A Carotenogenic Enzyme Aggregate in Phycomyces: Evidence from Quantitive Complementation

P oc. Na . Acad. Sci. USA Vol. 68, No. 9, pp. 2012-2015, Sep embe 1971 A Ca o enogenic Enzyme Agg ega e in Phycomyces: E idence om Quan i i e Complemen a ion (he e oka yons/mu an s/lycopene/phy oene) M. D. DE LA GUARDIA, C. M. G. ARAGON, F. J. MURILLO, AND E. CERDA-OL.MEDO Depa amen o de Gen6 ica, Facul ad de Ciencias, Uni e sidad de Se illa, Spain Communica ed by Max Delb uck, May 28, 1971 ABSTRACT Wild- ype Phycomyces blakesleeanus ac- cumula es ,8-ca o ene, while he mu an s ain C2 is un- able o syn hesize ca o enoids, and he mu an s ain C9 accumula es lycopene. He e oka yons con aining a p o- po ion, p, o C2 nuclei and (1 - p) o C9 nuclei accumula e lycopene, y-ca o ene, and (3-ca o ene in he ela i e amoun s (1 - p), p(l - p), and p2, espec i ely, o di e - en alues o p. I is shown ha hese esul s a e expec ed om he ope a ion o a ca o enogenic enzyme agg ega e ha wo ks as an assembly line and con ains wo copies o a cyclase, which is de ec i e in s ain C9, as well as o he enzymes. The biosyn hesis o ca o enoids is hough o p oceed ac- co ding o he scheme depic ed in Fig. 1. F om he i s 40- ca bon compound, phy oene, a se ies o ou dehyd ogena- ions leads o he p oduc ion o lycopene, wo u he cy- cliza ions con e i o 0-ca o ene. j3-Zeaca o ene subs i u es o lycopene as an in e media e in an al e na i e p oposal, which changes he o de o he las dehyd ogena ion and he i s cycliza ion (1, 2). The disco e y o mul ienzyme sys ems in he syn hesis o a y acids and o he compounds (3-5) has led o specula- ions (6) ha ca o enoid syn hesis migh be o ganized in an analogous way, bu no e idence has ye been ound o such a mul ienzyme agg ega e o ca o enoids. The wild ypes o he ungus Phycomyces blakesleeanus accumula e la ge amoun s o ,B-ca o ene, esul ing in b igh yellow mycelia, spo angiopho es, and young spo angia. Red mu an s (accumula ing lycopene) and whi e mu an s (ac- phy oene 1 + phy o luene 2+ -ca o ene 3+ 5 neu ospo ene :> -zeaca o ene 4+ 5 4 6 lycopene + 7-ca o ene + 0-ca o ene FIG. 1. Biosyn hesis o #-ca o ene om phy oene. Ve ical a ows indica e dehyd ogena ions, wi h o ma ion o a double bond; ho izon al a ows indica e cycliza ions. The hollow a ows ep esen an al e na i e p oposal. cumula ing phy oene o unable o syn hesize ca o enoids al oge he ) ha e been isola ed (7, 8). Phycomyces he e oka yons con aining wo ypes o nuclei, one ype om a s ain ailing o p oduce ca o enoids and he o he om a s ain accumula ing lycopene, a e o many di e en shades o colo , om whi ish o o ange, acco ding o he p opo ion o he wo ypes o nuclei (9). This obse a- ion led us o hink ha pe haps a mix u e o ca o enoids was esul ing om he ope a ion o an enzyme complex. We epo now on he esul s o quan i a i e analyses o he ca o enoids (lycopene, y-ca o ene, and (-ca o ene) p oduced by such he e oka yons. They coincide wi h he expec ed esul i he syn hesis is o ganized by enzyme complexes ope a ing as assembly lines, and pe mi he desc ip ion o ce ain ea u es o such complexes. MATERIALS AND METHODS P. blakesleeanus, s ain C2, ca ying he mu a ion ca -i (p e iously designa ed Alb. 5) p oduces a whi e myceliumn and whi e ui ing bodies and con ains only abou 1% o he usual concen a ion o (3-ca o ene and no o he ca o enoids. S ain C9, ca ying mu a ion ca R21 (p e iously R1) is b igh ed, because o lycopene accumula ion (8). Bo h we e isola ed om wild ype s ain NRRL1555, sexual ype (-) a e ea men wi h N-me hyl-N'-ni