Mar 10, 2016 - cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a ...
Vol. 260, No. 5, Issue of March 10,pp. 2675-2680,1985 Prrnted in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1985 by T h e American Societyof Biological Chemists, Inc.
Quantal Entryof Diphtheria Toxin to the Cytosol* (Received for publication, August 23, 1.984)
Thomas H. Hudson and David M. Neville, Jr. From the Section of Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20205
The rate-limiting step in diphtheria toxin (DT) intoxication of Vero cells has been determined utilizing cycloheximide as an inhibiter of the intoxication process. Cycloheximide is shown to inhibit the toxin catalyzed ADP-ribosylation of elongation factor 2 (EF-2). The inhibitionis blocked by puromycin thus establishing theribosome as the location of cycloheximide protection. Washing cells free of cycloheximide rapidly of proreverses the protectiveeffect. The initial rates tein synthesisinhibition observed after removal of cycloheximide from DT-intoxicated cells are 5 to 12-fold greter than rates observed in unprotected cells and are shown to reflectADP-ribosylation of EF-2 by cytosolic DT. Ten to thirty minutes after cycloheximide removal, the rate of protein synthesisinhibition abruptly changes to values identical to those of unprotected cells. Both the initial rates and extent of the initial rapid inactivationare directly relatedto toxin concentration and time of incubation withDT in thepresence of cycloheximide. We concluded that: 1) the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the earlier entry step of DT into thecytosol. 2) DT enters the cytosol as a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. 3)It is inferred from 1 and 2 that the first order inactivation rate exhibited by DT is the result of the probability of the release of a bolus of toxin to thecytosol of any cell in the population per unit time. Autoradiographic analysis of intoxicated cell populations support this twopopulation state model. The size of a single bolus or quantum of DT is calculated from data over the range of lo-” to lo-’ M DT and is found to remain constant. We suggest that the cytosolic entry mechanism of DT results from a unique ability of the internalized toxin molecules to destabilize the vesicular membrane resulting in a random releaseof a bolus of toxin into the cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a post-receptor packaging process, package size remaining a constant and package number increasing with receptoroccupancy.
dine residue, diphthamide (2). Protein synthesis inhibitionin D T intoxicated cell populations display first order kinetics over many hours (3). The bindingof DT toreceptors is rapid and not rate-limiting (4).The enzymatic activity of D T Achain is not saturatedat physiologic concentrations of either kinetics, then, havebeen EF-2 orNAD+ ( 5 , 6 ) .The first order explained by postulating a constanttoxinconcentration within the cytosol (steady state model (3)). Implicit in the steadystate model arethree requirements: 1) continuous influx of DT to the cytoplasm (7) must be balanced by removal of A-chain activity, 2) DT-catalyzed ADP-ribosylationis the rate-limiting step in the inhibition of protein synthesis by DT. 3) EF-2 andcytosolic D T within thecell population being assayed must be in a steady state within a single, large pool of enzyme and substrate.All available evidence indicates that cytosolic DT A-chain is neither removed from the cytosol nor destroyed (8,9). We show here that DT entry and not ADPribosylation of EF-2 is the rate-limiting step. Finally, this study also demonstrates that EF-2 exists in two pools within a cell population exposed t o D T one pool is susceptible to ADP-ribosylation by DT and the otherpool is not. Thus the steady statemodel is not tenable. Data presented here support a novel hypothesis for DT entry. The implication of these findings for illucidating the molecular events involved in toxin entry are discussed. EXPERIMENTALPROCEDURES
