THE JOURXAI, OF BIOLOGIC.~. CIIEMIS~P Vol. 246, No. 20, Issue of October 25, pp. 6144.6iti. 1071

Printed in U.S.A.

The Effect of Cycloheximide on Membrane

Transport in EugZena

A CO&IPARATIVE STUI1\- WITH SIGERICIT*

(Received for publication, February 18, 1971)

WILLIllM R. EVANS

WITH THE TECHNICAL ASSISTANCE OF CAROL SMITH

From the Charles F. Kettering Research Laboratory, Yellow: Springs, Ohio 45387

SUMMARY

Cycloheximide and nigericin have been found to inhibit the uptake of 2,4-dinitrophenol (DNP) and glucose in Eu- glena gracilis with the inhibition of uptake of DNP being dependent upon a prior incubation of the cells with the anti- biotics. The effect that cycloheximide exhibited upon the uptake of DNP and glucose was related to the growth phase of the cell. In log phase cells 3.6, 36, and 360 PM cyclo- heximide inhibited the uptake of DNP by 90%, whereas in carbon-starved cells the uptake of DNP was inhibited only by 5 to 15% by 360 PM cycloheximide but 3.6 pM cyclohexi- mide was as effective in the inhibition of uptake in starved cells as in log phase cells. Although 360 pM cycloheximide exhibited little effect upon the uptake of DNP and glucose in starved cells, the inhibition of protein synthesis was virtually complete. This observation coupled with the finding that after removal of the antibiotic the inhibition of protein syn- thesis and the inhibition of uptake of DNP exhibited different recovery patterns suggested that the inhibition of membrane transport by cycloheximide was not a consequence of the inhibition of protein synthesis.

A decrease in the polyribosome content, with a correspond- ing increase in the amount of monosomes, was observed when log phase cells were incubated with either 3.6 pM cyclo- heximide or 1.4 pM nigericin.

It was suggested that the inhibition of uptake of DNP by cycloheximide and nigericin was due either to a decrease in the pH gradient existing between the inside and outside of the cell or to an effect upon membrane function such that the un-ionized form of DNP was no longer permeable. The mechanism by which the inhibition of membrane transport was manifested by the two antibiotics was not viewed as being identical.

The toxicity of cycloheximide to a wide variety of eucaryotes such as fungi (l), higher plants (a), protozoa (3), and mammalian

* This is contribution 445 from the Charles F. Kettering Re- search Laboratory.

cells (4) has been well documented. This toxicity has been re- lated to the inhibition of protein synthesis associated with 80 S ribosomes (5-7). Cycloheximide has been shown to inhibit both growth and chlorophyll formation in Euglena (8). Smillie et al. (9) indicated that whereas these processes were inhibited, no inhibition of the light-induced synthesis of Fraction I protein and enzymes of the Calvin cycle was observed in the presence of the antibiotic. Kirk (10) suggest.ed that the only cellular process directly affected by cycloheximide in Euglena was protein syn- thesis due to the lack of an effect of the antibiotic on respiration or motility.

The present report describes studies which indicate that cyclo- heximide inhibits membrane transport, as well as protein syn- thesis, in Euglena and that these two effects on cellular metabo- lism operate independently of each other. The inhibition of membrane transport and the effect of cycloheximide on polgsome profiles was mimicked by the ionophore nigericin. A preliminary report of this work has appeared (11).

METHODS

Materials-[14C]DNP1 (1.76 C’ p m I er mmole) and L-[‘4C]leucine (344 mCi per mmole) were purchased from New England Nu- clear. n-[14C]Glucose (3.0 mCi per mmole) and L-[14C]phenyl- alanine (7 mCi per mmole) were purchased from Amersham- Searle. Cycloheximide was obtained from Nutritional Biochemicals. Nigericin was provided by Dr. J. M. 1IcGuire of Eli Lilly and Company, Indianapolis, Indiana. m-Cl(CC)P was provided by Dr. P. G. Heytler of E. 1. du Pont de Nemours and Company, Wilmington, Delaware. All other chemicals were of reagent grade and were used without purification.

Growth of Cells and Greening Conditions-Euglena gracilis, strain Z, was grown in the dark as a stationary culture at, 28” in the heterotrophic media of Hunter, Bach, and Ross (la), sub- stituting glucose (1 c/,) for sucrose. These cells have been main- tained in the dark for at least 2 years and are dark adapted. Dark-grown cells were harvested during midlog phase of growth, 2 to 3 X lo6 cells per ml, by low speed centrifugation, washed twice in a medium lacking carbon sources, and resuspended in the same medium at 2 X lo6 cells per ml. The cells were then placed on a reciprocating shaker and incubated overnight in thr dark.

1 The abbreviations used are: DNP, 2,4-dinitrophenol; m-Cl(CC)P, m-chloro(carbony1 cyanide)phenyl hydrazone.

6144

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Issue of October 25, 1971 W. R. Evans 6145

During this time the cell number doubled, the cell volume de- creased by almost a half, and no subsequent cell division occurred. All manipulations were carried out in a dark room equipped with a green safe light.

In the majority of the experiments the dark-grown cells were suspended in a medium composed of 0.72 m&f N(2-hydroxy- ethyl)cthy-lenediaminetriacetic acid, 1.1 mM KH,POb, 2.46 m&r RIgCln, 12.7 m&f N&IlC03, 0.5 mM CaC03, 14.2 rnM citric acid, 3.4 111~ Nai citrate, and 180 mg of trace metal Mixture 60 A per liter at pH 4 (Medium A). Medium C was identical with Me- dium A except that no citrate was added and 10 mM NH&l was used in place of NHJIC03.

