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Incorporation and distribution of ethionine-sulfur in the protein of ethionine-sensitive and ethionine-resistant yeasts
Incorporation
and
Distribution
Ethionine-Sensitive
of Ethionine-Sulfur and
Ethionine-Resistant
in the Protein
of
Yeasts
15thiollitie-resistant cultures of Saccharomyces cPieDLsiue and Cundida utilis \vere obtained by growing the normal yeasts in gradually increasing concentrations of Lethionine. The incorporation of et hionine-sulfur into the cell protein of cl hionincresistant S. cerevisiae u-as found to be less than that of normal cells, whereas thcrc was no difference bebeen normal and resistant cells of C. &/is. The uptake of ethioninr by suspensions of resistant cells of both yeasts was markctlly less than that of t hc normal yea&. Examination of the cell protein of yeasts grown ill the presence of 35S-labelled Lclthionine showed the presence of ethionine itself, which had replaced mrthionine residues, and also small amounts of lsbellcd cystinc and methionine. The labclling of protniwcystinc and -methionine was more rxtensive in ~11s grow, for prolonged periods in the presence of cthioninc, and was also greater in resist alit cells. The formation of cystine and methionine from ethioninc in thcsc yeasts is thollght to occlw through the intrrmcdiate formation of S-ad(~nos~-lcthio~~i~~~ and its convcrsioll to homocysteine. I31hionine resistance in these orgsnis~llS is characterized l)y a d~w+cesod :tl)ility to acc~m~~~late cthionine and an elthanccd abilii y to nlctaholizc~ ~h:tt amollllt of tlw amino acid ahich dots ent,cr t,he cells.
During a study of the uptake of various sulfur-c,ollt:liniIlg unlino acids by a brewer’s yeast (I), it was shomn that ethionine is accumulated by cell suspensions in the presenw of a suit.able energy source, part, of the sulfur of the amino acid appcnrillg ilk ccl1 prokin. Howcvcr, the form in which this incorpornt,ed sulfur was prcscnt~ was not) investigated. Data obtained with a variet,y of other organisms have established that cthionine per SCcan rcplwe methioninc rcsiducs in proteins (a-7), and there is aii~ple cvidencc that rthioninc can bc nlctjabolizcd by :I number of pathways (S, 9)) but, its wtuul conversion to other
transfer to ot.hcr amino acids occurs. X caomparison has also been n~(lo of ethioninc uptake and incaorporation into ethionincscnsitivc and cthionirlc-rtsititallt ycnsts.
Ytxsf and grouth wclia. The yeasts used were a (;uilnwss strain of Swcharowa?Jces c~erwisiae and a strain of Candida utilis (XCTC 357(i). Each organism was groxx from a small inocitlllm at 30” in a mcxdi\lrn containing 0.312 mhI ?;a$01 (rqllivalcnt to 10 mg S per liter) as dcscrilxd previously (I), and s\thcrlltluw wrc madr~ every 2-3 days.
period of 3 ntottths. These yeasts grew acll and showed :i normal appearance. Cho~ic~tla. l.-Et hiolrinc-“5s (5.0 mC per millimole) antI sodium srtlfate-::“$ (carrier-free) were ohtaincd from the l~adiochcmical Ccntcr, Amersham, l
for
eth,ioninP-S
incorporation
experi-
m2~t.s. The J-casts were grown in 25.ml conical flasks containing a total flttid volume of 10.5 ml, consisting of 5 ml of mcdirtm of twice the required strength and 5 ml of L-cthionine3%S diluted to 0.5 mC per millimole with thr tmlabelled amino acid in sterile water. The final concentration of rthionine was 0.78 111~ (equivalent to 25 mg S per liter). Two lcvcls of sulfat,e wcrc used in the medium: 0.156 mu. givitlg sttboptimtml growth, and 0.312 IBM, which was just adequate for optimum growth (10). 1’:ach flask was inoculated wit,h 0.5 ml of a sttspcnsion of salitrcwashcd yeast containing 0.3 mg dry weight of cells per milliliter, and the flask w-as agitntcd for 2 days at 30” in a shaking bath prc,viding 100 linear oscillations per minutc. Yeast growth was then determined turbidimetrically(lo), and a sttitable volume of ccl1 suspension it~sttally 57 ml) was cooled in ice and centrifuged. The resulting yeast pellet was rcsttspendcd in 10 ntl ice-cold 5:; (w/v) trichloroacetic acid for 2 hours with occasional st,irring. The prccipitnte was then ccntrifugcd, washed by two rcsuspcnsions in ice-cold saline, transferred with 10 ml water to a 30.ml hcaker, and ashcd in a furrtace at X0” with a crtpric nitr:ttc-potussirlrrl chlorate ntistllre, as descriljed previously (1). The lubcllcd stllfst,c so ohtaincd was made rtp to 10 ml with water containitrg 2.25 mg N&O, carrier, precipitated as \)ctrzidine sulfate, and counted with a thin-witalow Geigcr~RItiller tube (1). Samples of t)he origitral labelletl cthionine solrttion were also oxidized and assayed for 3% after sttitahle dilution. The cxtcnt of incorporntion of et,hioninr-S into protein was obtained from the percentage of 3% from t hc medium appearing in the trichloroacctic acidinsoluble fraction of the cells. E’rocedu,rc jar ethionined distribution ezperimerits. The yeasts were grown at, two sttlfatr concentrations, as in the previous scrtiott, the media in this cast Ireing supplemented w-ith 0.78 mu Lethionitt(,-“5S (5.0 mC per millimolc) rtndiltttcd with carrier ainino acid. Yeast growl h was dcfcrmined after 2 days, and samples of sttspcttsion contairring approximately C,mg dry weight, of cells wcrc rcmovcd and centrifuged. The sttpernatant mcditlm was retttrned to the flask and growl h was allowed to cant inoe ; furthrr sanrples corrc-
spending to fi nlg dry w-eight, of cells wcrc I hen taker1 at intervals. Each yeast pellet, was trcatcd with t richloroacetic acid for 2 hours at 0”, as in t hc previous section, atrd t hc saline-wash4 prccipi tntc was than oxidized with pcrformic acid atld hydrolyzed with ti N HCl as dcscribod clsewherc (11). The oxidized sulfrtr amino acids in each prcttcirt sample were separated try orlc-dilnensioll:Ll descending chromatography 011 a 1.5 inch-wide strip of Whatman 3MM paper in n-bllt:rrlol::tc.etic acid: water, 12:3:5 (v/v), for 20 hottrs at 18”, the solvent being allowed to drip off the paper. The dried paper st,rip was then scanned for radioactivity on a Forro radiochromatograph scanner fitted with a windowless gas-flow cormtcr. l~atlioact ivc bands located in this way were cut from the chromnt.ogram ami combusted separately by t hc Schijnigcr oxygen flask techniqtte, in one-liter flasks colttaming 5 ml water and 4 drops 3Oyn (w/v) hydrogun peroxide. The rrsttlting labcllcd srtlfatc was finally converted i o its benxidine salt, and assnycd for 3%. The activity of each band was cxprcsscd as a perccnt,age of the total activity on th chroma togram. and suljate b,y cell susI’ptake of L-ethionine pensions. The accumulation of L-ethioninc-““S and of lahelled sulfate by cells as :I function of time was exam&d by the procedure described in carlicr papers (1, la), ttsing washed yeast snspensions (final concentration in medium, a!:;, wet weight, pressed yeast, per voltmlc). LWS ULTS