Cells and Cell Culture-African green monkey kidney cells (Vero) were cultured in RPMI 1640 containing 2.4 mM NaHC03, 25 mM HEPES (pH 7.351, and 50 pg/ml gentamicin (culture medium) supplemented with 10% fetal calf serum. Culture conditions were humidified COz (5% in air) at 37 “C. Cells were trypsinized and seeded in Costar 96-well culture plates (8 X 10‘ cells/well in culture medium + fetal calf serum) 18 to 24 h prior to experimental manipulations. The S3 strain of HeLa cells was maintained and manipulated as detailed previously (6). Protein Synthesis Assay-Protein synthesis was assayed in cells maintained in 0.1 ml of leucine-free culture medium +0.1% BSA. The assay was conducted by addition of [“C]~-leucine(0.1 to 0.2 pCi/well) and fetal calf serum (10% final concentration) to wells for periods of 10 to 30 min. Cells were harvested onto glass fiber filters with a Titertek cell harvester, washed with water, dried, and radioactivity determined. In all experiments standard deviations were less than 15% of the mean; all points shown were at least twice the background counts/min with a counting error of 2 u = 2%; straight lines were determined by linear regression analysis (correlation coefInhibition of protein synthesis in mammaliancells by DT’ ficients and between -0.95 and -0.99) and controls were treated involves: (i) binding to plasma membrane receptors; (ii) trans- identically to experimental wells with the omission of DT. Assay for EF-2 and ADP-Ribosyl EF-2-HeLa cells were harvested porttothe cytosol compartment(entry);and(iii)ADPribosylation of EF-2, by D T A-chain (1) a t a modified histi- and resuspended in suspension MEM (Eagle’s) containing 0.1% BSA (2 X lo6 cells/ml). Four 100-ml aliquots of this suspension were * The costs of publication of this article were defrayed in part by maintained in 250-ml centrifuge tubes at 37 ”C in a humidified 5% the payment of page charges. This article must therefore be hereby COz/air atmosphere during the course of the experiment. At time 0, marked ‘‘duertlement” in accordance with 18 U.S.C. Section 1734 puromycin (600 p M ) was added to one tube. Twenty minutes later, cycloheximide (100 p ~ was ) added to thisand one other experimental solely to indicate this fact. M ) was added to these two The abbreviations used are: DT, diphtheria toxin; EF-2, elonga- tube. Twenty minutes later, D T (7 X tion factor 2; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic tubes and one other. All four aliquots were further maintained for 1 acid; BSA, bovine serum albumin; MEM, Eagle’s minimal essential h when the cells were harvested, resuspended, and washed by centrifmedium with modified Earle’s salts for suspension culture. ugation once with suspension MEM ? 0.1% BSA containing 100 p~
2675
Diphtheria Toxin Entry
2676
cycloheximide. The EF-2 extracts from HeLa cells were prepared as described in Ref. 6 except the homogenizing buffer contained 100 p~ cycloheximide. EF-2 and ADP-ribose EF-2 contents of the various HeLa extracts were determined in a 3-tube assay (6). Each tube in duplicate contained 250 pl of reaction mixture (50 mM Tris-C1, (pH 8.2), 1 mM EDTA, 40 mM dithiothreitol, 1.2 p~ [‘?]NAD+ (approximately 1 X 10’ cpm), and 25 to 50 pl of HeLa cell extract). To one tube was added 15 pg of DT A-chain and to another 15 pg of DT Achain and nicotinamide (0.1 mM). After a 60-min incubation at room temperature, the reaction was stopped by adding 0.25mlof 10% trichloroacetic acid and thesamples processed as described in Ref. 6. The calculation of EF-2 and ADP-ribose EF-2 contents were made as described in Ref. 6. Puromycin had no effect on EF-2 ADPribosylation in control or DT-treated cells (data not presented). Autoradiography-Vero cells (3 X 106 cells) were seeded on 25-mm glass coverslips and cultured overnight. The medium was removed and replaced with leucine-free RPMI 1640 + 0.1% BSA containing either no toxin or 7 X M DT. At 30,45, and 60 min after toxin addition, [“C]~-leucine(4 pCi/coverslip) was added for 15 min. At the end of the pulse the medium was removed, the cells washed 2X with complete RPMI 1640 at room temperature then 2X with icecold phosphate-buffered saline. The cells were fixedwith 4% paraformaldehyde in phosphate-buffered saline (pH 7.3) fixative for 20 min on ice. The coverslips were washed for 1 h in water, dried in air, coated with NTB-3 nuclear track emulsion, and stored at 4 “C for 24 to 48 h when the latent grains were developed. Fields of view were randomly selected from various sections of the coverslips. The location of individual cells was determined under phase-contrast optics. That cell was then viewed under bright field illumination to enhance the contrast of silver grains over that cell. The area of exposed silver grains over the individual cell was determined using the Optomax