Greening was carried out in 50-ml Erlenmeyer flasks contain- ing 5 ml of medium on a reciprocating shaker at 25-27” under Gro-Lux light equivalent to 150 footcandles of daylight fluores- cence light at 3.0 x lo6 cells per ml. The light-induced syn- thesis of chlorophyll has been shown to parallel both the rate at which chloroplast lamellae are formed and the rate of photo- synthesis in Euglena (13). Chlorophyll concentration was estimated according to Bruisma (14) on 80% acetone extracts. Cell counts were deterrnined with the model I3 Coulter Counter. Cycloheximide, DNP, and m-Cl(CC)I’ were added from aqueous solutions whereas nigericin was added in methanol to a final concentration of 0.5’Y$. This concentration of methanol had no effect on chlorophyll formation.

Assay for Uptake and Incorporation of Radioactive Compounds-

Uptake was determined on 0.5.ml aliquots of cells (7.5 to 8.0 X lo5 log phase cells or 1.5 X lo6 starved or stationary phase cells) pipetted onto &O-pm Millipore filter discs under low vacuum. Cells were immediately washed with 5 ml of the appropriate medium, placed on aluminum planchets containing 3 drops of 2’i; casein, dried, and counted in a gas flow low background counter. The amount of radioactivity adsorbed onto the filter and cells was determined on aliquots of cells which had previously been frozen in a Dry Ice-acetone bath and allowed to thaw. This freeze-thaw procedure disrupts the cell wall in Euglena (15). Determination of the radioactivity incorporated into protein was accomplished by first pipetting 0.5.ml aliquots of the cells into 5 ml of 5% trichloroacetic acid in methanol. The trichloro- acetic acid-methanol mixture was rernoved by centrifugation, and the pellet was suspended in 50/, trichloroacetic acid and heated for 15 min at 80-90”. After cooling, the samples were plated on O.&pm Millipore discs, washed with 5% trichloroacetic acid, placed on aluminurn planchcts containing 3 drops of 25;:/;, casein, dried, and counted. [IQDNP (1.76 mCi per mmole), n-[14C]glucose (3.0 mCi per mmole), and n-[14C]leucine (244 mCi per mmole) were used without addition of carrier. One nano- mole of [14C]DNP plated onto cells previously plated on a filter gave a count rate of 613 cpm. One nanomole of [14C]glucose plated and counted in the same manner as the [YJDNP gave a count rate of 814 cpm. One picomole of L-[14C]leucine plated on a planchet gave a count rate of 216 cpm. L-[12C]Phenylalanine was added to the L-[14C]phenylalanine (7.0 mCi per mrnolc) to give a specific activity of 0.9 mCi per mole. One nanomole plated on a planchet gave a count rate of 540 cpm.

Determination of Cations-Changes in Kf and Mg++ concen- trations of the medium were determined by at,omic absorption spectroscopy. The cells were centrifuged and the medium was decanted off and diluted 20.fold with dist,illed H20 prior to the assays.

Analysis of Polysomal Patterns-Thirty to 45 ml of log phase

cells (1.5 t,o 2.0 X lo6 cells per ml) were centrifuged at top speed for 15 to 20 set in a clinical centrifuge at room temperature, and then rapidly chilled by resuspension in 30 ml of 10 I~IM Tris-Cl, pH 7.7, 50 m&f KCl, and 5 rnM MgC12 (Buffer 1). The cells were centrifuged for 15 to 20 set and the pellet was suspended in 5.0 ml of Buffer 1 containing 0.5%) Triton X-100. The cells were then immediately broken in a French pressure cell at 1000 p.s.i. Cell membranes, nuclei, and mitochondria were removed by a IO-mm centrifugation at 25,000 x g. Aliquots of the super- natant were layered on a 35.ml 10 to 35’1; linear sucrose gradient in Buffer 1. After centrifugation at 27,000 rpm for 90 nun in the SW 27 rot.or, the gradients were analyzed with a continuous flow system by forcing the gradient up through the centrifuge tube with gravity-fed 40%, sucrose and through a 2-mm flow cell.

RFSULTS i

Preliminary studies of the effect of c>-clohexitnide and nigericin on E. gracilis indicated that concentrations of cycloheximide up to 3.6 ~.tivf and concentrations of nigcricin up to 1.4 PM had very little effect upon chloroplast development or respirat,ion. In addition, these concentrations of cycloheximide or nigericin were found to antagonize the inhibition of chloroplast formation by DNP or m-Cl(CC)P (Table I). Table II shows that the inhibi- tion of plastid development by these two uncouplers was de- pendent upon the pH of the extracellular medium as well as the cell concentration. In the majority of the experiments reported here the cell number was adjusted to 3.0 x lo6 per ml and the pH was maintained at 4. Under these conditions, concentrations of 8 to 10 ,ULM DNP or m-Cl(CC)P usually resulted in 80 to 100% inhibition of plastid formation. -1 45. to 60.min incubation with DNP resulted in as much inhibition of plastid formation as when the cells were incubated with t,he uncoupler for 20 hours. Under the same incubation conditions a 30.min incubation period in m-Cl(CC)P was required to produce the same degree of inhibition.

Effect of cycloheximide and nigwicin on inhibition of chloroplast jo~nzation by DYI’ and nl-Cl (CC)]’

One-day starved cells: 1)Nl (8 ~11) and m-Cl(CC)P (8 PM) were added after the cells had been incubated for 15 min in the presence of cycloheximide or nigericin. After 24 hours in the light t,he amount of chlorophyll formed was determined as described mrder “Methods.”

