sensitive to insensitive can also be studied by the use of semi-continuous culture in a chemostat.

Studies with the protein synthesis inhibitors cycloheximide and chloramphenicol indicate that protein synthesis on cytoplasmic ribosomes is required both for the acquisition and the loss of the rotenone-sensitive site. The acquisition of rotenone sensitivity is apparently associated with an increased phosphorylation efficiency, as evidenced by measurements of P/O ratios.

In a previous paper [1], it was reported that

7'orulWis utilis yeast cellfj harvested from an exponentially growing culture art@, insensitiv(.. to rote,none, and Piericidin A, inhibitors of mitochondrial eit@t,ron ti,anmpnrt,. lit,we-ver, respii,atioii of cells harv(@stt,,(] fti)m a culture which liaa reached stationary pliase (lue to del)letion of etlitinul (ttic, carbon motirce ust@(i iii the, ex@riinents report&d) and lias bcl-,ii in titim starved mtat(@ fc)r apl)l,t)ximat@y 15 min, i@ ot@t k3OO/, to 800/. I)y rota!rtone. lt was tils(, rtll)ort,o(l tliat if ad(litit)nal sul)8trate (ethanul) i@ administt@,rt@(I t(i th@@ starveti mtationary eultui,e, rot(,rione s(,@nBitivity is lost within 15 niin. In this coininunicatit)n w(@ 1)regent some fut,ther data on tiit@ I)I'oP(.rt@y ol' l'ott'none senritivity in l'orulo2iii.Y utilir, ace,king t(i c!xf)lain tlic sort of tran6itioll tiiat tak,, 13[ace wticn ii (!ulttir(,@ of' rotenonu-in orisitive yeast (!ells exiiaumt tllt!iw, carbon soui,ce aiid ente@ atatioiiai-y PliamL, tilt@s ac(luiriny the rotenone@"eilsitive conl@ ponent of tlic el(@ctroii transf)ort (',hain. AINT OCR A TRIP? RK 1997 Want to read this stuff? Call me. or email

Note from 1999: But I think it's worth pointing out that this paper states that there appeared to be a P/O ratio of more like 2 for the exponentially growing cells (exponential growth phase) and more like 3 for the stationary phase culture. One should note that these results were obtained with a nice piece of equipment we built that kept the oxygen tension nicely in the 5% to 20% range. RK

'dA'rk,'It[ALS ANI) Nil-@"rHol)STli(! tt@(!Iini(iuvH tise(t for cultiiring 7'. utilis were ILN LicHi@rib(!(t 1)rc%,iotiNly f I 1. R<)teiion(@ sltiuitivity was

assayed a4 it@ I I.]. @'t!ttst inittj(!Itf@n(tria wei,e I)repare(i i)Y tll(@ Mf@tlio(I 1 c@I)orce(i I)y @@l at. [2 1. l@/0 I'lit-lo@

in imolat(@(i tliitt)choji(tria were Iut(,xrniiied by the int-tlic)d of (7liari(@e atid Willianis 13]. Cyclo[iL@xiiytide an(I chlorainl)lieni(!ol wem obtairle(i fi,oni sigma. AI)sorbanoe was nicasure(i u8ing a Klett iiistrutilorit; tile unith are those (,n the instrument.

RESULTS

Ellert ut the Protein Synih@Qi8 Inhibitors C.ye,luhexi,in-ide und Chlora?iiphenicol on the ,40quiRitioyt and L@s o/ Roteiione Se?tsitivity

(','yeloh(iximide iiihibita de novo protein synthesis on cytol3lasinic ribosonies in eukaryotic organi8ms, inc-luding yeast (4). Tlie effect c)f cycloiieximide on the loss of rotonone sensitivity iA shown in Fig, 1, 750 mi ol' culturc rnedium containing 0. I O/, ettianol were inot!tila (i from an exponentially growirig preculture of 7'. utiliq. Tliroughout exponeiitial growtil there %va8 a jit%rly linear drop irl pH and tiie rat of culture oxygen c(,n8uml)tion inereaf3ed exponentially. Oxyger, consumption of sample4 of intapt e,?Ila r(,.moveci from the oultum during this time (froni zero time to 6 h) was not inhibited by rotenone even at concentrtttion,% of 5 niM. Shortly before growtli cc@ased clue to clepletion of ethanol the respiratory rate, of the c@tiltul,e f(lli, followed by a la.@t I)ilrmt (@f oxygen con8i4mption, and acid secretion atopped,