System IV image analysis system. This mode of analysis was used (rather than grain counts) to eliminate artifacts arising from a lack of grain resolution. The 0 to 5 reading (Fig. 4) corresponds to approximately 0 to 12 grains over a cell. Materials-Crude diphtheria toxin (Connought Laboratories) was purified as previously described (6). DT monomer was purified using a TSK 300 Spherogel column and Hewlett-Packard HPLC 1084-B. The monomer form of toxin wasexclusively used in this study. Cycloheximide, puromycin, and nicotinamide were purchased from Sigma. [I4C]~-Leucine and [3zP]NAD+were obtained from ICN. All other materials were of the highest grade commercially available. RESULTS AND DISCUSSION
The kinetics of DT inactivation of Vero cell protein synthesis (Fig. 1)were similar to those found in other cell systems (10). After a dose-dependent lag, first order inactivation rates
were established with values of -0.1, -0.5, -0.7 logs/h with lo-”, lo”’, and lo4 M DT, respectively. In order to determine whether entry of DT to the cytosol or the ADP-ribosylation of EF-2 by cytosolic DT A-chain was rate-limiting, we endeavored to study the kinetics of inactivation under conditions in which the rate observed would be due to ADP-ribosylation alone. The entry process and ADP-ribosylation were functionally separated by exploiting the finding that cycloheximide reversibly protected cellular protein synthesis during DT exposure (11). Cycloheximide inhibits protein synthesis by uncoupling the ribosome-binding and translocation functions of EF-2 resulting in a frozen polysome complex (12). Because EF-2 complexed to ribosomes is resistant to DT-catalyzed ADP-ribosylation (ll), itwas proposedthat theprotection of protein synthesis afforded by cycloheximide was a consequence of the inhibition of ADP-ribosylation of EF-2 by Achain. In order to verify this, we have utilized a modified method of Youle and Neville (6)for determining EF-2 and ADP-ribosylated EF-2 levels in HeLa cell cultures (see “Experimental Procedures” for details). After a 1-h exposure of cells to 7 x 10“ M DT, 97% (1 S.D. = 12%) of the total EF2 was in the ADP-ribosylated form. If 100 PM cycloheximide was included in the incubation with DT, no ADP-ribosylated EF-2 could be detected. With 600 p~ puromycin (an agent which competitively blocks tRNA-ribosomal binding and disrupts thepolysome complex (12)) included in the incubation with cycloheximide and DT, 67% (1S.D. = 11%)of the EF-2 was found in the ADP-ribosylated form. These findings indicate that cycloheximide inhibits ADP-ribosylation of cellular EF-2, and the inhibition is mediated at the level of the ribosome. Cells intoxicated with DT (thesame concentrations as Fig.
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FIG. 2. Kinetics of protein synthesis inhibition by DT after removal of cycloheximide protection.The medium of Vero cells cultured in 96-well plates was removed and replaced with culture medium + 0.1% BSA containing 20 p~ cycloheximide. After a 30min incubation under culture conditions toxin additions were made for final concentrations of 0 (O),lo-” M (0),lo-” M (O), and 10’ M (A). The cells were incubated under culture conditions for 1.5h when TIME (hours) the media were removed,the cells washed twice withculture medium FIG. 1. DT inhibitionof protein synthesisin Vero cells. Cells +0.1% BSA at room temperature (with the Nunc Immunowash), and (O), leucine-free culture medium (at room temperature) added (elapsed wereexposed to final DT concentrations of 0 (A),lo-” (O), or M (A) in culture medium + 0.1% BSA for 1 h on ice. The time: 8 min). The cells were incubated under culture conditions and media was removedand replaced with lucine-free culture medium (at protein synthesis was assayed as described in the legend to Fig. 1 at room temperature). After returning cells to culture conditions, protein the indicated times. The points represent the means of triplicates. synthesis was assayed as detailed under “Experimental Procedures.” Note because of the differences in incubation conditions before and Data points were plotted at the midpoint of the [“C]leucine pulse after the wash, extrapolations to zero time are not valid in Figs. 2,3, and represent the means of triplicates. The “C-labeled incorporation and 5. The I4C-labeledincorporation of no-toxin controls ranged from 1,654 to 3,707 cpm. of no-toxin controls range from 6,424 to 12,213 cpm.