Concentration Chlorophyll

N o additions DA-P m-CI(CC)P

u :g/cell x 10s

Cycloheximide 0.36 pLR1.. 4.93 1.92 0.9pM......... .._.. 4.38 3.36 1.8,uM............ 4.03 4.91 3.6pM... .._.......... 5.57 5.97

Xigericiu 0.175 jdvl. 3.411 0.55 0.35PLf. 3.9 4.75 0.7,uRI. 4.5 5.21 1.4@M... 4.55 4.4

Control. 3.16 I>NI’ ouly. 0.33 m-Cl (CC)P only.

1.07 2 .42 3.02 4.23

0.88 1.8

2.03 3.16

0.75

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6146 Inhibition of Membrane Transport by Cycloheximide Vol. 246, No. 20

TDLE II Effect oj extracellular pH and cell concentration on inhibition of

chlo?oplast formation by D,VP and m-Cl(CC)P

Effect of extracellular pH: l-day starved cells in Medium C were washed and resuspended in Medium C at pH 3.1 (6.0 X lo6 cells per ml). Equal volumes of Medium C, previously adjusted to pH 3.1, 5.15, 6.45, 7.45, and 8.15, were added and the resulting pH values recorded. DNP or m-Cl(CC)P, 6 PM, was added and after ZO-hour incubation in the light the chlorophyll content was determined as described under “Methods.” Effect of cell con- centration: l-day starved cells in Medium C were adjusted to 2.0, 3.0, and 4.0 X lo6 cells per ml and then 4 MM DNP or m-Cl(CC)P was added and after 20 hours in the light the chlorophyll content was determined as described under “Methods.”

Chlorophyll

Control DIVP m-CI(CC)P

la/ml

Initial extracellular pH 3.1 16.6 0.8 4.3 17.2 9.9 0.8 5.1 14.8 17.3 0.8 6.0 14.3 13.6 1.8 6.7 10.8 10.8 10.1

Concentration of cells X 106/ml 2.0 7.63 0.04 0.04 3.0 11.7 0.29 0.13 4.0 14.8 10.1 0.48

-

MINUTES PRE -INCUBATION WITH

CYCLOHEXIMIDE OR NIGERICIN

FIG. 1. Effect of time of prior incubation in cycloheximide (CH) ‘r nigericin (NIG) on the inhibition of chloroplast formation by

DNP. One-day starved cells: either 3.6 PM cycloheximide or 1.4 ,UM nigericin was added to the cells followed by the addition of 8.0 PM DNP at the times indicated. After 20.hour incubation in the light, t,he amount of chlorophyll formed was determmed as described under “Methods.” O---O, cycloheximide; O---O, nigericin.

The antagonism of the uncoupler-induced inhibition of chloro- plast formation by cyclocheximide or nigericin was dependent upon the time in which the cells were first incubated with the t,wo antibiotics (Fig. 1). I n all experiments the simultaneous addition of either uncoupler with cycloheximide or nigericin did not a1leviat.e the inhibition. Usually with 1% to 24-hour starved cells a lo-min prior incubation was sufficient for the complete

antagonism to be manifested. In other experiments a longer

3

L

MINUTES

FIG. 2. Effect of prior incubat,ion in 3.6 PM cycloheximide on t,he uptake of DNP. One-day starved cells: 8.0 ,UM [l*C]DNP (0.014 PCi per ml) was added either together with 3.6 PM cyclo- heximide, after a prior 20-min incubation of the cells in cyclohexi- mide, or to control cells alone. Aliquots (0.5 ml) of cells were plated at the times indicated after the addition of [I%]DNP and the radioactivity was determined as described under “Methods.” After 22-hour incubation in the light, the amount of chlorophyll formed was determined. Cells treated with the freeze-thaw pro- cedure gave a count rate of 110 cpm. 0-0 and 1, [W]DNP alone added; W-m and 2, [l%]DNP and cycloheximide added simultaneously; O--O and 4, [WZ]DNP added after a prior 20- min incubation with cycloheximide; 3, control chlorophyll, no ad- ditions.

prior incubation period was required, and the time required was a function of the time that the cells had been starved.

Experiments with [14C]DNP showed that the essentiality of the prior incubation of the cells in cycloheximide, in order for chloroplast formation to occur, was a reflection of the inhibition of uptake of DNP (Fig. 2). Other experiments performed in an identical manner with 1.4 PM nigericin produced similar results. Addition of either compound simultaneously with DNP result.ed in the same pattern of uptake of DNP as when DNP alone was added. Prior incubation of the cells with either antiobiotic inhibited the uptake by at least 90 %. In cells to which nigericin or cycloheximide was added t,ogether with DNP or in cells incu- bated with DNP alone maximum uptake occurred within 1 to 2 min followed by a decrease to a level which remained relatively constant for 10 to 30 min. Inhibition of DNP uptake was still evident after 20 hours of incubation in cells which had been previously incubated with the antibiotics. Cells which were in- cubated with DNP alone contained essentially no DNP after the 20-hour incubation period. The lack of radioactive m-Cl(CC)P prohibited the performance of the same type of experiments with this compound, but it would seem probable that the uptake of m-Cl(CC)P would also be inhibited.

In the presence of DNP or m-Cl(CC)P an increase in the pH of the medium was observed when cells were incubated in Me- dium C. The rat.e of this increase in medium pH was followed and was found to reach a maximal value 40 to 45 min after the addition of the uncoupler, and after 60 min the reaction was essentially complete. Analysis of the medium showed an in- crease in medium K+ when the cells were incubated with DNP alone or when the uncoupler was added at the same time as the antibiotics (Table III). Prior incubation of the cells in either cycloheximide or nigericin prevented the increase in extracellular K+ concentration. Identical results were obt,ained with m-Cl(CC)P. An increase in medium Mg++ was also observed with both uncouplers.