Incorporation
of ethionine-.sulj”ur info yeast
protein. Ethionirte-sensitive and ethionirrcresistant, cultures of H. ce~ez,isiae and C. utilis were grown for 2 days at 30” from small inocula in two media containing 0.156 and 0.312 IIM sulfate, respectively; both media were supplemented wi-ith 0.7s m.\r L-ethionine-35S (specific activity, 0.5 mC per millimole). The extent of incorporation of cthioninc8 into cell protein was ohtaincd from the ratio of the 36S prcscnt in the t,richloroacctic acid-insoluble frwtion of a known weight of ~11s to that’ initially present in t.he media. This was cxprcssctl as milligrams sulfur per gram dry weight of yeast. Table I gives the sumn~arixcd data obtained. In every case there w:ts :t dwreased incorporation of ethionitu-S at the higher sulfate conccntjrat ion, which mflcct s the incrcascd cornpetition for prot,cin formst,ion
of the prwious finditlgs of other workers on cthiotiilic inc9rporation, it was cxpwtcd that :u&sis of the yeast prot8cin ill the prwcnt c~xpcriiiients would reveal t hc prcwnw of I:~bcllctl cthioninc. To verify this, 3 ~i~~thod wts tl~vclopc~l to scpar:~tc ct~liioniiw front the norlnal sulfur corns. cerwkio~ J)OMTltS, cay&tie and ~ncthionilw. Satis0.1x 0.40 ~:thioJtitte-scttsitive f:ict.ory rwolutio~i was obt,airwd by first 0.:‘,12 0. -13 oxidizing nlixturcs of Ihc three amino acids 0.1x 0, :3:; 13csistaJtt IO 0.78 Illll with porfornlic wit1 and t,hw~ chromatjo0.312 0.27 cthiuttitte graphing the products on Whuttnan SAIL\1 0.1x 0.25 ICcsistnJtt~ to 7.8 tn.\1 paper with n-butanol: :ic:ct.ic wid : w’:~1w 0.1t; 0.312 cthiotlitw (12:3: 5, v/v) :LSsolvent. Treat~ncrlt of the c. 11ti1is ~hroinat,ogr:ui~s wit,h ninhydrill gave 1hrec 0.1x 0.48 Ethiottinr-rcnsit ive 0.312 0.27 bands with IiF values of 0.10, 0.25, ant1 0.33, 0.156 0.4 Rcsistntlt~ to 0.78 1tlM corrcspouding to cysttic acid, niethionine 0.312 0.24 cthiottinr sulfotlc, and ct,hiolline sulfonc, rc:spcc:t~ively. 0.13; 0.54 Rrsistattt tu 7.8 III&< Grwtly inlprowd sep:u‘at.iolr of’ the oxidaet hiortinr 0.312 0.2ci tion -product.t.s was obtninctl by using dewending c,tlrotli:Ltograplly for 20 hours and allonitlg the solwrlt. to run off the paper. Th(; yeasts wcrc grown in the two sulfatctby the sulfur amino wids formed through c~ontammg nlcdia referred 1.0carlicr, supplct,he normal sulfntc reduction pathway. The ment.cd with O.iS 111~ r,-ct hiotGr~e-R%, of sulfate effect WAR part,icularly nolkcablc spcGficL wtivity 5.0 mC per n~illimole. After with C. ulilis, :t decrease of 43-52 54:rcsult.ing 2 days samples containing about, 6 mg dry from :L doubling of the sulfate concentration weight of cells mcrc taken, and the wvashcd in the nrediunl. In S. cerevisiae an added trichlorowctic acid-insoluble: fraction was fcal.urr was a decrease in the incorporation oxidixcd with performic acid and hydrolyacd of cthioninr-S by the et,hioninc-resistalIt with HCI. The produck, together with 10 ~1 cult.ures, the effect; being lnarkcd in the of a mixture of cbysteic acid, methionc sulfonc, more highly resistant cells. So significant and etjhioninc~ sulfone (cnch 0.1 %, w/v), w(w differences in incorporation wre obtained then c,hromatographctl in the descending tliwith the c’. utilis cultures. as described above. The chromatoDcternlinations have hcen made of the r&ion grams wcw wntmcd for r:adioactivit,,v and tot’al sulfur contents of 1he yeasts when subscqucntjly trcnted with ninhydrin. grown for 2 days in t-he presence of 0.:312 ,111 c~hrw~lntogranw should a large zone mnI sulfate. The values obtained were, for c*oilwid(lllt with the nirlX. cerevisiae: 3.6, and for C. u?ilis: 4.2 mg per of radion&vity, hytlrirl-positive band of highw;t nlobility, gram c1r.yweight of ~11s. The corresponding that c*orrwponded to nt hioninc sulfone and ratios 01 protein-S per total-S were: found so c*onfirnlotl t,lw prcwnw of prootcin-bouttd to be SO and SO.6%, giving protein-S values for the t,w-0 yeasts of 238 3ncl 3.31 nlg S ct,liioniti(>. In :rddition, :IS seen ill Fig. 1, two sniallcr rcgiolis of radiowtivity c~oillcitlctl per gram dry xvcight’ of wlls, rcspwtiwly. with the qwtcic wit1 ant1 nlethionine sulfow Thcsc values were used lo calcul:~lc the bnnds, indiwting a Inbelling of protcinfra(+ons of protein-S dcrived from cthioninccyst inc and -nlcthionitlr. The total radioS in the cthionirlc-sensitive cultures grown in the presence of 0.312 ~1131 dfatc. These activif.y prewlt in the protein frwtion ~v:r;ls valuc~ wrc 14.9 ?: for S. cewvisiae md S.0 C(; confined to I 11~s~t,hrw hands. for C. ufilis.
ETHIONINE-SENSITIVE
CYSTEIC ACID
METHIONINE SULFON E
S. CEREVISIAE
ETHIONINE SULFONE
Grown for 7 doys t ? 6 v,
ds, :iJld growth was nllo\ved to c~ont.iJluciJJ the sanle nledium for further periods of up to 7 days; smnples wcrc tnlierl and trcat.ed as previously. l3xaminat.ion of the rem11iJJg chronJntmogranJsrevealed tZhat the act.ivities of the caysteic wit1 and nwt,hioninc sulfone bands had incrcascd rclativc t.o t.hat. of the cthionillc sulfor~ haJM1.This was n~~kcdly so in ihc wsc of the c~t.hioJJiJJc-rcxistsnl cult~ures (set I-& 2). ‘l’hc t,ric~\rloro:~~c!t,i(.a.(%-soluble fractions of lhc ycast.s, a.ft.cr rcJlJovs1 of the trichloroacct ic: acid by cthcr cxtract.ion, wrc conwJrtrat.,c:d, oxidized wit,h performic acid, and c*hrolJJat,ographed iJ1 a similar nwttlcr. Apwcrc prcciablc amounts of cthioninc-% found to be present, together with s111al1 mJounts of free labellcd cyst,inc ;III~ JJmthionilic, and also several sulfur-c~oJit:iirlirl(S nJctabolites as ye1 unident~ific~d. 01Jc of thcsc had a high mobility in the butanol::tcetic ncid:wuter eolvcnt, and wx5 present, in rclalively lnrgc MIJIOUIIIS.The spc~lt growth nw~liuni also c~oJJtaiJict1traws of labelled
nwthioninc olitc. Distribution
and the un1alow1J ~JJa,jornAsboJ ethioninr4
i/i
pwtein.
