Biochem. J. (1981) 196,403-410 403Printed in Great Britain

Growth-rate-dependent adjustment of ribosome function in the fungusMucor racemosus

Michael ORLOWSKIDepartment ofMicrobiology, Louisiana State University, Baton Rouge, LA 70803, U.S.A.

(Received 27 August 1980/Accepted 14 January 1981)

The dimorphic fungus Mucor racemosus was grown at rates between 0.043 and 0.434doubling/h while maintained as yeasts or at rates between 0.21 and 0.50 doubling/hwhile maintained as hyphae by altering the composition of the growth medium or thegaseous environment of the cells. Yeasts at the higher growth rates contained manymore ribosomes than did yeasts at the lower growth rates. They also had a higherpercentage of ribosomes active in protein synthesis and a faster rate of poly-peptide-chain elongation than did the slower-growing cells. Hyphal cells at faster growthrates also contained many more ribosomes and showed a faster rate of poly-peptide-chain elongation than did slower-growing cells. However, the faster-growingcells had a substantially lower proportion of ribosomes active in protein synthesis thandid the slower-growing hyphae. Pulse-chase experiments failed to provide any evidenceof protein turnover, which might otherwise invalidate the values calculated for thepeptide-chain elongation rates.

There are essentially only two ways in which a cellcan regulate the overall rate of protein synthesis: (i)adjustment of the rate of nascent polypeptide-chainelongation and (ii) adjustment of the number ofnascent polypeptide chains. The rate of peptide-chain elongation as a function of cell growth rate hasbeen studied extensively in bacteria. Most of theearly work suggested a constant value for thisparameter (Coffman et al., 1971; Forchhammer &Lindahl, 1971; Dalbow & Young, 1975; Young &Bremer, 1976). This contention has been chal-lenged, and a more pliable relationship between therate of translation and the rate of cell growth,especially at slow growth rates, has been proposed(Forchhammer & Lindahl, 1971; Martin & Ian-dolo, 1975; Young & Bremer, 1976).

There has been relatively little work done relatingpeptide-chain elongation rates and cell growth ratesof eukaryotic micro-organisms. What little has beenpublished on the subject abounds with contra-dictions. Boehlke & Friesen (1975), using a methodbased on the recovery of active polyribosomes,determined that the peptide-chain elongation ratevaried linearly with the growth rate over a 5-fold

Abbreviations used: TMK buffer, 5OmM-Tris/HCI(pH 7.25), 10mM-magnesium acetate, 500mM-KCl;TMKC buffer, TMK buffer containing 200,ug of cyclo-heximide/ml.

range of doubling times in the fungus Saccharo-myces cerevisiae. Bonven & Gull0v (1979) corrob-orated these findings in S. cerevisiae, using amethod by Gausing (1972) based on the kinetics ofradioactive labelling of nascent and completedpolypeptides. In contrast, Waldron et al. (1977),using the same organism and the same method asBonven & Gull0v (1979), concluded that the rateof amino acid addition to growing protein chainswas constant. Alberghina & Sturani (1975) studiedNeurospora crassa, using methods similar to thoseof Boehlke & Friesen (1975); however, they foundlittle variance in the peptide-chain elongation rateover a 7-fold range of growth rates. Orlowski &Sypherd (1978a), using both types of methodology,reported a 4-fold increase in the rate of pep-tide-chain elongation during yeast-to-hypha con-version in the fungus Mucor racemosus. Thismorphogenetic process is accompanied by a 3-foldincrease in the cellular growth rate.

Adjustment of the percentage of ribosomesactively engaged in translation is the major alter-native mechanism for regulating the overall rate ofprotein synthesis. This parameter has also beenstudied as a function of growth rate in theabove-mentioned fungal systems. Boehlke & Friesen(1975), as did Alberghina & Sturani (1975), foundthe percentage of active ribosomes to be constantover a wide range of growth rates. To the contrary,

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M. Orlowski

Waldron et al. (1977) and Bonven & Gull0v (1979)reported a greater percentage of active ribosomes infaster-growing cells than in slower-growing cells,whereas Orlowski & Sypherd (1978a) found ahigher proportion of active ribosomes in the slower-growing than in the faster-growing cells.