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Issue of October 25, 1971 W. R. Evans 6147

Effect of prior incubation of cells in cycloheximitle or nigericin on D1I.P.induced loss oj cellular K+

Effect of time of prior incubation in cycloheximide upon inhibilion of uptake of [‘T]DA’P in log phase cells

One-day starved cell,q: I)NP, #.O~M, was added eithersim\llt,ane- or~ly with 3.6 PM cyrloheximide or 1.4 MLM nigericirr, or after a prior 15.min incubatiotl of ihe cells with the antibiotics. After 2% hour incubatiorl, :diqt:ots were removed for chlorophyll and K+ determinxtiolls :‘s dcFcrihed lulder “Methods.”

IIlC&~

DNP addition

K+ in media ( lhlorophyll after 22 hr after 22.hr incubation incubation

min ??%M aglml 1.2 10.8 1.64 1.3 1.2 16.4 1.64 0.3

1.26 15.0

1.12 14.2

1. ($4 0.3

1.14 12.2

Log phase cells were harvested by low speed centrifugation, washed once with Medium A, and resrlspended in the same medium to 1.50 X lo6 cells per ml. Cells were incubated in cycloheximide for the minutes indicated prior to the addition of 10 pM [lG]L)NP (0.018 PCi per ml). Aliquots (0.5 ml) of cells were plated at 1, 2, and 3 min after addition of t,he [14C]I)NP. Uptake of [14C]I)NP was calculated as in Table IV. Control cells gave a count rate of 1092 cpm, 1.79 nmoles, corrected for a freeze-thaw control of 131 cpm.

Concentration of cycloheximide

/ Time of prior

incubation Uptake of [“CIDNP Cont,ro. DNP Cycloheximide Cycloheximide + DNP. Cycloheximide + 1)NP.. Nigericin. Nigericin + 1)NP.. : Nigericin + 11NP.

Inhibition

nntoles/7.5 x 105 cells

1.79 1.49 1.08 0.12

0.82

0.6

%

0 13

40

93

54

67

0.08 96

0.74 61

0.48 73

0.17 92

0.06 97

15

min

1

2

4

G

1 2

4

6

1

2

4

G

P’M

3.6

36

360

0 15

0 15

-

Efecl of cyclohexinzide and nigericin on inhibifion of the upluke of [14C]DA%vP

Two-day starved cells were previously incktbated for 30 min with cycloheximide prior to the addition of 10 PM [‘“C]DNP (0.018 PCi per ml). Three-day starved cells were previously incllbated for 30 min with nigcricin prior to the addition of 8.0 PM [lJC]l)NP (0.014 &i per ml). In both experiments, 0.5.ml aliquots of cells (1.5 X lo6 cells) were plated on 8.0-p Millipore filter discs at 2, 4, and 6 min after the addition of DNP. The radioactivity was de- termined as descrilxd Ilnder “Methods” and the connts in each sample were corrected for the freeze-thaw control, totaled, and averaged, and the nanomoles of Ii4C]l>NP taken up were calcu- lated with this value. The control cells gave a count rate of 1214 cpm and the freeze-thaw control gave a count rate of 84 cpm.

T.zn~n VI

Changes in inhibition of 7cptuke of [‘“C]DA4’P by cycloheximide as junction of growth phase of cell

Cells in the various phases of growth were harvested by low speed centrifugation, washed once, and resuspended in Medium A to 3.0 X lo6 cells per ml. Cells (0.4 ml) at the end of log phase and 0.5 ml of the stationary and starved cells were plated (as de- scribed under “Methods”) 1, 2, and 3 min after the addition of 10 .UM [l(C]DNP (0.018 &i per ml). Control cells at the end of log phase gave a count rate of 1065 cpm after correction for a freeze- thaw control of 117 cpm, the corresponding values for the P-day stationary and 2-day starved cells were 1390 cpm, 107 cpm and 1589 cpm, 110 cpm, respectively. The nanomoles of [l%]DNP taken up were calculated as in Table IV. Values for the cells at the end of log phase were corrected to nanomoles per 1.5 X lo6

cells.

Jptake of [‘4C]DNP Inhibition

~des/l.5 x 10” cell

2.12

0.71

0.24

1.65

1.98

l.G 1.46

0.14

0.43

0.68

Concentration

Cycloheximide 0.0/m... 0.36p~.. 3.BpLM..

36.0,,&........ 360,0pM........

xigericin O.O~IM....... O.l4jLM.......

1.4pM..........

14.0/.&M....................

70.0pM... ,.._......,.....

I IP %

66

89

22

7

9

02

i3

58

- Uptake of [‘TIDNI’

COlICen- tration of

CyClO- heximide

After prior incubation in cycloheximide of Cell growth phase

Control

5 min 1 10 min / 20 min

I”‘+f 3.6

3G

360

3.6

36

300

3.6

36

360

In t,he st.udies described thus far Iv-e were primarily interested

in demonstrating the correlation bet,ween the uptake of the two

uncouplers and the inhibition of a metabolic process, such as the

formation of chloroplasts. Experiments were then directed toward defining conditions which influenced the uptake of DNP.