Entry
Toxin
Diphtheria
1)in the presence of cycloheximide were washed free of both cycloheximide and DT and thekinetics of protein synthesis inhibition observed(Fig.2). Inactivation was seen to be governed by two rate constants. The initial slopes were -5, -3, and -1 logs/h forlo-', 10"O, and lo-" M DT, respectively. These slopes are 5 to %fold greater than the corresponding slopes observedin Fig. 1.The rapid initial ratesof inactivation are interpreted to be due to the ADP-ribosylation of newly unprotected EF-2 by A-chain which had entered the cytoplasm during the protective incubation with cycloheximide. Support for this interpretation comes from the fact that the initial rates of inactivation extrapolate to 100% of control values at the washout of cycloheximide.Toxin entering from the cell surface exhibited significant lag periods (10"O and lo-" M DT (Fig. 1) yet no lag was seen after the washout of cycloheximide. The initial rates of protein synthesis abruptly changed to lower values, 15 to 20 min after cycloheximide washout.The later slopes were identical to the firstorder inactivation rates observed in control cells treated identically except for the absence of cycloheximideduring DT intoxication (Fig. 3). The rapid initial rate of inactivation after cycloheximide washout was eliminated by the inclusion of puromycin with cycloheximide and DT (Fig. 3, inset). Thus, the rapid rates were a consequence of cycloheximide protecting EF-2 from ADPribosylation at the level of the ribosome. We conclude that ADP-ribosylation of EF-2 by cytosolic A-chain is the rapid step and not therate-limiting step in protein synthesis inhibition by DT. For protection to be observed, cycloheximideand DT must have simultaneous access to the ribosome/EF-2 pools. The protection afforded by cycloheximide was only effective ona portion of the protein synthesizing apparatus in the cell
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2677 population (Fig. 2).The size of this portion is determined by the extent of inactivation occurring under the initial rapid rate constant. The portions protected (i.e. the portions subsequently rapidly ribosylated)were dependent on DT concentration (45%, 87%, and 298% protection with lo-", lo-", and lo-' M DT, respectively (Fig.2)). The levels of cycloheximide usedin thisstudy were sufficient to inhibit greater than 97% of protein synthesis (data not shown). Cycloheximide must, then, have accessto virtually all of the ribosome/EF-2 in the cell population. This leads to theconclusion that only a subpopulation of the protein synthesis apparatus (ie. EF2) present in the culture had been exposed to DT A-chain during the incubation with cycloheximide. One way to divide the EF-2 in a tissue culture experiment into subpopulations is at the cellular level. In thismodel, after the cycloheximide washout, EF-2 is rapidly inactivated, not homogeneously throughout the cell population but only in a subpopulation of cells. These cells are defined as containing DT A-chain in their cytosol. The second, slowerinactivation rate, which is a function of entry, represents the rate of conversion of cells from the population containing no toxin to the population containing toxin in their cytosol. In this model, toxin entry is the release of a quantum2of DT A-chain molecules to the cytoplasm large enoughto rapidly inactivate all EF-2 in that cell. This model predicts that the cells in a DT intoxicated population will exhibit, in themain, either no protein synthesis or control levels of protein synthesis. Autoradiographic analysis of DT intoxicated cultures at early times (Fig. 4) support this model. As intoxication progresses from 20 min to 1 h the subpopulation of cells exhibiting control levels of protein synthesis decreases and the population exhibiting no protein synthesis increases. The steady state hypothesis predicts the shifting of a single population of cells toward lower and lower mean levelsof protein syntheS ~ S The . ~ steady state hypothesis can be formally related to experimental data as follows: P = Po e" P In- = -kt
PO
k
= ckp
Pois the rateof protein synthesis in theabsence of toxin and P is the rateof protein synthesis at any time ( t )after the first order rate is established. The first order inactivation rate constant ( k ) is obtained by plotting the log fractional rate of protein synthesis uersus time and is equal to the slope. k is I proportional to both the cytosol DT A-chain concentration 5 ! 3 I 2 0 ( c ) and the apparent turnover number of DT A-chain (b). w4w TIME (hours) Varying receptor occupancy changes k, see Fig. 1 (3, 7). The FIG. 3. Comparison of kinetics of protein synthesis inhibi- steady state model incorporates this finding by predicting tion by DT in unprotectedor protected cells after washout of that c is a function of A-chain entry and efflux (or degradacycloheximide.Vero cells in 96-well plates were incubated with (0) or without (0)cycloheximide for 30 min as detailed in the legend to tion) from the cytosol withentry being a function of receptor I
I
I
6
Fig. 2. Toxin additions were made for final toxin concentrations of 0 (0)and 10"' M (0,0). The cells were incubated under culture conditions for 1h, washed, and protein synthesis assayed at thetimes indicated as described in the legend to Fig. 2. The points represent the means of triplicates. The "C-labeled incorporated of no-toxin controls ranged from 982 to 5790 cpm. Inset: Vero cells in 96-well plates were incubated with (m) or without (A)100 p~ puromycin under culture conditions for 15 min. Cycloheximide was then added to all wells and theincubation continued under culture conditions for 30 min as in the main figure. DT was added for final concentrations of 0 (0)and 10"' M (A,m). The cells were incubated under culture conditions for 1h, washed, and protein synthesis assayed at thet i e s indicated as in the main figure. The points represent the means of triplicates. The 'C-labeled incorporation in the no-toxin controls range from 968 to 1023 cpm.