o-N-ni osoguanidine (7). A he e oka yon C2 * C9, con aining nuclei o bo h s ains was cons uc ed a i icially. The nuclei o he he e oka yon a e andomly dis ibu ed in o mul inuclea e spo es, which can gi e ise o ei he ype o homoka yon o o he e oka - yons o di e en nuclea p opo ions. A he e oka yon may be p opaga ed wi h a cons an nuclea p opo ion by pla ing a. small piece o young mycelium on o a new medium (7). Mycelia a e g own on glucose-aspa agine-yeas solid medium (9) a oom empe a u e (18-23°C) unde no mal dayligh condi ions. Fo chemical analysis, he mycelium g own 4 days in a Pe i dish is sc aped o wi h a spa ula, ca e ully cleaned wi h weeze s o bi s o aga and young spo angiopho es, and s o ed in he da k a -20°C i he analysis is no done immedia ely. The mycelium is hawed a oom empe a u e, d ied wi h il e pape , cu o small pieces, and di ided in o- wo weighed po ions, one o de e mina ion o d y weigh by exposu e o 1050C o 1 h and he o he o ca o enoid ex ac ion. The la e po ion (usually 0.2 g d y weigh ) is blended in a So all Omni-Mixe wi h 20 ml o me hanol and 2012 P oc. Na . Acad. Sci. USA 68 (1971) 20 ml o pe oleum e he (boiling poin 50 70'C) o 3 min; he ope a ion is epea ed wice a e changing he pe oleum e he laye , and he h ee esul ing pe oleum e he ac- ions a e mixed and cen i uged a 2000 pm o emo e any g oss pa icles. The supe na an is concen a ed o abou 5 ml unde a s eam o ni ogen, and is ch oma og aphed on a MgO-dia omaceous ea h column (8). The spec a, aken wi h a Beckman DK-2A eco ding spec opho ome e , and he ch oma og aphic p ope ies o lycopene, -y-ca o ene, and j-ca o ene coincide wi h published da a (10, 11). The con- cen a ions o hese pigmen s a e calcula ed om hei ab- so p i i ies (12); hei sum is aken as o al ca o enoid con- en since no o he ca o enoids we e ound in app eciable amoun s. The nuclea p opo ions in a he e oka yo ic mycelium a e deduced om he p opo ions o whi e (C2), ed (09), and yellow (he e oka yo ic) mycelia a ising om he spo es o such a he e oka yon by a p ocedu e desc ibed p e iously (7, 9). The ubes a e sco ed as con aining yellow (alone o mixed), ed, whi e, ed and whi e, o no mycelium. The a e age concen a ion o C2 and C9 homoka yo ic spo es is de e mined om hei Poisson dis ibu ions in ubes no con- aining yellow mycelia. The alue o he p opo ion o C2 nuclei in he he e oka yon is hen ob ained om he heo- e ical cu e ep oduced in Fig. 2. RESULTS The esul s o gene ic analyses o nuclea p opo ions a e gi en in Fig. 2. The a e age o se e al independen de e mi- na ions is aken as he p opo ion o C2 nuclei in he he e o- ka yon. Table 1 gi es quan i a i e analyses o ca o enoids in he e oka yo ic mycelia wi h di e en nuclea p opo ions; each analysis e e s o ma e ial om a di e en Pe i dish. INTERPRETATION The esul s i he expec a ions om he hypo hesis ha ca o- enogenesis is pe o med by a linea ly-o ganized enzyme agg ega e wo king unde he ollowing ules: Each enzyme ecei es i s subs a e om he p e ious one and passes i s p oduc o e o he nex enzyme. In e media e subs a es canno be ans e ed om one agg ega e o an- o he , no aken up om he su ounding cy oplasm. The mu an genes in s ains C2 and C9 p oduce de ec i e enzyme molecules ha subs i u e o he no mal ones in he agg ega e. I one o he enzyme molecules is de ec i e, he co esponding in e media e subs a e is eleased om he agg ega e. The gene p oduc s o he many nuclei p esen in a my- celium combine andomly o o m he agg ega es. Each agg ega e con ains wo copies o a cyclase, pe o ming