In the present study I have grown the fungus M.racemosus in either the yeast or hyphal formexclusively over a wide range of growth rates bycontrolling the nutrients in the medium or thegaseous environment of the cells. I was thus able tostudy the regulation of the overall rates of proteinsynthesis in this organism uncoupled from cellularmorphogenesis.

Materials and methodsOrganism and growth conditionsMucor racemosus (A.T.C.C. 1216B), a leucine

auxotroph (leu-2A) derived from this strain (Peters& Sypherd, 1978), or a mutant strain (coy) that cangrow only as a yeast in the absence of addedmethionine (Sypherd et al., 1979), were used in theseexperiments. Several different growth media wereused. YPG medium contained 0.3% (w/v) yeastextract, 1.0% (w/v) Bactopeptone (Difco, Detroit,MI, U.S.A.) and 2.0% (w/v) glucose. All of thedefined media contained the following vitamins andbasal salts (per litre): 1 mg of biotin, 1 mg of thiamin,0.5 g of KH2PO4, 0.5 g of Na2HPO4, 0.5 g ofMgSO4, 20mg of MnSO4, 10mg of CaCl2, 10mg ofZnSO4, 10mg of FeCl3 and 10mg of CUS04. Allyeast cultures were supplemented with 1.5 g ofsodium glutamate, 1 g of (NH4)2SO4 and 2 g ofglucose (all per litre). Hyphal cultures were supple-mented with 2 g of glucose or 2 g of mannitol as acarbon source and 1 g of (NH4)2SO4 or 1 g of urea asa nitrogen source (all per litre). Cultures of theleucine auxotroph were supplemented with lOO1g ofL-leucine/ml. All media were adjusted to pH 4.5 withconc. H2SO4. All cultures were inoculated with5 x 105sporangiospores/ml. The spores were pre-pared as previously described (Orlowski, 1979) andwashed with distilled water before inoculation. Allcultures were grown at 220C with shaking. Cultureswere sparged with water-saturated air, 100% N2 or100% CO2 as appropriate. Growth was monitoredby measuring the A600 of the cultures. Concomitantwith each absorbance measurement, samples ofculture were collected and stored frozen for latermeasurement of protein and RNA. All cultures wereallowed to grow to an A600 of 0.4-0.6 before use inany experiment. At such time all yeasts had multiplebuds, and all hyphal cells had short germ tubesapprox. 2-10 spore diameters in length.

Determination ofactive ribosomesThe procedure of Orlowski & Sypherd (1978a)

was used to measure the proportion of ribosomesactive in protein synthesis. Cycloheximide (200,g/ml final concn.) was added to the culture medium.After 2min the cells were collected on a membranefilter (Millipore Corp., Type AA, pore size 0.8,um),washed briefly with cold TMKC buffer and groundunder liquid N2 with a mortar and pestle. The brokencells were resuspended in TMKC buffer andcentrifuged at 15 000g for 10min at 40C. A volumeof the supernatant fraction containing 4.0 A260 unitswas layered on a 10-40% (w/w) linear sucrosegradient (in TMK buffer, 11.0 ml) resting on a 60%(w/w) sucrose cushion (0.8 ml). Gradients werecentrifuged in an SW4 1 rotor (Beckman) at150000g for 80min at 40C. The gradients werescanned at 254nm with an ISCO model 640density-gradient fractionator. Previous analyses(Orlowski & Sypherd, 1978a) of the material on thegradients indicated properties diagnostic of poly-ribosomes, 80S, monoribosomes and 60S and 40Sribosomal subunits. Altering the centrifugal force(5000g to 27 000g) in the preparatory step ortreatment of the broken cell suspensions with TritonX-100 or deoxycholate did not change the ribosomeprofiles obtained on the gradients. Further extrac-tion of the 15 OOOg pellets with buffer or theaforementioned detergents liberated only smallamounts of polyribosomes, which had the same sizedistribution as found in the initial supernatantfractions. Treatment of the broken cell suspensionswith ribonuclease A, which converts all polyribo-somes into 80S particles, did not change theobserved subunit/active-ribosome ratios. Though theexistence of a potential population of membrane-bound or very heavy polyribosomes not quanti-tatively recovered in these procedures is possible, theabove observations minimize this possibility. Theprocedure of Forchhammer & Lindahl (1971) waspreviously used to show that all polyribosomes andall 80S monoribosomes in Mucor are active intranslation (Orlowski & Sypherd, 1978a). Thecorrect determination of active ribosomes thusincludes the sum of all polyribosomes plus all 80Smonoribosomes.