Midway through this final bur8t of respiration, the fir8t ceils af)pear(,d hich were partially inhibited

by roten(ine. This acnsitivity to rotenone inerease(i

to approxiinately 500/, with subsequent aliquotf3, Ttic

culture waa kept in thib @rbon-starved atate for approximately 3 h. At thin point, cyclohexjmide wm added at a final concentration of 100 ILM. I 5 min later, othanc)l was added (final concentratioti 0, I G/.). In (@c)ntramt to tlie experiments mported in I I ], where rottinone gengitivity was lost within 15 min aftl'r the addition of ethanol, rotenone sensitivity decre@@d only elightly for 2 li, and never maehed zert).

A gecond experiment was perf()rmed to dt@@rinine the effect of cyclohomimi(le on the ac(luisition of rotc,none 8ensitivity, raking advantago of oui- ciir(tot roadout,of culture oxygen constiml)t.ion,eyeloheximide was ad(led immediately after tlie ratcl of culture i)xygen consumptic)n first decreaned- As Bliown in Fig.2, the exl)i@c!tc@(i flnlil I)tirst of respiration (lid not occur, an(i rotonolle @cn'3itivity did not appear. A(lditic)tl of etlianol (final concentration 0.10/,) to tliis cycl()iieximide-inhibitod culture produced st)me litioar growtli, a sniall (irop in pff, and @v perio(I of aiioinaloti@ly low 1'(@4I)ii,atic)n siniilar to that in tlie CNI)f@rimL-iit sliuwti in Fig. 1. Rotcnt)ne slnsitivity retnatineit zero tlirottgliOLit.

i@ en iniiibitor of iiiitocil()Ildrial sylitlic@sis in yeast (4). Am sliown in Fig.;I,

e,liforiiiiil)lit,ni(-Ol (;i 1)(,r rnl) wiw a(inliniStk'L'L(1 to a (!Illttiit@ wliic@ii liad been iri tlio gtarn,c(l stationary Stilt(@ f,@l, @il)l)i'oxiiiiately 3 li an(I was al)l)i,t)xiiii@@tely 50"/, selisiti%,t, tc, rutcrtont,, During thc@ ').5 11 iii(lit-

))Iltioll tilt- (ii,iig, rc)t(@ric@n(@ Kt,il@itivity

to 700/,. ll'ollowitig ttlis inctil)iltioll

1)('Iio(l, (@til@ilit)l (0.1 O/,) %t,ah Ad(lect, Growth, ial)id

ux.@, exi an(i acid slcretion

IloIL-1101IL' st!nsit!Nrity fl'll to zel,o. Followilig

(,f et-linni)], iotenont, gonsitivity r(,@-

Ell,,c@ (if (@'ftjillh Lio?titaiio?b by SubvtrateA Oili(@r

than Ethanut

t)ti (@tiecose, Fig. 4 sliows the efft@,ot t)f grtjwth oti g[uluso i,@ttlier tlian ethanol. Tlie grt)wth @urve, ILH IZIC'ftHlil'i@(i t)3, ttirbictity, indioate8 that some (iiaiixic @rowtli took place. Ttie seoond ancl IC66er inereasc in ttirbitlity waf3 aceompanied by a dect-ea@d but stable rate of culture oxygen coniiumption. All samplo8 takeii from the actively growing culture were inat@tisitive to rotenone. Following the ce8sation of growtli and tlie final drop in respiratory ra , there was an elisci(le of alight rotenone sensitivity (20 O/, or less), followed by a period of approxima ly 3 b of rotenone insensitivity. Following this was a second episodc of rotenone 8ensitivity (30-400/. inhibition) which persisted for 4 h, whereupon the experiment waa terminated,

rowth m glycerol (not illui3trated). Samples removed fl'om a culture of P, tili, with glycerol aa carbon and energy source during exponential growth were ingensitive to rotenone. Samples taken during

otationary pham were mnsitive to rotenone, although tbe percentage of inhibition of rut3piration wao always quite small, between 20 O/. and 30 Q/..