The first experiments in this regard were concerned with the effect of relatively high concentrations of the antibiot,ics on

uptake of DNP in st,arvcd cells. Table IV shows that at 360

End of log

2.day stationary

2.day starved

2.18

2.28

2.G

1.27

0.66

0.89

1.02 0.08

0.15 1.02

0.31 0.45

0.18 0.0

0.17 0.13

0.95 0.89

1.47 0.19

1.31 0.87

2.2 2.2 ELM cycloheximide the uptake of DNP m-as inhibited by only 5cjL -

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6148 Inhibition of Membrane Transport by Cycloheximide Vol. 246, No. 20

compared to the 86%;, inhibition which occurred at 3.6 PM cyclo- these cells the uptake of DNP was 50 to 607; inhibited by 36 and heximide. Increases in nigcricin concentration above 1.4 pM 360 PM cyclohrximide before significant inhibition of uptake of also resulted in less inhibition of uptake of DNP but the differ- DNI’ by 3.6 PM cycloheximide could be observed. After a 6-min ences were not as pronounced as those observed with cyclohexi- prior incubation period all three concentrations of the anti- mide (Table IV). I f the prior incubation period in 360 PM cyclo- biotic inhibited the uptake by at least 90%. Thus, compared hesimide was extended to 180 min before the addition of DNP, to the starved cell a shorter prior incubation period was required uptake of the uncoupler was then inhibited by 807,. Prior in order for the complete inhibition of uptake to be expressed. incubat,ion in 3.6 PM cycloheximide for either 20 or 180 min Table VI shows the changes in t.he effects that 3.6, 36, and 360 resulted in the same degree of inhibition of uptake of DNP. It pM cycloheximide exert upon t.he uptake of 2,4-DNP during the should be noted that in starved cells 1.4 pM nigericin inhibited transition from a log phase cell to a starved cell. In cells which the uptake of DNP in the presence of 360 PM cycloheximide, and had just reached the end of log phase Iittle difference was ob- 3.6 l.11~ cycloheximide inhibited the uptake of DNP in the pres- served between the three concentrations in the extent of the ence of 14 pM nigericin. inhibition after a lo- to 20-min prior incubation with the anti-

The contrasting effects of the low (3.6 PM) and high (360 pM) biotic, although both 36 and 360 PM cycloheximide exhibited concentrations of cycloheximide noted upon DNP uptake in greater inhibition after a 5-min prior incubation. Little differ- starved cells were reversed in log phase cells (Table V). In ence was observed in the extent of the inhibition between 3.6 and

30 60 90 - 0 20 40 60 0 IO 20 30 MINUTES MINUTES MINUTES

FIG. 3 (Zefl). Effect of 3.6 and 360 PM cycloheximide upon the uptake of glucose in starved and log phase cells. A, l-day starved cells, D-[l%]glucose (0.25 PC1 per ml) T\vas added to cells either after a 15.min prior incubation in either 3.6 or 360 pM cyclohexi- mide or at the same time as the antibiotic. Aliquots (0.5 ml) were plated and washed with 5 ml of Medium A containing 5 mM glucose at the l,imes indicated. Radioactivity taken up was deter- mined as described under “Methods.” B, log phase cells, cells xvere first a-ashed three times wit,h Medium A by low speed cen- trifugation and resuspended in the same medium to 1.6 X lo6 cells per ml. Prior incubation conditions and plating procedures were identical to those of starved cells. O-0, control cells; A---A, previously incubated with 3.6 PM cycloheximide; a---a, no prior incubation, 3.6 pM cycloheximidc; M--I, previously incubated with 360 .uM cycloheximide; O-0, no prior incubaiion, 360 PM cycloheximide.

FIG. 4 (center). Effect of 3.6 and 360 PM cycloheximide on the uptake and the incorporation of rj-j14C]phenylalanine into protein. One-day starved cells were previously incubated for 20 min in cycloheximide prior to the addition of L+C]phenylalanine (0.05 &i per ml). Aliquots (0.5 ml) of cells were plated and washed with 5 ml of Medium A conlaining 5 m&f DL-phenylalanine at t,he times indicated. Determination of incorporation of radioactivity

into protein was as described under “Methods.” O-O, uptake control; A---A, uptake, 3.6 PM cycloheximide; +H, uptake, 360 .UM cycloheximide; O-O, incorporation, control; a---n, incorporation 3.6 PM cycloheximide; U--U, incorporation 360 pM cycloheximide.

FIG. 5 (right). Effect of 3.6,36 and 360 PM cycloheximide on the uptake of [lIC]DNP and the incorporation of [14C]leucine into protein in 40.hour starved cells. A, uptake of [l(C]DNP was determined on cells previously incubated for 10, 20 and 30 min in the three concentrations of cycloheximide. Aliquots (0.5 ml) of cells were plated at 1,2 and 3 min after addition of 10 fiM [l”C]DNP (0.018 .&i per ml). Per cent, inhibition of uptake was calculated as described in Table IV. Control cells gave a count rate of 1589 cpm corrected for a freeze-thaw control of 117 cpm. B, incorpo- ration of [l%]leucine (0.1 PC1 per ml) was determined on cells which had been previously labeled for 20 min with the radioactive leucine. The three concentrations of cycloheximide were then added and 0.5-ml aliquots removed at 10, 20 and 30 min, and the radioactivity incorporated into protein was determined as de- scribed under “Methods.” O-0, control; A-A, 3.6 PM cycloheximide, w-,-m, 36 p~ cycloheximide; O-+, 360 PM

cycloheximide.