Protein andRNA measurementsTotal cellular protein was measured by the

procedure of Lowry et al. (1951). Total cellularRNA was measured by the method of Cheung et al.(1974). The proportion of total cellular RNArepresenting rRNA was determined by fractionatingpurified RNA on sucrose-density gradients by themethod of Orlowski & Sypherd (1978b).

Cellular ribosome densityThe number of ribosomes per g of total cellular

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Ribosome function in the fungus Mucor

protein was calculated from the following rela-tionship:

RNA x % ofrRNA x Avogadro's number

Protein x mol.wt. ofrRNA

Protein, RNA and the % of rRNA were determinedas described above. The combined molecularweights of rRNA species from M. racemosus asreported by Lovett & Haselby (1971) was 2.04x106. Avagadro's number is 6.02 x 1023.

Calculation ofpeptide-chain elongation ratesPeptide-chain elongation rates were calculated as

described by Forchhammer & Lindahl (1971). Theparameters measured were those necessary tocomplete the following relationship:

obtained were plotted and interpreted as describedby Scornik (1974).

of ribosomes/g of protein

Assayfor protein turnover

Portions (2ml) of a culture were transferred tosmall test tubes (13mm x 100mm), where bubblingwith the appropriate gas was continued. A radio-active amino acid (L-[14C]valine, L-[A4C]proline orL-[14C]leucine) was added to a final concentration of2.5,uCi/ml. Portions (100,l) of the culture wereremoved at intervals and placed into 1 ml of 10%(w/w) trichloroacetic acid. Samples were heated for20min at 900C and then chilled for 30min at 0°C.

(Protein! 120) x ,u x ln 2= no. of amino acids/s per ribosome

(rRNA/mol.wt. ofrRNA) x % of active ribosomes x 3600

where p is the growth rate in doublings/h, In 2 isfrom the growth-rate equation, 3600 is the numberof s/h and 120 represents the average molecularweight of the 20 naturally occurring L-amino acids.Total cellular protein, rRNA and the percentage ofactive ribosomes were measured as described above.The molecular weights of the rRNA species from M.racemosus were previously reported (Lovett &Haselby, 1971).

Transit-time determinationsFor this, 200ml of leucine-auxotroph cells in

mid-exponential growth (A 600 = 0.4-0.6) were col-lected on a membrane filter and resuspended in 50 mlof fresh medium contained in a large syringe barrelfixed vertically and fitted with a three-way stopcock.The medium was sparged with the appropriate gas(N2, C02 or air). Then 50,uCi of L-3H]leucine wasrapidly injected into the culture and 5 ml portionswere drawn at intervals into 5 ml of an ice-coldsolution of cycloheximide (500,ug/ml) and NaN3(10mM). The labelled cells were collected on amembrane filter, washed with cold TMKC bufferand broken under liquid N2. The broken cells wereresuspended in TMKC buffer and centrifuged at15000g for 10min at 40C. The supernatant fluid(S-15) was recovered and a portion of it centrifugedat 150000g for 3h at 40C, which yielded a secondsupernatant fraction (S-150). This centrifugationstep separated labelled nascent polypeptides (ribo-some-bound, in the pellet) from labelled completedprotein molecules (released into the S-150 fraction).Protein in the S-15 and S-150 fractions wasprecipitated in ice-cold 10% (w/w) trichloroaceticacid, collected on glass-fibre filters (Reeve-Angel,Clifton, NJ, U.S.A.), dried under a heat lamp andassayed for radioactivity by liquid-scintillationspectroscopy (Orlowski & Sypherd, 1977). The data

Vol. 196

The precipitates were collected on glass-fibre filters,washed with ice-cold 10% trichloroacetic acid, driedunder a heat lamp and assayed for radioactivity byliquid-scintillation spectroscopy (Orlowski & Syp-herd, 1977). After 20min, a chase of 200-fold excessunlabelled amino acid was added to the culture andthe assay of acid-insoluble radioactivity continued asabove.