Grwth on Ethanol; RmgimuWim wiih Glycerol and Acetic Acid. As shown in Fig.5, an othan(i@limited culture of 2'. u4iii8 wa8 allowed tAi gmw into a r()tenone-wnsitive gtationary phase. Growth wm matimulated with first glycerol, and then @when tlie glycerol was exhauated) acetic acid. The addition of glycerol (final concentration 0,10/,) resulted in illcreased rotenone 8Lnuitivity in the samplea takeli. To the giyeerol-depleted culture, an addition of glacial acetic acid (final concentration 0.101,) was made, allowing growth to resume for approxiinately 1.5 li. While there was t5ome decrease in i-otenone sensitivity, at no time. di(i rotenone 6ensitivity fall t(, zert) (as im the came with control cultilrea guing t[irough uuch feeding-starving cycle8 with t,,thanol).

(@'rowth on r@,lhanol Li7o4ited by Iron. Iii thin 8eries Of t-xl)erinionts, 300 mi sliake-flauks C-.Ontaiiiing 50 mi (,f inedium ieplaced ttie tiaual clabol,ato cultum@ appaiatu8, to facilitate coml)letion of an ciitire irt)n titration siniultatleounly, As Btl()wn iii Fig.6, at all the iron concentrations te8ted (including those wliere finnl eell denfjity, as indica@,cl @)y absorl)anc(!, waN (!(@i,tairily limited by iron), all %amplee tak(!n during

gfowth werc inbensitive to ruten(@]lti, takc-,Il

froin Ntati@)nary phase culttires (I)resumably (I(,pluted (if iron, except for the 50 [LM Pe culture) ore Bensitiv(@ to rt@tt@none, albeit to varying ext(@nts and in diff'e,rent I)atterns. It should be 1)ointed out that in th(!Se exljeriments shake-flask oulture@, although nioi,e convenient and i,eadily available, yield some. wtiat less reproduciblo results than the clabor8Lte culture appai,atum aiied in the rest (@t'tlie studies reported here and in [ I

Chemostat Experimenta

Tile transition of 7'. utilis from rotenone-insensiti%'o to -scnsitive and vice versa can alao I)e studied (anci more conveniently) by the use of semicontinuou8 culture ill a chemostat, In the initial stages of chemootat operation, when thc! culture was not, I)c-ing dilutt@d but was allowed to grow from an inoculuni as a batch culture, cells removeci fr()m the culture wer(,, insensitive, t, rotenone, Aft,, a Uitabl@ i@ll (lensity was reached, the veasel was diluted at a rate of 0.2 ti-', with ethanol as tht litniting subeti'ate (final concentratio,, 0. IO/,). Wlien the stea(lystat(, cell dengity was reached, it was found that the clell-4 grown under tliese steady-8t&te (@hemostat (@oii(litiong were &pl)roximately 609/, sensitive to rotc-rione. This aequence was repeated by inereas;ing

the dilution rate to 2.0 h- I until approximately holf the colla werc washed out, whereupon the dilution ra@ waa @t back to 0.2 h-1. In the tranaient period wliile the culture waq returning to the stk&dy tate, the ceII8 were inwnsitive to rotenone. When steady@ state conditions prove6i[ed once more (as indiested by a constant cell density) the cells were sensitive,

Mitochondria Studies

To test the pos8il)iiity that the transition from rotRnone-insensitive t() -sensitive w&A merely a change in whole-cell permeability to rotenone, mitochon(Iria were prepared frum 1', utili@ cells harvested during exponential phasu; cells harvcst,,d from a stationary phase culture; and also cells harvested from the chemostat clescribeti above, operating at a (iilution rate of 0.2 li-1 with etlianol as limiting Bubstrate. The Table slic)wh tht) vitrious types of mitocliondria prepare(l, tlieir respirat()ry coritrul an(i P/0 ratios, and sensitivity to r tonono and flierici(tin A. 'I'lic roten(ine-st@nsiti%,it@y of tile isolate(i mito(@h()nfiria paralleled tilat@ ol' tilk@ (!Clls froiil wliieh they were iso-