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Issue of October 25, 1971 W. R

T.\HLI,: VII TABLE VIII

Effect of prior inczbbalion in 360 p.v cyclohexirnide on inhibition of protein synthesis and uptake of DSP in absence oj cyclohexiwzide

Forty-hour starved cells were first incltbated for 30 min with 360 .UM cycloheximide. The cells were then washed twice with Media A at 28” by low speed centrifugation (20 set at top speed in clinical centrifuge). At 0, 30, 60, and 120 minutes after resuspen- sion the uptake of [14C]DNP and the incorporation of [14C]leucine were determined. The amount of DNP taken up was determined by plating 1.5 X IO6 cells (0.5 ml) at 1, 2, and 3 min after addition of 8 PM D?JP (0.014 ,&i per ml) as described in Table IV. Control cells gave a count rate of 816 cpm corrected for a freeze-thaw con- trol of 40 cpm. The rate of incorporation Ij-[14C]leucine (0.1 PCi per ml) was determined on 0.5.ml aliquots of cells plated at 0, 10, 20, and 30 minutes after addition of the radioactive leucine as described under “Methods.”

Effect of a d/t-hour incubation in cyclohexinzirle on uplalce of

llT]D~VP and incorporation of [14C]phenyZaZanine inlo protein Log phase cells were harvested by low speed centrifugation,

washed once, and resuspended in Media A to 1.5 X 10” cells per ml. Cycloheximide was added and after 24 hours of incubation the uptake of [14C]DNP and the incorporation of [‘4C]pherlylala- nine were determined. Aliquots (0.5 ml) of cells were plated I, 2, and 3 min after the addition of 10 FM [‘%]DNP (0.018 &i per ml) and the amount of [14C]DNP taken up was calcldated as de- scribed in Table IV. Control cells gave a count rate of 1423 cpm corrected for a freeze-thaw control of 100 cpm. Incorporation of [14C]phenylalanine was determined on 0.5-ml aliquots of cells .at 30, 60, and 90 min after the addition of [14C]phenylalanirle (0.05 &i per ml) as described under “Methods.”

Time after removal of Rate of incorporation of cycloheximide [Wjleucine Inhibition

- min ~molesjhr/lO~ cells %

0 10.3 64 30 12.8 55 60 19.3 33

120 21.2 26 Control 28.7

Uptake Of / Inhibition [W]DXP

?VdCS/O.5 ml cells Y*

Control. 2.33 3.6 ,UM cycloheximide. 2.14 8 36 pM cycloheximidc. 0.02 92

Time after removal of cycloheximide Uptake of [‘JCIDNP Inhibition

min nmole/lS x 106 cells %

0 0.600 32

30 0.024 97 60 0.038 96

120 0.560 37 Control 0.890

36 PM cyclohcximide in 2-day stationary phase cells, but 360 PM cyclohcximide did not inhibit uptake to the same extent. In 40.hour starved cells, a 20.min prior incubation in 3.6 ,UM cyclo- heximide was required in order for 90% inhibition of uptake to be observed, whereas at 360 PM cycloheximide uptake was in- hibited by only 15% after a lo- to 20.min prior incubation and 36 ~11 cycloheximide did not inhibit. to the same extent as 3.6 ,UM cycloheximide.

Since unpublished experiments had indicated that starved cells, as compared to log phase cells, were more susceptible to growth inhibition by cycloheximide when glucose was used as the carbon source, we also determined the effect of cycloheximide on glucose uptake. As shown in Fig. 3, the uptake of glucose was affect’ed by cycloheximide in a similar manner as the uptake of DNP. In st’arved cells, 3.6 PM cycloheximide inhibited the uptake of glucose whereas 360 ,LLM cycloheximide actually en- hanced the uptake. When the starved cells I\-ere not previously

incubat,ed with 3.6 PM cyclohesimide, little inhibition of uptake of glucose was noted during t,he first fen minutes after the addi- tion of glucose. Prior incubation of the cells in 360 PM cyclohexi- mide did not show any effect on the uptake of glucose over t.hose cells which were not previously incubated. Nigericin, 1.4 PM, was found to inhibit the uptake of glucose in starved cells in an identical fashion as 3.6 PM cycloheximide. In log phase cells both 3.6 and 360 PM cycloheximide inhibited t,he uptake of glucose to the same degree (Fig. 3).

and incorporation of [14C]phenylalanine into protein in starved cells was investigated in order to substantiate the hypothesis that GL Go protein synthesis was inhibited by the high conccn- tration of cyclohcximide which had little rffect upon the uptake of DNP and glucose. Figure 4 shows t,hat the uptake of phenyl- alanine was also not inhibit,ed by 360 pal cyclohesimide (the difference in upt,ake between the control and 360 PM cl-clohexi- mide was due to the inhibition of protein synthesis), but 3.6 ,LLM cycloheximide did inhibit uptake. No difference was observed between the two concentrations in t’he incorporation of phcnyl- alanine into protein. In another experiment the cffcct of 3.6, 36, and 360 PM cycslohcxirnide on the in vivo incorporation of [%]leucine into protein was st’udicd (Fig. 5). In t’his experiment the cells Tvere previously labeled wibh the isot’ope for 20 min prior to the addition of cyclohesimidc. Cyclohcsimide, 36 and 360 PM, inhibited protein synthesis to the same extent, but 360 KM cycloheximide inhibited the uptake of DNP by- cnly 155; whereas 36 PM cgclohesimide inhibited the uptake of DNP by 60 to SOT;. Although inhibition of protein synthesis by 3.6 FM cycloheximide was not as pronounced, the inhibition of the up t.akc of DNP \yas 90 ‘;/; inhibited.