ChemicalsAll biochemicals were obtained from Sigma

Chemical Co., St. Louis, MO, U.S.A. All inorganicchemicals were purchased from MallinckrodtChemical Works, St. Louis, MO, U.S.A., and wereof analytical grade. L-[3,4,5-3HlLeucine (118.3 Ci/mmol), L-[U-14C]leucine (289 mCi/mmol), L-[U-"4Civaline (25OmCi/mmol) and L-[U-14C]proline(238mCi/mmol) were obtained from New EnglandNuclear Corp., Boston, MA, U.S.A.

Results

Manipulation ofcellular growth rateFigs. 1(a) and 1(b) respectively show the growth

response of Mucor yeasts and Mucor hyphae undera variety of nutritional and atmospheric conditions.Growth rates, expressed in mass doublings/h, werecalculated from the slopes of the curves in Figs. 1(a)and l(b) and are displayed alongside the appro-priate growth curves. The yeasts showed a 10-foldvariance in growth rate, whereas the hyphaeexhibited only a 2.5-fold range for this parameter.

Cellular ribosome population and rate of pep-tide-chain elongation

Tables 1 and 2 respectively show how the growthrate of Mucor yeasts and Mucor hyphae correlatewith the RNA/protein ratio and with several other

405

M. Orlowski

Table 1. Polypeptide-chain elongation rate and other measured parameters as a function of growth rate in Mucoryeast cells

For full details, see the Materials and methods section. 'YPG' is yeast-peptone-glucose medium (see the Materialsand methods section). Glc-Glu-NH3 medium contains vitamins and salts as described in the Materials and methodssection, glucose (Glc) as carbon source, and glutamate (Glu) and (NH4)2SO4 (NH3) as nitrogen source.

Growth conditions

Medium AtmosphereYPG N2YPG CO2Glc-Glu-NH3 CO2Glc-Glu-NH3 Air*

p(doubling/h)

0.4340.1790.0500.043

RNA/protein(w/w)0.490.370.1340.142

No. ofribosomes perg of protein12.5 x 10169.4 x 10163.4 x 10163.6 x 1016

* Conditional yeast (coy) mutant grows as a yeast in air in the absence of methionine.

Polypeptide-chainPercentage of elongation rate (amino

active acid molecules added/ribosomes s per ribosome)

85.9 5.582.0 2.275.5 1.871.5 1.9

Table 2. Polypeptide-chain elongation rate and other measuredparameters as afunction ofgrowth rate in Mucor hyphaeFor full details, see the Materials and methods section. 'YPG' is yeast-peptone-glucose medium (see the Materialsand methods section). The other media contain vitamins and salts as described in the Materials and methods section,glucose (Glc) or mannitol (Man) as carbon source, and (NH4)2SO4 (NH3) or urea as nitrogen source.

Growth conditions

MediumYPGYPGGlc-NH3Glc-ureaMan-NH3

AtmosphereAirN2AirAirAir

Ja(doubling/h)

0.500.430.330.240.21

RNA/protein(w/w)0.470.470.340.330.22

parameters each having the potential for altering theoverall rate of protein synthesis. Examples of thequality of RNA resolution and ribosomal resolutionachieved in the present systems have been published(Orlowski & Sypherd, 1978a,b). The percentage ofrRNA was a relatively constant 85.7 + 2.3%(mean + S.E.M., n = 8) over a wide range of growthconditions. Cells of relatively fast-growing yeastsand hyphae contained more ribosomes and hadfaster rates of peptide-chain elongation than didrelatively slow-growing cells. When these para-meters were plotted versus growth rate, a linearrelationship was observed in both cases (Figs. 2aand 2b). The percentage of ribosomes active inprotein synthesis did not relate to growth rate insuch a straightforward manner. In yeasts there wasan increase, though not a linear one, in theproportion of ribosomes active at increasing growthrates (Table 1). However, in hyphae nearly theconverse was true. Some slower-growing cells had ahigher fraction of ribosomes active in protein syn-thesis than faster growing cells (Table 2).