latt@d, ev(@n when stibstrato of NAI)H wt@rt

etyiploye,il, Ttic one (@.Net@ptic)ti %vas for iiiiwcliondria, resi)iring on etliatit)l, in wliieli ca.4t@ rott@iione or l'it@ri(ti(litiA at liigii C!onot!@ltl';@tioii.4 iiivtli,iably in, tlil)itc,cl V(@SI)il'ation, wi'Lttit@ tll(- iiitivi@t (!ell,% were @t,lisitive t)r t)ot,. 'rliis (liserlpancy lias 1)(@eii (lis(,@ussL-d I)rclviously I I 1. Tiiesu 6ttt(lies itt viti,o tnako it Iiiglily Linlikely tliat tli(@ a(,(Itti,@ition t)f roten()ri(@ sensitivity iii intact elll% is citle to an ititc-@,vil I)Lrin(lability to rot(@nont.

With initocliont.1i'ilt @tli,ea(ly 1)rcparLd, it wo,@ of intLreNt to a68ay tlie plit)s@@ltor3,lation efficienoy (P/0 ratio) of ttie liai,vesteci froni thu@e thrte

difrurent LLIltUCC Coii(litions, i. 0, exl)ont@ntial l@hase

c!ells, @tatit)ilary phase OLIIS, and

othanol-limit4@d cheino-4tat cells. For both chemostat celig anci mtationar3,-pliase C(,@its, the isolated mitocliondria were eoul)led (res@)iratory control ratio 2,0 or greater) aiici exiiibite(i P/0 ratios of approximately 3, However, mitocliondria isola d by the same procedure in parallel experiments from exponential phase cells were nearly completely un. coupled, with a respiratory control ratio of approximately 1,0.

To assay phoijphorylation efficiency in the cells, data were oolleoted on total oxygen uptake and total

increment in culture dry weight for veral I h inter-

vals, during the growth of 0, typical T. usili8 culture with ethanol as carl)on and energy source. Making uae of the Eladen "Congtant" for YATP (5], the following relationhip, were d,ri,ed nd umd in

evaluating a P/0 ratio i, vivo;

g dry weight produftd/h

YATF @- ...

m l@l P prod-u -@-/h--

10, the E18don "const-ant" [5) (1)

moles ATP prodti@/h

-- f@@ (i .. - L@ I'/O ratio, (2)

2, mo 126cilb

arranging Eqn. (2),

mt)les ATII E)rodLic!e(]/h =7 (I'/O) 2 X moles 0,

iitilized/h (:t)

Substituting Eciii, (3) iii Eqn, (1).

,lr5, weight prodticed

nioieso,titilized 10. (4)

ll(!art-titiging F(in, (4),

g drt, @veigilt pix)(luce(i

x flioie 6.

s Litilized P/0 .

Dry weiglit %vas ineastice(i by i-eiiiaving 25 iiil sllmf)IC8 froin tht,, culture, %@a.411ing the C('l[S in clistill(,(t wa@', reHt@41)cn(ling in di@tille(I wata!i, Kjici firying ov,@rniglit at 10,7)' C. Total 0. eonstiniptioti wa.4 measure(i by rec-orcling t[kc arnount of digsolve(i oxygen wliit@li liarl to be ft(itniigtere(I to rilairitaiii t@l(! level (If'Lli6S,)l v(,-(] oxygell btween I 3 O/, art(i 25 O/,

of ox c , h t,

,Yge@l s@itui,atioll (Hot, [ I 1). In thr,, I in ,v,l,,

the vallies of II/O ratio.% c)btaine(i b@@, thib inetlit)(I wi@i,e 1.3, 1.6, aticl 1.4,

1)18(.',L78@ION

As rc!i)o@ted iiiiij, 7'orulopgis utitig yeast c@118

growing (@x[)onei)tially witli eth@@not a@ sole carboyi aii(i (,@nergy ar, ir'sen,itiv, t(, ilotLriont@; ill)o@l

entLiirkg an thanol-(Iepletecl @tationai, - N, phase, th(@ cells ac(luiil, tli(, rotonoiie-senuitive (@oinponent (@f' the ele(!tron traiispe)rt chain. The results of the seril@s t)f (-,xperimenti3 %@itli the I)rotein synthesis inhibitoi,A cYclot)eximi(If@ and chloram[,ht,@nicol imply tliat I)rotein i3ynthesis is requii,ed for the trallsition fr,m "c)tenont@-sen8itiN,e tc) -inseii@,itivc,, and vice wrsa. rhib 8ynthesi6 I)resumably tak(,s place c)n cytoplasmic ribosomes, not on mitochon(irial ribosoiiies,