he cycliza ion s eps. This enzyme is de ec i e in s ain C9. The agg ega e may also con ain one copy o he enzyme ha is de ec i e in s ain C2 and is in ol ed in an unknown s ep be o e he o ma ion o phy oene. Fig. 3 diag ams he expec ed ac i i y o he ca o enogenic agg ega es in homoka yons C2 and C9 and he e oka yons C2 * C9. Agg ega es blocked be o e he o ma ion o phy- oene, because hey ecei ed he aul y enzyme om C2, a e unable o syn hesize ca o enoids. The o he agg ega es syn- hesize lycopene i he i s cyclase is copied om C9 (i - espec i e o whe he he second is copied om C2 o C9), e-ca o ene i he i s is copied om C2 and he second om C9, and a-ca o ene i bo h a e copied om C2. The expec ed C2I .6 *2 a l e .2 .1 2p 0 0 L* 0 .5 1 C9 FIG. 2. P opo ions o C2 and C9 homoka yo ic spo es p oduced by C2 * C9 he e oka yons. Iden ical symbols e e o gene ic analyses pe o med on di e en samples o he same he e oka yon. The con inuous cu e ep esen s heo e ical ex- pec a ions and i s sidelines poin o co esponding alues o he p opo ion o C2 nuclei in he he e oka yon, p. p opo ions o hese pigmen s in he o al ca o enoid con en as a unc ion o he p opo ion o C2 nuclei, p, a e (1 -p) o lycopene, p(l-p) o y-ca o eine, and p2 o a-ca o ene. The p edic ed and he expe imen al alues a e shown in Fig. 4. The conco dance is excellen and cons i u es s ong sup- TABLE 1. Quan i a i e analyses o ca o enoids in he e o- ka yo ic C2 * C9 mycelia wi h di e en p opo ions o C2 nuclei, p (a e aged om Fig. 2) To al ca o enoid con en Rela i e p opo ions (pg/g d y p weigh ) Lycopene y-Ca o ene 6-Ca o ene 0.000 415 1.0 0.0 0.0 300 1.0 0.0 0.0 560 1.0 0.0 0.0 500 1.0 0.0 0.0 0.167 311 0.82 0.11 0.06 337 0.78 0.15 0.07 337 0.81 0.12 0.07 491 0.86 0.10 0.04 0.290 204 0.70 0.16 0.14 422 0.72 0.17 0.11 221 0.68 0.18 0.14 0.513 54 0.48 0.27 0.25 77 0.45 0.26 0.30 0.580 201 0.37 0.26 0.36 0.695 73 0.31 0.22 0.48 62 0.32 0.22 0.47 63 0.36 0.26 0.38 0.775 171 0.26 0.18 0.56 0.923 43 0.20 0.19 0.61 Ca o enogenic Agg ega es 2013 2014 Gene ics: De La Gua dia e al. C2 no ca o enoids C9 lycopene x E , .w Ea C2* C9 p I I1 no ca o enoids FIG. 3. Ope a ions pe o med by he ca o enogenic agg e- ga es o homoka yons C2 and C9 and he e oka yons C2 * C9, indica ing he expec ed p opo ion o each p oduc acco ding o he ules speci ied in he ex . Whi e symbols ep esen ac i e enzymes, black symbols, de ec i e enzymes, do ed symbols, ei he ac i e o de ec i e. po o he p oposed model o cycliza ion. The o al ca o - enoid con en should be p opo ional o (1 -p), in he ab- sence o unknown egula ions o uncon olled en i onmen al in luences. The expe imen al esul s (Fig. 5) sugges he in- eg a ion in he agg ega e o he enzyme de ec i e in C2. 0~~~~~~~~~ 0 C 0 b. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ o .55 0 CL 0 - 0 nuclea p opo ion FIG. 4. A e age p opo ions o lycopene, y-ca o ene, and jB- ca o ene in he o al ca o enoid con en o he e oka yo ic mycelia as a unc ion o he p opo ion o C2 nuclei in he he e oka yon. Ci cles indica e expe imen al esul s; con inuous lines, he heo- e ical expec a ions om Fig. 3. Lycopene, 0-; e-ca o ene, - -, and #9-ca o ene, 0..... nuclea p opo ion FIG. 5. To al ca o enoid con en in he he e oka yo ic mycelia as a unc ion o he p opo ion o C2 nuclei in he he e oka yon. DISCUSSION The esul s de ini i ely elimina e many simple models o ca o enogenesis, pa icula ly he andom ac ion o ee enzymes. They es ablish he sequen ial ac ion o wo copies o a cyclase, which a e pa o a mul ienzyme agg ega e. Each