Transit time ofmRNA translationThe transit time is the period of time required for a

No. ofribosomes perg of protein12.0 x 101612.0x 10168.6 x 10168.3 x 10165.6 x 1016

Polypeptide chainPercentage of elongation rate (amino

active acid molecules added/ribosomes

60.845.786.084.175.3

s per ribosome)6.77.74.33.34.8

ribosome to bind to an mRNA molecule, completetranslation and release a finished polypeptide. Thekinetics of transfer of radioactive amino acids fromnascent peptide chains to completed proteins aredirectly related to the transit time (Haschemeyer,1969; Fan & Penman, 1970; Scornik, 1974).Centrifugation at 150000g was used to separatebroken cells into a ribosome fraction, where labellednascent peptides were located, and a supernatantfraction, where labelled completed proteins werefound (see the Materials and methods section). Thedistribution of label between these two fractions aftera pulse of L-T3H]leucine was measured over shorttime intervals and plotted as the fraction ofradioactivity in nascent protein (N/T). Transit timeswere calculated for Mucor yeasts and hyphae atseveral growth rates from the decay kinetics of N/T(Fig. 3) as described by Scornik (1974). Thecalculated transit-time values are presented in Table3 (column 5). Assuming that there is no majorchange in the molecular-weight distribution ofproteins made at the different growth rates, thepeptide-elongation rates and transit-time valuesshould change in a strict relationship with oneanother, expressed in the following equation:

Transit time x peptide-elongation rate x 120 = average protein molecular weight

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Ribosome function in the fungus Mucor

measurements accurate. Such calculations have beenmade by using the appropriate values of thepeptide-elongation rate (column 6 of Table 3) takenfrom Tables 1 and 2. The calculated values appear incolumn 7 of Table 3 and are relatively constant.Thus the data from the two methods correlate quitewell.

Protein turnover0.3 - One important assumption made, which must be

0.050 true in order for the calculated peptide-elongation0.2 - .043 rates to be valid, is that protein turnover does not

occur to any significant degree. Mucor cells pulse-labelled with a radioactive amino acid under a wide

0.1 28 variety of different growth conditions never released0 4 8 12 16 20 24 28 a significant amount of radioactivity after a chase

with unlabelled amino acid. Figs. 4(a) and 4(b) showrepresentative results for hyphal cells growing in

(b) various media under air with L-[ 14C]valine or=0.50 43 L-[14C]proline respectively as the label. Identical

1.0 _ / 033 .24 results were obtained for yeast cells under CO2 or0.8 N2 with these amino acids or L-[ 4CIleucine or L-0.7 -021 [14C]lysine as the label. These and other previously0.6 published experiments (Orlowski & Sypherd, 1977)0.5 strongly suggest that little or no protein turnover0.4 occurs in actively growing Mucor cells.

24

Fig. 1. Growth kinetics of Mucor yeasts and hyphaeunder various nutritional and gaseous environmentsGrowth rates (,), expressed in mass doublings/h,were calculated from the slopes of the growth curvesand are displayed alongside the appropriate curves.(a) Yeasts. Symbols: 0, YPG medium, N2 atmos-phere; O, YPG medium, CO2 atmosphere; 0,defined medium (see the Materials and methodssection) with glucose, glutamate and (NH4)2SO4,CO2 atmosphere; *, defined medium as above, airatmosphere, coy mutant strain (all other experi-ments performed with strain 1216B). (b) Hyphae.Symbols: 0, YPG medium, air atmosphere; E, YPGmedium, N2 atmosphere; *, defined medium withglucose and (NH4)2SO4, air atmosphere; A, definedmedium with glucose and urea, air atmosphere; 0,defined medium with mannitol and (NH4)2SO4, airatmosphere. All experiments were performed withstrain 1216B.