The effect of 360 and 3.6 ~LRI cycloheximide on both the uptake

Additional observations which suggested that the effect that

cycloheximide exerted upon the uptake of DNP was not directly related to the inhibition of protein synthesis arc presented in Tables \‘I1 and VIII. In Table VII the effect of a prior incuba- tion of starved cells in 360 /*M cyclohcximide upon the inhibition of protein synthesis and the uptake of DNP afber removal of t’he drug are compared. The degree of inhibit.ion of protein synthesis continued to decrease after removal of the antibiotic, but the uptake of DXP did not show a similar pattern. Both immcdi- ately aft.er removal of the drug and 120 min later the upt,ake of DXP was inhibit,ed by 300/. However, at 30 and 60 min after resuspension in the cycloheximide-free medium the upt-ake of DNP was inhibited by over 950/O. Table VIII shows the results of an experiment in which cells were incubated with 3.6 and 76 PM cycloheximide during the initial 24.hour starvation period, and subsequently the effect of this prolonged incubation upon

I

Incorpora- ,$,~&l- Inhibition

alanine ~ -- fmoles/hr

0.5 ml cells YO

640 123 81 x0 88 I

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6150 Inhibition of Membrane Transport by Cycloheximide Vol. 246, A-o. 20

01 r 3.5% - 10% 35%- 10%

SUCROSE

FIG. 6. Effect of 3.6 PM cycloheximide and 1.4 PM nigericin on the ratio of monosomes to polysomes. Log phase cells were incu- bated with either 3.6 PM cycloheximide or 1.4 pM nigericin for 20 min prior to the analysis of the ribosomal pattern as described under “Methods.” Left panel: - - -, control; m, 1.4 1~ nigeri- tin; right panel: - - -, control; ---, 3.6 /LM cycloheximide.

the uptake of DNP and protein synthesis was examined. The uptake of DNP was inhibited by 92% and the incorporation of phenylalanine into protein was inhibited by 88%> in cells which had been incubated with 36 pM cycloheximide, whereas in cells incubated with 3.6 pM cycloheximide the inhibition of protein synthesis was inhibited by 81% but the upt’ake of DNP was inhibited only by 8%.

The effect of 1.4 PM nigericin and 3.6 PM cycloheximide on the ratio of monosomes t.o polysomes in Euglena was investigated since the in vivo ratio in other eucaryotes can be altered in the presence of cycloheximide (16, 17). An increase in the amount of monosomes was invariably observed when log phase cells were incubated with either 3.6 ~.RI cycloheximide or 1.4 P”M nigericin (Fig. 6). St.arved cells could not be utilized in this type of ex- periment as the majority of the ribosomes were present as monosomes.

DISCUSSION

The present experiments indicate that cycloheximide and nigericin inhibit the uptake of DNP, and presumably m-Cl(CC)P, under conditions in which there appears to be little effect of the antibiotics on respiration. Previous work in fungi has shown that toxic concentrat.ions of cycloheximide had no effect or only partially inhibited respiration (B-20). Greig, Walk, and Gibbons (21) report’cd that the antibiotic inhibited fermentation of glucose in Streptomyces cerevisiae, but no inhibition was ob- served in broken cell preparations and the inhibit.ion was related to a surface phenomena. Freeman’s personal communication to hshwell and Work (22) indicated that a concentration of at least 100 pg per ml was required to show any inhibition of bovine heart mitochondrinl NADH oxidase. It was recently shown that cycloheximide acts as a typical uncoupler in cert,ain plant tissues but fails to stimulate respiration in other types and in many instances the antibiotic was found to inhibit ion uptake without an apparent effect upon oxygen uptake (23). Reilly, Fuhrmann, and Rothstein (24) found that the drug inhibits the uptake of phosphate and K+ in yeast, but the uptake of phosphate in Euglena is unaffect.ed by cycloheximide (25).

Nigericin can either induce respiratory inhibition or have no effect, depending upon the substrate and the concentrations of substrates and cations in the external medium, on mitochondrja

in vitro (26, 27). When succinate or fi-hydroxybutyrate is the oxidizable substrate, nigericin induces release of alkali metal cations and contraction of the mit,ochondria without inhibiting respiration. The primary action of the antibiotic has been shown to be the promotion of alkali metal cation-proton exchanges across membranes (28) and the ionophore can abolish the uptake of alkali metal cations induced by valinomycin in mitochondria (29, 30). In chloroplasts (31, 32) and chromatophores (33) nigericin inhibits the light-induced uptake of protons and in chlo- roplast uncouples photophosphorylation. Henderson, hlcGivan, and Chappell (34) indicated that proton-carrying agents such as DNP, methylamine, or ammonium increased the rate of .K+ extrusion across membranes to maximal values in the presence of gramicidin, but not for nigericin or dianemycin. This differ- ence in activity, which was suggested to be the rate at which protons can traverse the membrane, becomes limiting for grami- cidin, but not for nigericin. In our studies dianemycin was found to be as effect,ive as nigericin in inhibiting the uptake of DNP. This observation would seem to preclude the involvement of an induced ATPase in t,he inhibition of uptake as dianemycin does not induce XTPase activity in mitochondria under condit.ions in which the activity is induced by nigericin (35). The fact that prior incubation in either cycloheximide or nigericin pre- vented the increase in extracellular pH and the loss in intracel- lular cations was in all probability a consequence of the inhibition of uptake of the uncoupler. Moreover, the observat’ion that when cells were treated w&h nigcricin alone there was no ap- parent change in extracellular K+ concentration suggests that the antibiotic does not induce a proton-alkali metal cation ex- change across the cell membrane of Euglena, at least not, under the conditions used in these experin1ent.s.