cyclase canno be a mul ime wi h iden ical subuni s coming om di e en nuclei. Va ious s e ic designs a e compa ible wi h he da a, o example, b anching agg ega es wi h se e al wo-cyclase eams connec ed o a single chain o ea ly enzymes. Fu he suppo o he hypo hesis comes om ou obse a- ion ha 2-(4-chlo ophenyl hio) ie bylamine hyd ochlo ide (13) and 2-(p-die hylaminoe hoxibenzal)p-me hylace ophe- none ( ecei ed om D . H. Yokoyama) p o oke in he wild ype he accumula ion o lycopene, y-ca o ene, and P-ca o- ene. The ac ion o bo h compounds may be in e p e ed as cyclase inhibi ion. The dehyd ogena ions may be explo ed by he analysis o he ca o enoids in he e oka yons whe e one componen is blocked in a dehyd ogena ion. P elimina y esul s wi h he e o- ka yons o C5 (which accumula es phy oene) and C6 (which p oduces no ca o enoids) indica e ha phy oene, phy o- phy oene phy oene nyopn 6-zeaca oe 0-ca o ene B-ca o ene FIG. 6. Hypo he ical ep esen a ion o he ca o enogenic agg ega e (le ) indica ing ha a sligh s uc u al modi ica ion ( igh ) migh al e he sequence o he eac ions and ha e 3- zeaca o ene as an in e media e ins ead o lycopene. IP oc. Na . Acad. Sci. USA 68 (1971) P oc. Na . Acad. Sci. USA 68 (1971) luene, -ca o ene, neu ospo ene, and ,8-ca o ene a e p esen in he p opo ions expec ed i he agg ega e con ains ou copies o a dehyd ogenase ha is de icien in C5. I has long been deba ed whe he he ,-ca o ene pa hway passes h ough lycopene o P-zeaca o ene. Ou esul s a e easie o accommoda e wi h he i s sugges ion and explain some obse a ions supposed o a o he second. In Rhizo- phlyc i8 osea, an aqua ic ungus, he lycopene made in he ea lie pa o i s li e has been shown no o be a p ecu so o he la e syn hesis o 7y-ca o ene (1). We would p edic ha he ea lie ca o enogenic agg ega es ha e no cyclase and p oduce lycopene; la e a single copy o cyclase is added o each agg ega e, and he new agg ega es make y-ca o ene. Howe e , hey canno use he p e iously made lycopene as a subs a e, jus as lycopene made in cyclase-de icien ag- g ega es in Phycomyces is no ans e ed o o he agg ega es. I he spa ial s uc u e o Phycomyces agg ega es esembles he one depic ed in Fig. 6, le , a sligh s uc u al change may lead o he s uc u e ep esen ed in Fig. 6, igh , which will p oduce j3-zeaca o ene as an in e media e. Pe haps he de- hyd ogenase inhibi o , diphenylamine, induces such a change, esul ing in he obse ed accumula ion o P-zeaca o ene and o he in e media es (1). As sugges ed abo e o Rhizophlyc i8, he s uc u e o he agg ega es may depend on he species and e en on he de- elopmen al s age. Quan i a i e complemen a ion s udies should be easible in Neu ospo a and o he ungi, and he cen al poin , p = 0.5, should be eadily obse ed in he e o- zygous diploids. The esul s epo ed he e depend on he andom asso men o gene p oduc s o o m he agg ega e and he lack o ans e o in e media e subs a es. These ea u es may no be uni- e sal, and hey seem no o apply o he a om agg ega es in Neu ospo a (14). In ou case, i is likely ha wa e -soluble compounds, like he cyclase inhibi o s, ha e access o he agg ega es om he cy oplasm, while he wa e -insoluble ca o enoids do no . I is hen di icul o p edic he use- ulness o quan i a i e in e genic complemen a ion in s udy- ing he o ganiza ion o o he cell unc ions. Phycomyces is pa icula ly well sui ed o such s udies because o he s a- bili y o he he e oka yons and he easy de e mina ion o nuclea p opo ions. The hospi ali y o M. 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