Since the calculated average protein molecularweights are a product of the two measured para-meters, they should not vary significantly from oneanother if the methods used are reliable and the

Discussion

The present data indicate that Mucor yeasts andhyphae can effect regulation of the overall rate ofprotein synthesis by adjusting both the number ofnascent polypeptide chains and the rate of nascent-polypeptide-chain elongation. The number of nas-cent peptide chains in the cell is the product of thenumber of cellular ribosomes and the percentage ofribosomes active in translation. Changing either ofthe latter two parameters, of course, alters the first.Both ribosome numbers and percentage of activeribosomes were measured directly in this study. Thevelocity of translation was determined in two differ-ent ways. The number of cellular ribosomes, theproportion of ribosomes active in protein synthesisand the velocity of ribosome movement along themRNA all varied with the cellular growth rate. Allof these parameters appeared to change indepen-dently of one another, with the net rate of proteinsynthesis apparently representing a synergy of allthree mechanisms. The results of this work agreemore with the findings of Bonven & Gull0v (1979)than with any of the other studies cited in theintroduction. Bonven & Gull0v (1979) found thatboth the velocity of translation and the percentage ofribosomes active in translation varied with thegrowth rate. As mentioned above, the other studiesusing fungal material reported a change in only one(Boehlke & Friesen, 1975; Waldron et al., 1977) ornone (Alberghina & Sturani, 1975) of these para-meters as the cellular growth rate changed.

(a)

1.0

0.80.70.6

0.5

0.4

0.3

0.1 L-

o 4 8 12 16 20Time (h)

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407

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M. Orlowski

S.~0'UCi)la

0

co

0._

E 1-

0

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al , I , . 1 .0 0.1 0.2 0.3 0.4 0.5 0 0.1 0.2 0.3 0.4 0.5 0.6

, (doublings/h)Fig. 2. Polypeptide-chain elongation rate (a) and cellular ribosome density (b) as afunction ofgrowth rate

Data are taken from Tables 1 and 2.

Table 3. Calculated transit times ofmRNA translation in Mucoryeasts and hyphae at various growth ratesFor full details, see the Materials and methods section. 'YPG' is yeast-peptone-glucose medium. The other mediacontain vitamins and salts as described in the Materials and methods section, glucose (Glc) as carbon source, andglutamate (Glu) and (NH4)2SO4 (NH3), or urea, as nitrogen source.

CellmorphologyYeastsYeastsYeastsHyphaeHyphae

Growth con(

MediumYPGYPGGlc-Glu-NH3YPGGlc-urea

Peptide elongationditions rate (amino acid

I I" Transit molecules added/sAtmosphere (doubling/h) time (s) per ribosome)

N2Co2Co2AirAir

0.4340.1790.0500.5000.240

150366450114228

5.52.21.86.73.3

Averageproteinmol.wt.9.9 X 1049.6x 1049.7 x 1049.2x 1049.2x 104

Alberghina & Sturani (1975) observed a con-sistently low (50%) proportion of active ribosomesin N. crassa, whereas Boehike & Friesen (1975)reported a constant high percentage (90%) of activeribosomes in S. cerevisiae. The latter authors usedlysed spheroplasts to make this determination, acondition leading to spuriously high values, asdemonstrated by Bonven & Gull0v (1979). TheNeurospora study (Alberghina & Sturani, 1975)was the only work other than studies on Mucor thatused a filamentous fungus. Physiologically andmorphologically normal cells were broken underliquid N2 in each case. Previous studies with Mucorhave shown that the percentage of active ribosomesin hyphal cells can be as low as reported for N.crassa, but the values are closely correlated with thestage of hyphal development (Orlowski & Sypherd,1978a,c). The general pattern was for the fastest-growing hyphae with well-developed germ tubes tohave the lowest percentage of active ribosomes. Thisis why cell morphology was so rigorously controlled