The distribution of labelled sulfur between the c-ystine, methionine, and cthionine of (~11 protein was assessed quantitat ivrly by m1t.tiJJg out t,he radioact~ivc bandy from each c*hrolJJatogram, colrlbust,irlg then1 to sulfate, :~rtl tlrterriiinirJg the Y3 of cnch hrd iJJ t,hc for111 of bcrizidinc sulfate by ~w-~vintIow cmmting. The activity of cac*h band was cxpreseed as a. perwJJtnge of the sum of tht: :wt,ivit~itrs of the three balids on wc*h chronwtograrn. -1s seen from the sunmmizctl rcaults in Ttlhlcs II and III, sulfur tmrsfcr was greatly increased after a longer grow-th period jn the c,thiorJinc-corltnilliJlg nledium, and was also substantially higher in ~11s adaptcad to ct hionirw prior to tlw growi,h cxperimwt. Thus in C. utilis the fraction of incorporated sulfur prcscnt a~ cyatinr + ni($hioJii~Jc row from sonic 1.55 to about 50% on prolonged grow-i-tli, a11t1 in iho
ETHIONINE SULFONE t a “3 ‘r 2 u
ETHIONINE-SENSITIVE
z 0
CYSTEIC ACID
Grovn
METHIONINE
C. UTILIS
for ‘2 days
5 0 E c t, >
b-
ETHIONINE-SENSITIVEC. Grown
UTILIS
for 7 days
6 E 2 >”
.; ki a m ; a v
+ ETHIONINE-RESISTANT Grown
C. UTILIS
for 7 days
2
z
‘2 :i i 2
0.15li 0.312 0. 1X
0.312 0.1% o.?J12 0.15Cl 0.312
0.5
4. fi 11.” 7 5
24.8 1x.1 15.8 11.5
10. :i 8.2 I(i.3
12.4 29.5 26.5 15.8 19.0
X3.2
Xi.2 7” .5 x0.1
O..5ti 0.60
45.7
55.4 08.4 09 .5
L? FE
0.68 0.61
c,tltiottitto-rclsislattt straiti roscbto 90 I;‘. I’rottt the values ohtxitied for the prot.citt-S (aotttent of the yeasts, quotml in XI e:trIior SWtion, logcthcr with the ratios for cystinc-S/ in ntethiortittc-% it, protein detrrniitictl previous ctxpcriiim~ts (1 1, 13)) t,hc ctst.cttt. of replacement of proteirt-ttlcthiorrirlc by ethionine iu t,he lwo cthiortittc-sensitive c.ulturcs grown in the presence of 0.312 rmt sulfate for 2 days was dctertttittcd. In S. cmvisiae the rcplac~cmelt~ W:LS21.0 ‘,‘; under these condit.ions, and in C. utilis, 12.2 % Calculations were also made of thcl pcrcmtagc~ rcpl:mnmlt of the total caystitie and uiethionine of cell protein by the cystitta and mcthiotlinc derived exc~lusively front ct~hionine-S. The values obt:~ined wcrc, for S. c’ej.eUisjae: cyslinc 13 “;, tttet,hiottittc 2.9 :‘;I; for (1. utilis: cystitte 1.1 ‘:;, nxthiortit~c 1.5 c/c.Those rc~prcsent,titinitttuni rcplamttctits, the values for 3 7-day growth period bci~ig ;tt. Ic:M three 1inx.s as great. Tables II and III also show l,hat the ratios of c*yst.inct-S derived fro111 ethioninc-S t’o n~cthionim-S derived frottl t,he same sourm rangod frottt 0.56 to 0.6s (average 0.61) for A’. ce~visiac and 0.61-0.79 (:~vcrsgc>0.70) for C. utilis wheu Ihe yea& were grown in the presmc*c of 0.312 nm sulfate, i.e., in the lmxmce of art adequate supply of sulfur. It, is signifirnnt that, t,hesc ratios arc c4ose to those whi(ah have been obtained for proteincysl6ue and -mct,hioninc derived front sulfatcl nan~ly, 0.63 :tttd 0.79, 01 nttrthiortinr, rcspe~t ivcly (11, IS). The sittti1arit.y between the two sets of data would he :~cY~outlt~ed for
if ethiottitte wcrc to be t~~c~t:tholizctl to ;m itttc1rtttcdiat.e which is a (‘onttttott prt~c~utsorof both cystine and nlethiottittrl rind which is also on the pat.hwv:ly of Ittct~hiottinc synthesis frottt sulfat,c. The c~onrpountl considered tttost likely to fit the role of itttc~tmlcdintc is horttoc~ystrine. C’l,tde
o\ dhimine
hy ccl1 s~~spensions.
The differert~cs itt ittc.orpotxt iott and distribution of ethionitto-8 iIll- protciti shown b? ctthiottitlc-scttsitivc arid c~tliiotijnc-resistallt mltures protttpted an esatttinntiott of the nmutlulat,iott of the anritto :tc*itl by t,hesc yeasts. _As shomti in Fig. 3 tn:uked diffetmiccs iii 1tie rate of uptake n-cre fnutid for hot h S. wcisiae and C. utilis. In o:ic~h case t,licrc~ was a very rapid ittitixl :tc~c,ttttiulatiott of t hc lahclled ntiiino acid by that et hiotiitictsctisitive (*ells, followed b?- :t rel~me itito tlte uicdiuni of ;I, small p:trt of th(a Mx~llctl sulfur, probably in -the forttt of t~1e!.aboIit,es of cthioniiic, whicli wcrc’ also dclcc*tcd in thcl ~11 fluids, as refcrrctl to itt lhc previous soc4ou. The cthiottinc~-rcsist:tttt m~l~ttrcs;,011 the other hand, iook up the mttitto Llt*id only very slowly. Fur/her exanGzation of cthiotl it/e-resistant cultures. Sitnilar uptake cxpcriiim~t s ott
labellrtl sulftttc: revealed :I considerable differcrtc~e it1 acmtrtulatitlg ability bet-wcetr ethiotlittc-sc,tlsii ive and etlliotlittc~-~esistatlt; cult,ures in the cast of C. utilis, although this was not as well tmrkcd in X. ccr~iszke (SW Fig. 4). The storage of glycaogen by the *J’.cerccisiue cultures was cxnnGned by Dr. IV. E. Chest.cr
Uptake img S/w dry
Uptake (w S/w dry
E-sensitive I------
5. CEREVISIAE
C. UTILIS
1
/
/ 0.5
1 Time (hr.)
1.5
0.5
2
1 Time (hr.)
1.5
2
uptake hs V’gm d wt.
Uptake img ‘%‘gm dry st)
2
I
C. UTILIS
1
1
2 Time (hr.)
3
4
1
2 Time (hr.)
3
FIG. 4. Uptake of NaPSO by ethiorlille-serlsitivc and 0.78 IBM ethioIli]le-rcsist,allt yeasts. Temperat lws L:rMlcd s111fate in rnedirlrn 0.312 xn~. l’cssts 27i (ml wt. pressed ycwt/vol.) 30".