I'h(- uRe of (,arbc)n source@ otht,.r tlian ethallol, @uch as glucom And glycerol, shows tilat the efft-,et of growth plia,8e n Ictc@n,,,, ,n,iti,ity is tiot ali (@ffc'c@t pt@tiliar to etl)anc)l growtii. Wilen growth iN limi d by 4 non-carbon substra@, c. g. irun, it is observed that the transitic)ii to statioriary I)hamtpr<)(luces roteno,,,-"18itill lelim, in a faqliiun quitc@ similar to otlianol cicl)letion, liowever, the effect ot' t!thanol during acti,e gro,,t], in producing roten(,nt@ insen8itivity i, Ipparently 1)e@uliar to etl,,n,l am,,,g the subf3trates test(!(I. Whcii grc)wth ig i-estiniiiiate(i with glycerol or acetic acici, for example, rotenotit@ insensitivity does not reapl)ear. Yet if tile culture has not gone througli a periocl of acquisitio,l of r,ten,n, Oonsitivity, tlk,, gr,wth on gly,,rol and glueoge, for example, still procluces insen@itive cells.

@ent resultd obtained in this laboratory [6] implictite ant)ther factor in ont,ol ,f the gynthosis

of tb(-. i-ot(@none-mnaitive site. Cellf3 gro",n on k@,thanol

ulicl(!r, (t(@nditions of insufficient aeration, where the

st(@n(ly .4t&te coneuntratioti of (lissoll-eci oxygn i,i tlie cuIttirL, ig noar zero, Art' sensitive. to i't)@n(,ne. Thim iN ill 'Contrast to the experim,,,t, (I(@S(.-,,ib,d pri@vioti@ly, where th concelitration )f (lissolv(!(i L@xygtln waa maintained between 150/, snd 250/, )f H4ttit-ation by an apl)rol)riate sei'vtjmeeliallimin (oxy8tat).

'Pli(I stu(iies pt@rformed to estimate the. I'/O ratic) in vii,o, or tile P/0 ratio with isoloted ftom (!(@lls in difret(,nt growtb "I)hases", i. e. ex[)t)ll(!nt,ial pliii@t@, stationary phase, aii(i frim tli, h,,n(,,t,,t, I)ermit a goo(I basia i)f compa,-i,on it)i th, ,,,,ul@ (,t'otliei,s worlting witti tht, l,inenergetic, f 71. utili,. V@triotiN workera [2,71 til@ve rel)ot, (i I,it, ,n 1)o.Rsibi(@ C!of rel@ttiorlm I)L-twto(i r(@t(@nono (,,- l'i,,,i(ti(lin A mt@nBitivit,y an(I tlit@ pres(@n(!(@ t)r at)9(@lloo of (@iit,rgy c-oul@lirig (lih(]F,;[)Iiol.ylation) @it sitk, I )f' tl,, ,,,)il,at()ry (!Ilhill

loss of, L)<)th rot4@nc)t)e g(,ii,@itivity aild 8ittt 1 131)08I)Itoryl@itioll was i,(,I)ortecl tt) frolli iron lirylita-

tioji (luiiii@ growtli. l,lli8 Clot'@4 iii,t apt)(-ar too it, tll(, c,f' bat(!Il (@llitutog [7 1. %vhere ii,t)n lin)itat.it)n (7otil(i Clilly OLICIUI' at, tlie en(j of,gi-owth ailci at

t,lie tim(@ wlit,ii r,)ti,non(! st,,nmitivity lii tlic

(")t)tillllt)kls cliltul't, [')], l'otc-lion(@ is only ori(@ of t,lit, ilitilly ittiii-k4

I)y ttiu ( lli'mc@st'Lt, "'itit it, fli 11 I , n,i gl.(,U,tlL

1)lia%c". 3 tj tkl,@l

T")Kul