in the present study. Whether it was even con-sidered in the Neurospora study is not known.The relationship between growth rate and the

active ribosome population in Mucor yeasts wasqualitatively similar to that reported for Saccharo-myces yeasts; however, the very low values reportedin slow-growing cells (Waldron et al., 1977; Bonven& Gull0v, 1979) were never seen. Bonven & Gull0v(1979) reported 36% active ribosomes in S. cere-visiae growing at 0.17 doubling/h, and Waldron etal. (1977) indicated 50% active ribosomes in thesame organism growing at 0.3 doubling/h. Mucoryeasts growing at much slower rates had a sub-stantially higher percentage of active ribosomes(Table 1).Though the Bonven & Gull0v (1979) study agrees

with the present findings on an adjustable rate ofpeptide-chain elongation, the work of Waldron et al.(1977) does not. Both groups would like to suggestthat slow-growing yeasts, as previously reported forbacteria (Koch, 1971), have a large excess of

1981

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Ribosome function in the fungus Mucor 409

1.00.9 -0.80.7 -

0.6 -

0 0 0 1

0.5

0.4 - 0F- Az 0.3 a

0.2 - 0

0.1I0 2 4 6 8 10 12

Time (min)

Fig. 3. Kinetics of-flow of radioactive amino acids fromnascent polypeptides to completed and released protein

moleculesResults are represented as the rate of decay of thefraction of total incorporated label occurring in theribosome-bound (nascent protein) fraction (N/T).Symbols: 0, yeasts grown under CO2 in YPGmedium; 0, yeasts grown under CO2 in the definedmedium with glucose as carbon source and glu-tamate plus (NH4)2SO4 as nitrogen source; *, yeastsgrown under N2 in YPG medium; O, hyphae grownunder air in the defined medium with glucose ascarbon source and urea as nitrogen source; A,hyphae grown under air in YPG medium. Strainleu-2A was used in all these experiments.

inactive ribosomes present in a 'standby' status untilpressed into immediate service by a sudden im-provement in the environment. The immediatemobilization of these excess ribosomes, rather thanan increase in the speed of translation, would initiatethe increased rate of protein synthesis upon ashift-up. The support for such a notion is not veryimpressive for Mucor yeasts, and non-existent forMucor hyphae. One priority for future work with theMucor system will be nutritional shift-up experi-ments. If previous C02-to-air shifts (with accom-panying morphogenesis) (Orlowski & Sypherd,1978a) prove to be the equivalent of a nutritionalshift-up, the organism will respond to the improvedconditions by immediately increasing the peptide-chain elongation rate, but only gradually increasingthe number of cellular ribosomes. The percentage ofactive ribosomes may not change very suddenly orvery much. Certainly much remains to be donebefore a complete understanding of the mechanismscontrolling protein synthesis in eukaryotes, ormerely in fungi, is possible. Indeed, it still remains tobe determined whether different fungi use alter-

10 (a) 100

8 80

6 60

4 040

2 20

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100 100

80 K 80

60 60

~40 40~20 20

8

0X

0 20 40 60 20 40 60

Time (min) ,

08 8 01

Cd (b)c: 6 6 cx x

4 60 4

2 2

C 0

10 20

8 16

6 012

4 8

2 4

00 20 40 60 20 40 60

Time (min)Fig. 4. Pulse-chase assayfor protein turnover

Results for hyphae grown aerobically in variousmedia are shown. The pulse with a radioactiveamino acid was begun at zero time. The chase withunlabelled amino acid was commenced at 20min(arrows). (a) L-[14C]valine label; (b) L-44C]prolinelabel. Symbols: 0, YPG medium; O, definedmedium (see the Materials and methods section)with glucose and (NH4)2SO4; A, defined mediumwith glucose and urea; 0, defined medium withmannitol and (NH4)2SO4. Strain 1216B was used inall experiments.

native strategies or one universal mechanism toregulate the rate of protein synthesis.

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410 M. Orlowski

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