4
NOW
Sulfate (0.312 mhl) L-Ethionine (0.78 nlM) Sulfate (0.312 mu) + L-ethioninc (0.78 IIIM)
2.2 56.4 2.2
1.7 57.2 2.0
3. 1 2.9 50.0 ~ 5;:; 3 .4
30.6
5-1.8
32.4
44.0
a Growth expressed as mg dry wt. cells per 10 ml medium. 6 Grown for 2 days at 30”. c Grown for 3 days at 30”. d Yeasts resistant to 0.78 1n.q r,-ethioninc. of this Laboratory. The applic~ation of an iodine staining t’ec*hnique irldicat,ed that ethionine-rcsist.ant cells grown in the cthioniIle-supplemented medium cont,ained only negligible quantities of the polysaccrhsride, cells to et,hionir~c-scllsitivc iu contrast in the normal sulfat,e-c~ont,ninirlg grown medium. This difference in glycLogen storage may reflect a defic*ienc*y in ATE’ available for glucose ut’ilization in cells grown in the presence of ethioninr, due to the ATPt,rapping action of the alnitlo acid. In view of the ability of the et,hionineresist,ant yeasts to fornl caystine alld Ill&Coniue from et~hioniu&, ctShionine was tested as a source of sulfur for growth. The resistant~ cells grew well in the presence of a nlixture of L-ethionine and sulfate (Table IV), but failed to grow when sulfate was omitted. The synthesis of rystine and nlethionine by this pathway thus appears inadequate to rncet growth requiremrnts. I>ISCIJSSION Evidence is available of several pathways for the nlet.abolism of ethionine. So far, how ever, there is no calear proof of the existence of erlaynles specific for ethiouine breakdown, :UI~ this mrlirlo acid irl its various reac4ons
Sol~ic yearn ago Shun arid I,r\vis C11) obscrvcd dranliriatioll of eihiorrino to the comespondjng cu-keto uc.jd ilr the al1i11ral. C’lcav:~gc ac*iti to ethatlt$hiol by live1 of thtl ~III~IIO extnicts wtls 1:ttc:r Irportec! by Hiulilcy (15). In atldit.ioll, Sttkol and FVciss (16) found tdlat “Wabellctl cthioninc atlnlirlistc~rc~d together with bro~~~obmxerlc to the rat rcsultcd in tl~ uriilary cbxc*rotjiorl of “%labellcd p-bronlopllerryllller(.~~~)turi(~ ac*id, mhicah inlplied that ethiotlilic-Yulfur was :~vnillthlc for rysteine syii thesis. Alore recently cthionine has been shown to reuc+ with ,IITI’ in the prescmc of an ac+ivating systtrljl from several aninittl species and also yeasts, the producbt being S-aderiosylcthiollirIc (17mm21). The cauzymc involved appears to bc the mcthionincartivatiug systelll (17, 21). Further Illrltnbolites derived fro111 S-adcrlosylethioilillc, illelude S-adcriosylhol~lo(~.ysteille, following loss of the ct.hyl group to au ;rppropriatje :W c,cptor rilolet*ulc (19, 2), alid also T,‘-ethylt.hioaderlosine (19, 23, 23) and 5’.cthylt,hioiriosine (25). The biochemical simi1arit.y of et,hionine to methionine is further apparent fro111 the ability of the ethyl analogue to replacse methioninr residues to a substantial exknt in thr protcjns of tllany specks (2- 7). The experiments described here extend this finding to the ye&s 8. cerevisiae and c’. ufiks. The present. work, together wit)h that of others (26, 27)) shows that yeasts coin be adapted to grow in the presenchc of high concaentrations of ethionine. Vronl the satisfnc.t,ory growth obtained it is apparent, that t’hc which are prokiiis clthioliirle-corltaillirlg synthesized untlcr these caontlit ions arc not noticeably toxic t.o the orgznnism, as has been suggest,ed (2S), :u~d this is in c*onformity with the observation of Yoshida (5, 6) t)hat, a-anlylascl fro111 Bacillus subtilis, in which one-third of the nlethioninc residues had been replaced by ethionillc, posstxssed tht propertics of Ihc Ilortlial crtzy~t~c*. The present st.udies have rtave:dcd :L further pathway for (,t hioninc nlrtaholislll in
yeasts, ~tanlely that whott ihwc orgattistw are g-on-n itt the presetiw of etl~iotriric, part of t,he sulfur of the atnitto ac*id appears in the cystine and tl~et’hiottitte of the caytoplasttt and of the protein. It is sigttifkattt that the (Lystinc and tttcthioninc dcrivetl frotti cthiottinc arc prcwtlt in the protein in tlrc: WIN proportions as ;tre the csystitie and tttcthionine derived frottl sulfate or tttethiottittc. This 11.ould be cqx1c.t cd if the t,ratlsfcr of &ionine-sulfur to thcw attlitto acids tool; pl:w~ via sonw cottittwti internxdinte, atid if this cotnpound w-erc also on the ttonttal pathway of sulfur :u~~itio wit1 synthesis front sulfate. Hotnocysteitw is suggested as the ~ttost likely intcrnwdiatc, arid it,; fortiiatioti frotti ethioninc is hclirtvcd to take pl:wc~ by the following txwtiott scqwitw: Etllionine
ATI’ --I
S-Adellos!lrthiotliIlc
With the fortnation of hontoc*ystcinc, cottversion to cystcittcs atitl niethionirte iSteps IV and T’) would then prowed by the n-cllS-Adenosylethionine -
l’arlis (19) to produw :qqxec~iable quatttitics of tttrthiottittc. Thcl following tncchanisrn was suggest,cd :
S-Adenosylhomocysteine Homocysteine
wt.ablished pathways of’ traits-sulfuratiott and nxthylntion, respwtivcly. Reaction I has been identified it1 scwral species and in yeasts (17-21). Step II, a transethylntion, has also bwn well nuthcttt,icated in ninny spwies iii rewtit years (16, 29-33). Furthcrntorc, it1 yeasts hottlocaystcitle itself can nest as the ethyl acceptor, giving et,hionine (19, 22). The cleavage of B-ndettosylhotnoc~yst&te t.o hotnoc*ysteine (Sirp III) has not as yet been satisfactorily confirttlrd and nierita further study. Duerrc and Schlcnk (34) ohtaitted ittdiwtion of sottt(L bwalxlowr, I)urticul:zrly it1 C’. ufilis. Howcvcr, thcw workers faikd to ohsorw t 11~
_
- S-Adenosylmethionine Methionine
X-Xtlcrtosylcthiottiti(~ was ctivisaqgd :ts uudergoittg dc-cthylnt’ion to S-adetwsylhomoc*yst~eittc~,mhicsh is nlethylat~cd to the tact-hiottitte derivative, whic~h acts as tnct’hyl dottor to hotttocyst~eitw. It is possible that, this squcttw was operative under the in vitro c*ottdit,ious chosen, but it’ could not :wwuttt. for the ttwthiottinr: synthesis frown cthiottittt iti whole ~11s described in this paper, sittcc itt I’arl~s’ whence t.he sulfur of the ilwthiotlino would obviously originate front c~ridogeriously fortitctl honioc~ystc~int: and not, frotn ethiotritic.
25. ‘LCi.
2. 27.
3 2s.
*5
!I. 10. 11. 12. 1:s.