The observation that the degree of inhibition of plastid forma- tion by DNP and m-Cl(CC)P increases with decreasing extra- cellular pH suggests that the permeat,ing species is the un-ionized form of the acid. The degree of uptake will then be determined by the distribution of the ionized form of the acid between the outside and inside of the cell, assuming the unionized form to be freely permeable and equally distributed on both sides of the membrane. This dist,ribution will be governed by the pH gradient existing between the outside and inside of the cell as well as by the pK of the acid (36). Consequently, any process which would lead to a diminution of this pH gradient should decrease the uptake of DNP. The distribution of DNP has been used to calculate intracellular pH values in the giant axon of the squid (37) and in yeast (38). The absence of DNP inhibi- tion at pH values at which inhibition was not,ed with m-Cl(CC)P was probably due to the lower pK of DNP (3.95) as compared to the pK of m-Cl(CC)P (5.95).

One tent,ative hypothesis which might account for the in- hibit.ion of upt,ake of DNP by cycloheximide and nigericin could be that upon addition of these antibiotics a redistribution of intracellular ions is induced which results in a decrease in the pH gradient between the inside and outside of the cell. An impor- tant feature of both nigericin and cycloheximide action, in the relationship to these experiments, is the observation that nigeri- tin can induce movement of ions across the mitochondrial mem- brane wit.hout exhibiting an effect upon oxygen uptake and that cycloheximide can inhibit ion uptake without affecting oxygen uptake. Therefore, in vivo an alteration in the int.racellular ion distribution could presumably occur in the absence of any effect upon respiration. It should be emphasized that, althou.gh nigeri- tin may induce a decrease in Dhe nH gradient, t,hrouch a.n alkali

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Issue of October 25, 1971 I/V. Ii. Evans

metal catiorl-proton exchange, me do not propose that cyclo- hesimide also promotes such a reaction. Presumably cyclo- hesimide could induce a decrease in the pH gradient t.hrough some type of anion exchange mechanism similar to the anion exchange reaction catalyzed by the bisdichloroacetamides in mitochondria in vitro (39). We would suggest that as DNP enters the cell it passes through a region or compartment which is distinct from the cytoplasm and the inhibition of upt,akc of DNP may be due to a decrease in the pH of this cellular compart- ment.. Another tentative hypothesis would be that the inhibi- tion of uptake is the result of some effect upon membrane func- t.ion per se. Since the ua-ionized form of DNP appears to be the permeating species one must presume that after treatment with cycloheximide or nigericin the ml-ionized form of DNP is no longer permeable.

Whatever mechanism is involved in the inhibition of uptake of DNI’ by cycloheximide and nigericin, the following observa- tions indicate that the uptake of glucose is affected in a similar fashion. (a) In log phase cells both 3.6 and 360 HM cyclohexi- mide inhibit the uptake of DNP, and glucose upt.ake is similarly inhibited. (b) In starved cells 3.6 PM cycloheximidc inhibits the uptake of both l)NP and glucose whereas 360 MM cyclohexi- mide fails to inhibit the uptake of either compound. (c) In starved cells nigericin inhibits the uptake of both DNP and glu- cose.

Cycloheximide, 3.6 MM, invariably induced a decrease in the polyribosome content with the most pertinent observation being that’ 1.4 p>l nigericin induced identical changes in the polyribo- some profile as 3.6 ,uM cyclohesimide, concentrations which were effect,ive in inhibiting the uptake of DNP. Stanners (16) found that with low concentrations of cycloheximide nearly all the free ribosomes in hamster cells became attached to polysomcs whereas high concentrations of the drug had virtually no effect on the polysome profile. In reticulocytes 4 PM cycloheximide caused a light increase in the polyribosome cont’ent and 0.1 mM caused breakdown of the polyribosomes, whereas no breakdown was observed when the recticulocytes were incubated with 15 1nM

cycloheximide (17). The finding that in starved cells 360 PM cycloheximide pro-

duced essentially no inhibition of DNP uptake whereas protein synt,hesis was completely inhibit,ed suggests that the inhibition of membrane transport was not a consequence of the inhibition of protein synthesis. The observation that the inhibition of protein synthesis and the inhibition of uptake of DNP exhibited completely different patterns of inhibition after removal of the antibiotic also suggests that these two phenomena are not directl> related. Furthermore, the fact that incubation in 3.6 PM cyclo-

heximidc, during the initial 24.hour starvation period, resulted in the nearly complete disappearance of the inhibition of uptake of DXP whereas protein synthesis Teas still 805; inhibited offers additional evidence as to the two independent actions of the anti- biotic.

We do not mean to imply that the mechanisms responsible for t.he inhibition of membrane t,ransport by cycloheximide and nigericin in Euglena are necessarily identical, but the following observations suggest that t’he consequences of this act,ion, pos- sibly a decrease in pH gradient between bhe inside and outside of the cell, are quite similar. (a) Both antibiotics inhibit,ed the uptake of DNP and the requirement for a prior incubation period in order for the inhibition to be observed applied to both com- pounds. (b) The uptake of glucose was similarly inhibited. (c) The addition of eit,her compound to Euglena resulted in an

increased ratio of monosomes to polysomes. At present we have no indication as to how the growth phase of the cell determines the effect that cycloheximide exerts on membrane transport.

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William R. Evans and With the technical assistance of Carol SmithCOMPARATIVE STUDY WITH NIGERICIN

: AEuglenaThe Effect of Cycloheximide on Membrane Transport in

1971, 246:6144-6151.J. Biol. Chem. 

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