14. 15. Iti. li. 1X.
I!). 20. 21.
and
Distribution
Ethionine-Sensitive
of Ethionine-Sulfur and
Ethionine-Resistant
in the Protein
of
Yeasts
15thiollitie-resistant cultures of Saccharomyces cPieDLsiue and Cundida utilis \vere obtained by growing the normal yeasts in gradually increasing concentrations of Lethionine. The incorporation of et hionine-sulfur into the cell protein of cl hionincresistant S. cerevisiae u-as found to be less than that of normal cells, whereas thcrc was no difference bebeen normal and resistant cells of C. &/is. The uptake of ethioninr by suspensions of resistant cells of both yeasts was markctlly less than that of t hc normal yea&. Examination of the cell protein of yeasts grown ill the presence of 35S-labelled Lclthionine showed the presence of ethionine itself, which had replaced mrthionine residues, and also small amounts of lsbellcd cystinc and methionine. The labclling of protniwcystinc and -methionine was more rxtensive in ~11s grow, for prolonged periods in the presence of cthioninc, and was also greater in resist alit cells. The formation of cystine and methionine from ethioninc in thcsc yeasts is thollght to occlw through the intrrmcdiate formation of S-ad(~nos~-lcthio~~i~~~ and its convcrsioll to homocysteine. I31hionine resistance in these orgsnis~llS is characterized l)y a d~w+cesod :tl)ility to acc~m~~~late cthionine and an elthanccd abilii y to nlctaholizc~ ~h:tt amollllt of tlw amino acid ahich dots ent,cr t,he cells.
During a study of the uptake of various sulfur-c,ollt:liniIlg unlino acids by a brewer’s yeast (I), it was shomn that ethionine is accumulated by cell suspensions in the presenw of a suit.able energy source, part, of the sulfur of the amino acid appcnrillg ilk ccl1 prokin. Howcvcr, the form in which this incorpornt,ed sulfur was prcscnt~ was not) investigated. Data obtained with a variet,y of other organisms have established that cthionine per SCcan rcplwe methioninc rcsiducs in proteins (a-7), and there is aii~ple cvidencc that rthioninc can bc nlctjabolizcd by :I number of pathways (S, 9)) but, its wtuul conversion to other
transfer to ot.hcr amino acids occurs. X caomparison has also been n~(lo of ethioninc uptake and incaorporation into ethionincscnsitivc and cthionirlc-rtsititallt ycnsts.
Ytxsf and grouth wclia. The yeasts used were a (;uilnwss strain of Swcharowa?Jces c~erwisiae and a strain of Candida utilis (XCTC 357(i). Each organism was groxx from a small inocitlllm at 30” in a mcxdi\lrn containing 0.312 mhI ?;a$01 (rqllivalcnt to 10 mg S per liter) as dcscrilxd previously (I), and s\thcrlltluw wrc madr~ every 2-3 days.
period of 3 ntottths. These yeasts grew acll and showed :i normal appearance. Cho~ic~tla. l.-Et hiolrinc-“5s (5.0 mC per millimole) antI sodium srtlfate-::“$ (carrier-free) were ohtaincd from the l~adiochcmical Ccntcr, Amersham, l
for
eth,ioninP-S
incorporation
experi-
m2~t.s. The J-casts were grown in 25.ml conical flasks containing a total flttid volume of 10.5 ml, consisting of 5 ml of mcdirtm of twice the required strength and 5 ml of L-cthionine3%S diluted to 0.5 mC per millimole with thr tmlabelled amino acid in sterile water. The final concentration of rthionine was 0.78 111~ (equivalent to 25 mg S per liter). Two lcvcls of sulfat,e wcrc used in the medium: 0.156 mu. givitlg sttboptimtml growth, and 0.312 IBM, which was just adequate for optimum growth (10). 1’:ach flask was inoculated wit,h 0.5 ml of a sttspcnsion of salitrcwashcd yeast containing 0.3 mg dry weight of cells per milliliter, and the flask w-as agitntcd for 2 days at 30” in a shaking bath prc,viding 100 linear oscillations per minutc. Yeast growth was then determined turbidimetrically(lo), and a sttitable volume of ccl1 suspension it~sttally 57 ml) was cooled in ice and centrifuged. The resulting yeast pellet was rcsttspendcd in 10 ntl ice-cold 5:; (w/v) trichloroacetic acid for 2 hours with occasional st,irring. The prccipitnte was then ccntrifugcd, washed by two rcsuspcnsions in ice-cold saline, transferred with 10 ml water to a 30.ml hcaker, and ashcd in a furrtace at X0” with a crtpric nitr:ttc-potussirlrrl chlorate ntistllre, as descriljed previously (1). The lubcllcd stllfst,c so ohtaincd was made rtp to 10 ml with water containitrg 2.25 mg N&O, carrier, precipitated as \)ctrzidine sulfate, and counted with a thin-witalow Geigcr~RItiller tube (1). Samples of t)he origitral labelletl cthionine solrttion were also oxidized and assayed for 3% after sttitahle dilution. The cxtcnt of incorporntion of et,hioninr-S into protein was obtained from the percentage of 3% from t hc medium appearing in the trichloroacctic acidinsoluble fraction of the cells. E’rocedu,rc jar ethionined distribution ezperimerits. The yeasts were grown at, two sttlfatr concentrations, as in the previous scrtiott, the media in this cast Ireing supplemented w-ith 0.78 mu Lethionitt(,-“5S (5.0 mC per millimolc) rtndiltttcd with carrier ainino acid. Yeast growl h was dcfcrmined after 2 days, and samples of sttspcttsion contairring approximately C,mg dry weight, of cells wcrc rcmovcd and centrifuged. The sttpernatant mcditlm was retttrned to the flask and growl h was allowed to cant inoe ; furthrr sanrples corrc-
spending to fi nlg dry w-eight, of cells wcrc I hen taker1 at intervals. Each yeast pellet, was trcatcd with t richloroacetic acid for 2 hours at 0”, as in t hc previous section, atrd t hc saline-wash4 prccipi tntc was than oxidized with pcrformic acid atld hydrolyzed with ti N HCl as dcscribod clsewherc (11). The oxidized sulfrtr amino acids in each prcttcirt sample were separated try orlc-dilnensioll:Ll descending chromatography 011 a 1.5 inch-wide strip of Whatman 3MM paper in n-bllt:rrlol::tc.etic acid: water, 12:3:5 (v/v), for 20 hottrs at 18”, the solvent being allowed to drip off the paper. The dried paper st,rip was then scanned for radioactivity on a Forro radiochromatograph scanner fitted with a windowless gas-flow cormtcr. l~atlioact ivc bands located in this way were cut from the chromnt.ogram ami combusted separately by t hc Schijnigcr oxygen flask techniqtte, in one-liter flasks colttaming 5 ml water and 4 drops 3Oyn (w/v) hydrogun peroxide. The rrsttlting labcllcd srtlfatc was finally converted i o its benxidine salt, and assnycd for 3%. The activity of each band was cxprcsscd as a perccnt,age of the total activity on th chroma togram. and suljate b,y cell susI’ptake of L-ethionine pensions. The accumulation of L-ethioninc-““S and of lahelled sulfate by cells as :I function of time was exam&d by the procedure described in carlicr papers (1, la), ttsing washed yeast snspensions (final concentration in medium, a!:;, wet weight, pressed yeast, per voltmlc). LWS ULTS
Incorporation
of ethionine-.sulj”ur info yeast
protein. Ethionirte-sensitive and ethionirrcresistant, cultures of H. ce~ez,isiae and C. utilis were grown for 2 days at 30” from small inocula in two media containing 0.156 and 0.312 IIM sulfate, respectively; both media were supplemented wi-ith 0.7s m.\r L-ethionine-35S (specific activity, 0.5 mC per millimole). The extent of incorporation of cthioninc8 into cell protein was ohtaincd from the ratio of the 36S prcscnt in the t,richloroacctic acid-insoluble frwtion of a known weight of ~11s to that’ initially present in t.he media. This was cxprcssctl as milligrams sulfur per gram dry weight of yeast. Table I gives the sumn~arixcd data obtained. In every case there w:ts :t dwreased incorporation of ethionitu-S at the higher sulfate conccntjrat ion, which mflcct s the incrcascd cornpetition for prot,cin formst,ion
of the prwious finditlgs of other workers on cthiotiilic inc9rporation, it was cxpwtcd that :u&sis of the yeast prot8cin ill the prwcnt c~xpcriiiients would reveal t hc prcwnw of I:~bcllctl cthioninc. To verify this, 3 ~i~~thod wts tl~vclopc~l to scpar:~tc ct~liioniiw front the norlnal sulfur corns. cerwkio~ J)OMTltS, cay&tie and ~ncthionilw. Satis0.1x 0.40 ~:thioJtitte-scttsitive f:ict.ory rwolutio~i was obt,airwd by first 0.:‘,12 0. -13 oxidizing nlixturcs of Ihc three amino acids 0.1x 0, :3:; 13csistaJtt IO 0.78 Illll with porfornlic wit1 and t,hw~ chromatjo0.312 0.27 cthiuttitte graphing the products on Whuttnan SAIL\1 0.1x 0.25 ICcsistnJtt~ to 7.8 tn.\1 paper with n-butanol: :ic:ct.ic wid : w’:~1w 0.1t; 0.312 cthiotlitw (12:3: 5, v/v) :LSsolvent. Treat~ncrlt of the c. 11ti1is ~hroinat,ogr:ui~s wit,h ninhydrill gave 1hrec 0.1x 0.48 Ethiottinr-rcnsit ive 0.312 0.27 bands with IiF values of 0.10, 0.25, ant1 0.33, 0.156 0.4 Rcsistntlt~ to 0.78 1tlM corrcspouding to cysttic acid, niethionine 0.312 0.24 cthiottinr sulfotlc, and ct,hiolline sulfonc, rc:spcc:t~ively. 0.13; 0.54 Rrsistattt tu 7.8 III&< Grwtly inlprowd sep:u‘at.iolr of’ the oxidaet hiortinr 0.312 0.2ci tion -product.t.s was obtninctl by using dewending c,tlrotli:Ltograplly for 20 hours and allonitlg the solwrlt. to run off the paper. Th(; yeasts wcrc grown in the two sulfatctby the sulfur amino wids formed through c~ontammg nlcdia referred 1.0carlicr, supplct,he normal sulfntc reduction pathway. The ment.cd with O.iS 111~ r,-ct hiotGr~e-R%, of sulfate effect WAR part,icularly nolkcablc spcGficL wtivity 5.0 mC per n~illimole. After with C. ulilis, :t decrease of 43-52 54:rcsult.ing 2 days samples containing about, 6 mg dry from :L doubling of the sulfate concentration weight of cells mcrc taken, and the wvashcd in the nrediunl. In S. cerevisiae an added trichlorowctic acid-insoluble: fraction was fcal.urr was a decrease in the incorporation oxidixcd with performic acid and hydrolyacd of cthioninr-S by the et,hioninc-resistalIt with HCI. The produck, together with 10 ~1 cult.ures, the effect; being lnarkcd in the of a mixture of cbysteic acid, methionc sulfonc, more highly resistant cells. So significant and etjhioninc~ sulfone (cnch 0.1 %, w/v), w(w differences in incorporation wre obtained then c,hromatographctl in the descending tliwith the c’. utilis cultures. as described above. The chromatoDcternlinations have hcen made of the r&ion grams wcw wntmcd for r:adioactivit,,v and tot’al sulfur contents of 1he yeasts when subscqucntjly trcnted with ninhydrin. grown for 2 days in t-he presence of 0.:312 ,111 c~hrw~lntogranw should a large zone mnI sulfate. The values obtained were, for c*oilwid(lllt with the nirlX. cerevisiae: 3.6, and for C. u?ilis: 4.2 mg per of radion&vity, hytlrirl-positive band of highw;t nlobility, gram c1r.yweight of ~11s. The corresponding that c*orrwponded to nt hioninc sulfone and ratios 01 protein-S per total-S were: found so c*onfirnlotl t,lw prcwnw of prootcin-bouttd to be SO and SO.6%, giving protein-S values for the t,w-0 yeasts of 238 3ncl 3.31 nlg S ct,liioniti(>. In :rddition, :IS seen ill Fig. 1, two sniallcr rcgiolis of radiowtivity c~oillcitlctl per gram dry xvcight’ of wlls, rcspwtiwly. with the qwtcic wit1 ant1 nlethionine sulfow Thcsc values were used lo calcul:~lc the bnnds, indiwting a Inbelling of protcinfra(+ons of protein-S dcrived from cthioninccyst inc and -nlcthionitlr. The total radioS in the cthionirlc-sensitive cultures grown in the presence of 0.312 ~1131 dfatc. These activif.y prewlt in the protein frwtion ~v:r;ls valuc~ wrc 14.9 ?: for S. cewvisiae md S.0 C(; confined to I 11~s~t,hrw hands. for C. ufilis.
ETHIONINE-SENSITIVE
CYSTEIC ACID
METHIONINE SULFON E
S. CEREVISIAE
ETHIONINE SULFONE
Grown for 7 doys t ? 6 v,
ds, :iJld growth was nllo\ved to c~ont.iJluciJJ the sanle nledium for further periods of up to 7 days; smnples wcrc tnlierl and trcat.ed as previously. l3xaminat.ion of the rem11iJJg chronJntmogranJsrevealed tZhat the act.ivities of the caysteic wit1 and nwt,hioninc sulfone bands had incrcascd rclativc t.o t.hat. of the cthionillc sulfor~ haJM1.This was n~~kcdly so in ihc wsc of the c~t.hioJJiJJc-rcxistsnl cult~ures (set I-& 2). ‘l’hc t,ric~\rloro:~~c!t,i(.a.(%-soluble fractions of lhc ycast.s, a.ft.cr rcJlJovs1 of the trichloroacct ic: acid by cthcr cxtract.ion, wrc conwJrtrat.,c:d, oxidized wit,h performic acid, and c*hrolJJat,ographed iJ1 a similar nwttlcr. Apwcrc prcciablc amounts of cthioninc-% found to be present, together with s111al1 mJounts of free labellcd cyst,inc ;III~ JJmthionilic, and also several sulfur-c~oJit:iirlirl(S nJctabolites as ye1 unident~ific~d. 01Jc of thcsc had a high mobility in the butanol::tcetic ncid:wuter eolvcnt, and wx5 present, in rclalively lnrgc MIJIOUIIIS.The spc~lt growth nw~liuni also c~oJJtaiJict1traws of labelled
nwthioninc olitc. Distribution
and the un1alow1J ~JJa,jornAsboJ ethioninr4
i/i
pwtein.
The distribution of labelled sulfur between the c-ystine, methionine, and cthionine of (~11 protein was assessed quantitat ivrly by m1t.tiJJg out t,he radioact~ivc bandy from each c*hrolJJatogram, colrlbust,irlg then1 to sulfate, :~rtl tlrterriiinirJg the Y3 of cnch hrd iJJ t,hc for111 of bcrizidinc sulfate by ~w-~vintIow cmmting. The activity of cac*h band was cxpreseed as a. perwJJtnge of the sum of tht: :wt,ivit~itrs of the three balids on wc*h chronwtograrn. -1s seen from the sunmmizctl rcaults in Ttlhlcs II and III, sulfur tmrsfcr was greatly increased after a longer grow-th period jn the c,thiorJinc-corltnilliJlg nledium, and was also substantially higher in ~11s adaptcad to ct hionirw prior to tlw growi,h cxperimwt. Thus in C. utilis the fraction of incorporated sulfur prcscnt a~ cyatinr + ni($hioJii~Jc row from sonic 1.55 to about 50% on prolonged grow-i-tli, a11t1 in iho
ETHIONINE SULFONE t a “3 ‘r 2 u
ETHIONINE-SENSITIVE
z 0
CYSTEIC ACID
Grovn
METHIONINE
C. UTILIS
for ‘2 days
5 0 E c t, >
b-
ETHIONINE-SENSITIVEC. Grown
UTILIS
for 7 days
6 E 2 >”
.; ki a m ; a v
+ ETHIONINE-RESISTANT Grown
C. UTILIS
for 7 days
2
z
‘2 :i i 2
0.15li 0.312 0. 1X
0.312 0.1% o.?J12 0.15Cl 0.312
0.5
4. fi 11.” 7 5
24.8 1x.1 15.8 11.5
10. :i 8.2 I(i.3
12.4 29.5 26.5 15.8 19.0
X3.2
Xi.2 7” .5 x0.1
O..5ti 0.60
45.7
55.4 08.4 09 .5
L? FE
0.68 0.61
c,tltiottitto-rclsislattt straiti roscbto 90 I;‘. I’rottt the values ohtxitied for the prot.citt-S (aotttent of the yeasts, quotml in XI e:trIior SWtion, logcthcr with the ratios for cystinc-S/ in ntethiortittc-% it, protein detrrniitictl previous ctxpcriiim~ts (1 1, 13)) t,hc ctst.cttt. of replacement of proteirt-ttlcthiorrirlc by ethionine iu t,he lwo cthiortittc-sensitive c.ulturcs grown in the presence of 0.312 rmt sulfate for 2 days was dctertttittcd. In S. cmvisiae the rcplac~cmelt~ W:LS21.0 ‘,‘; under these condit.ions, and in C. utilis, 12.2 % Calculations were also made of thcl pcrcmtagc~ rcpl:mnmlt of the total caystitie and uiethionine of cell protein by the cystitta and mcthiotlinc derived exc~lusively front ct~hionine-S. The values obt:~ined wcrc, for S. c’ej.eUisjae: cyslinc 13 “;, tttet,hiottittc 2.9 :‘;I; for (1. utilis: cystitte 1.1 ‘:;, nxthiortit~c 1.5 c/c.Those rc~prcsent,titinitttuni rcplamttctits, the values for 3 7-day growth period bci~ig ;tt. Ic:M three 1inx.s as great. Tables II and III also show l,hat the ratios of c*yst.inct-S derived fro111 ethioninc-S t’o n~cthionim-S derived frottl t,he same sourm rangod frottt 0.56 to 0.6s (average 0.61) for A’. ce~visiac and 0.61-0.79 (:~vcrsgc>0.70) for C. utilis wheu Ihe yea& were grown in the presmc*c of 0.312 nm sulfate, i.e., in the lmxmce of art adequate supply of sulfur. It, is signifirnnt that, t,hesc ratios arc c4ose to those whi(ah have been obtained for proteincysl6ue and -mct,hioninc derived front sulfatcl nan~ly, 0.63 :tttd 0.79, 01 nttrthiortinr, rcspe~t ivcly (11, IS). The sittti1arit.y between the two sets of data would he :~cY~outlt~ed for
if ethiottitte wcrc to be t~~c~t:tholizctl to ;m itttc1rtttcdiat.e which is a (‘onttttott prt~c~utsorof both cystine and nlethiottittrl rind which is also on the pat.hwv:ly of Ittct~hiottinc synthesis frottt sulfat,c. The c~onrpountl considered tttost likely to fit the role of itttc~tmlcdintc is horttoc~ystrine. C’l,tde
o\ dhimine
hy ccl1 s~~spensions.
The differert~cs itt ittc.orpotxt iott and distribution of ethionitto-8 iIll- protciti shown b? ctthiottitlc-scttsitivc arid c~tliiotijnc-resistallt mltures protttpted an esatttinntiott of the nmutlulat,iott of the anritto :tc*itl by t,hesc yeasts. _As shomti in Fig. 3 tn:uked diffetmiccs iii 1tie rate of uptake n-cre fnutid for hot h S. wcisiae and C. utilis. In o:ic~h case t,licrc~ was a very rapid ittitixl :tc~c,ttttiulatiott of t hc lahclled ntiiino acid by that et hiotiitictsctisitive (*ells, followed b?- :t rel~me itito tlte uicdiuni of ;I, small p:trt of th(a Mx~llctl sulfur, probably in -the forttt of t~1e!.aboIit,es of cthioniiic, whicli wcrc’ also dclcc*tcd in thcl ~11 fluids, as refcrrctl to itt lhc previous soc4ou. The cthiottinc~-rcsist:tttt m~l~ttrcs;,011 the other hand, iook up the mttitto Llt*id only very slowly. Fur/her exanGzation of cthiotl it/e-resistant cultures. Sitnilar uptake cxpcriiim~t s ott
labellrtl sulftttc: revealed :I considerable differcrtc~e it1 acmtrtulatitlg ability bet-wcetr ethiotlittc-sc,tlsii ive and etlliotlittc~-~esistatlt; cult,ures in the cast of C. utilis, although this was not as well tmrkcd in X. ccr~iszke (SW Fig. 4). The storage of glycaogen by the *J’.cerccisiue cultures was cxnnGned by Dr. IV. E. Chest.cr
Uptake img S/w dry
Uptake (w S/w dry
E-sensitive I------
5. CEREVISIAE
C. UTILIS
1
/
/ 0.5
1 Time (hr.)
1.5
0.5
2
1 Time (hr.)
1.5
2
uptake hs V’gm d wt.
Uptake img ‘%‘gm dry st)
2
I
C. UTILIS
1
1
2 Time (hr.)
3
4
1
2 Time (hr.)
3
FIG. 4. Uptake of NaPSO by ethiorlille-serlsitivc and 0.78 IBM ethioIli]le-rcsist,allt yeasts. Temperat lws L:rMlcd s111fate in rnedirlrn 0.312 xn~. l’cssts 27i (ml wt. pressed ycwt/vol.) 30".
4
NOW
Sulfate (0.312 mhl) L-Ethionine (0.78 nlM) Sulfate (0.312 mu) + L-ethioninc (0.78 IIIM)
2.2 56.4 2.2
1.7 57.2 2.0
3. 1 2.9 50.0 ~ 5;:; 3 .4
30.6
5-1.8
32.4
44.0
a Growth expressed as mg dry wt. cells per 10 ml medium. 6 Grown for 2 days at 30”. c Grown for 3 days at 30”. d Yeasts resistant to 0.78 1n.q r,-ethioninc. of this Laboratory. The applic~ation of an iodine staining t’ec*hnique irldicat,ed that ethionine-rcsist.ant cells grown in the cthioniIle-supplemented medium cont,ained only negligible quantities of the polysaccrhsride, cells to et,hionir~c-scllsitivc iu contrast in the normal sulfat,e-c~ont,ninirlg grown medium. This difference in glycLogen storage may reflect a defic*ienc*y in ATE’ available for glucose ut’ilization in cells grown in the presence of ethioninr, due to the ATPt,rapping action of the alnitlo acid. In view of the ability of the et,hionineresist,ant yeasts to fornl caystine alld Ill&Coniue from et~hioniu&, ctShionine was tested as a source of sulfur for growth. The resistant~ cells grew well in the presence of a nlixture of L-ethionine and sulfate (Table IV), but failed to grow when sulfate was omitted. The synthesis of rystine and nlethionine by this pathway thus appears inadequate to rncet growth requiremrnts. I>ISCIJSSION Evidence is available of several pathways for the nlet.abolism of ethionine. So far, how ever, there is no calear proof of the existence of erlaynles specific for ethiouine breakdown, :UI~ this mrlirlo acid irl its various reac4ons
Sol~ic yearn ago Shun arid I,r\vis C11) obscrvcd dranliriatioll of eihiorrino to the comespondjng cu-keto uc.jd ilr the al1i11ral. C’lcav:~gc ac*iti to ethatlt$hiol by live1 of thtl ~III~IIO extnicts wtls 1:ttc:r Irportec! by Hiulilcy (15). In atldit.ioll, Sttkol and FVciss (16) found tdlat “Wabellctl cthioninc atlnlirlistc~rc~d together with bro~~~obmxerlc to the rat rcsultcd in tl~ uriilary cbxc*rotjiorl of “%labellcd p-bronlopllerryllller(.~~~)turi(~ ac*id, mhicah inlplied that ethiotlilic-Yulfur was :~vnillthlc for rysteine syii thesis. Alore recently cthionine has been shown to reuc+ with ,IITI’ in the prescmc of an ac+ivating systtrljl from several aninittl species and also yeasts, the producbt being S-aderiosylcthiollirIc (17mm21). The cauzymc involved appears to bc the mcthionincartivatiug systelll (17, 21). Further Illrltnbolites derived fro111 S-adcrlosylethioilillc, illelude S-adcriosylhol~lo(~.ysteille, following loss of the ct.hyl group to au ;rppropriatje :W c,cptor rilolet*ulc (19, 2), alid also T,‘-ethylt.hioaderlosine (19, 23, 23) and 5’.cthylt,hioiriosine (25). The biochemical simi1arit.y of et,hionine to methionine is further apparent fro111 the ability of the ethyl analogue to replacse methioninr residues to a substantial exknt in thr protcjns of tllany specks (2- 7). The experiments described here extend this finding to the ye&s 8. cerevisiae and c’. ufiks. The present. work, together wit)h that of others (26, 27)) shows that yeasts coin be adapted to grow in the presenchc of high concaentrations of ethionine. Vronl the satisfnc.t,ory growth obtained it is apparent, that t’hc which are prokiiis clthioliirle-corltaillirlg synthesized untlcr these caontlit ions arc not noticeably toxic t.o the orgznnism, as has been suggest,ed (2S), :u~d this is in c*onformity with the observation of Yoshida (5, 6) t)hat, a-anlylascl fro111 Bacillus subtilis, in which one-third of the nlethioninc residues had been replaced by ethionillc, posstxssed tht propertics of Ihc Ilortlial crtzy~t~c*. The present st.udies have rtave:dcd :L further pathway for (,t hioninc nlrtaholislll in
yeasts, ~tanlely that whott ihwc orgattistw are g-on-n itt the presetiw of etl~iotriric, part of t,he sulfur of the atnitto ac*id appears in the cystine and tl~et’hiottitte of the caytoplasttt and of the protein. It is sigttifkattt that the (Lystinc and tttcthioninc dcrivetl frotti cthiottinc arc prcwtlt in the protein in tlrc: WIN proportions as ;tre the csystitie and tttcthionine derived frottl sulfate or tttethiottittc. This 11.ould be cqx1c.t cd if the t,ratlsfcr of &ionine-sulfur to thcw attlitto acids tool; pl:w~ via sonw cottittwti internxdinte, atid if this cotnpound w-erc also on the ttonttal pathway of sulfur :u~~itio wit1 synthesis front sulfate. Hotnocysteitw is suggested as the ~ttost likely intcrnwdiatc, arid it,; fortiiatioti frotti ethioninc is hclirtvcd to take pl:wc~ by the following txwtiott scqwitw: Etllionine
ATI’ --I
S-Adellos!lrthiotliIlc
With the fortnation of hontoc*ystcinc, cottversion to cystcittcs atitl niethionirte iSteps IV and T’) would then prowed by the n-cllS-Adenosylethionine -
l’arlis (19) to produw :qqxec~iable quatttitics of tttrthiottittc. Thcl following tncchanisrn was suggest,cd :
S-Adenosylhomocysteine Homocysteine
wt.ablished pathways of’ traits-sulfuratiott and nxthylntion, respwtivcly. Reaction I has been identified it1 scwral species and in yeasts (17-21). Step II, a transethylntion, has also bwn well nuthcttt,icated in ninny spwies iii rewtit years (16, 29-33). Furthcrntorc, it1 yeasts hottlocaystcitle itself can nest as the ethyl acceptor, giving et,hionine (19, 22). The cleavage of B-ndettosylhotnoc~yst&te t.o hotnoc*ysteine (Sirp III) has not as yet been satisfactorily confirttlrd and nierita further study. Duerrc and Schlcnk (34) ohtaitted ittdiwtion of sottt(L bwalxlowr, I)urticul:zrly it1 C’. ufilis. Howcvcr, thcw workers faikd to ohsorw t 11~
_
- S-Adenosylmethionine Methionine
X-Xtlcrtosylcthiottiti(~ was ctivisaqgd :ts uudergoittg dc-cthylnt’ion to S-adetwsylhomoc*yst~eittc~,mhicsh is nlethylat~cd to the tact-hiottitte derivative, whic~h acts as tnct’hyl dottor to hotttocyst~eitw. It is possible that, this squcttw was operative under the in vitro c*ottdit,ious chosen, but it’ could not :wwuttt. for the ttwthiottinr: synthesis frown cthiottittt iti whole ~11s described in this paper, sittcc itt I’arl~s’ whence t.he sulfur of the ilwthiotlino would obviously originate front c~ridogeriously fortitctl honioc~ystc~int: and not, frotn ethiotritic.
25. ‘LCi.
2. 27.
3 2s.
*5
!I. 10. 11. 12. 1:s.
14. 15. Iti. li. 1X.
I!). 20. 21.