* The terms quantum and quantal are used here in their neurophysiological sense. Byquantum we mean the number of effector molecules which are rapidly released (compared to other time constants in the system) from one compartment to a second compartment containing effector targets. The size of the quantum determines the size of the response (ie. miniature end plate potential), and the effects of several quanta may or may not be cumulative depending on the state of other variables in thesystem. Previously Eiklid and co-workers (13) demonstrated two populations of cells, those synthesizing protein and those not at a late time during the intoxication process, 15 b. The intoxication rate constant of protein synthesis was zero or positive at this time. Data at this time is irrelevant to distinguishing between the two different models under discussion here.
Diphtheria Toxin Entry DT must undergo an acid requiring processing event before it efficiently enters thecytoplasm (15). This event is blocked by NH4Cl presumably through raising the pHof intracellular vesicles (16). Fig. 5 demonstrates that theaddition of NH&l after cycloheximide washout has no effect on the subsequent kinetics of inhibition. The replacement of leucine-free RPMI at pH 5.8 or 7.35 after the cycloheximide washout resulted in no differences in the subsequent kinetics (data not shown). We conclude from these findings 1)the pHsensitive processNo Tar8n ing step occurs early in the incubation with DT; 2) NH4Cl and pHof the medium have no effect on ADP-ribosylation of EF-2 by cytosolic A-chain; 3) toxin molecules do not necessarily enter the cytoplasm directly after undergoing the pH sensitive processing step. Ratherthe toxin enters via a cryptic, &ea 01 S~lve,Gram Over Cell IAlbttrary Unllrl pH insensitive compartment as hasbeen suggested by Marnel FIG. 4. Kinetics of protein synthesisinhibition by DT: anal- et al. (10). Evidence for the latter conclusion is the fact that ysis at the cell population level. Vero cells were exposed to DT the second slower rate of inactivation (which represents the and [14C]leucine,washed, fixed, prepared for autoradiography, and conversion of cells with no cytoplasmic A-chain to cells with analyzed as described under "Experimental Procedures." The ordinate A-chain) is not sensitive to either pH changes or NH,Cl (Fig. represent arbitrary ranges of silver grain density over individual cells. 5). The columns represent the number of cells with that grain density in Toxin binds receptors and entersendocytotic vesicles (17). the population expressed as theper cent of total cells counted (-200/ Acidification of these vesicles by proton pumps (18,19) occurs condition). approximately 3 to 4 min after receptor binding (10) and allows the acid requiring processing step to proceed. This step occupancy (3, 7). In studies presented here, cycloheximide probably results in the insertion of DT into the membrane. blocks $. The firstorder rate of inactivation should return to Acid dependent insertion of DT into artificial membranes has been reported to result in the translocation of the A-chain the original (pre-blocked) value and remain constant until inactivation is complete. The experimental data in Figs. 2, 3, nucleotide binding site (20, 21) and glycohydrolytic activity and 5 do not support these predictions of the steady state (22) to theopposite side of the membrane. The present study demonstrates that if A-chain movement through the memmodel. An estimate of the range in number of molecules of A-chain brane does occur immediately following vesicular acidificaneeded to rapidly inactivate all EF-2 within the susceptible tion, it does not necessarily result in the immediate susceptpopulation cell can be derived from Fig. 2. By using a turnover ability of cytosolic EF-2 to ADP-ribosylation. This is apparent from the two populations of cells seen in Fig. 4 which are number of EF-2 ADP-ribosylation by DT A-chain (5) and adjusted to cytosol pH (14), the initial rapid rate constant of M DT (Fig. 2) corresponds to 300 moleinactivation at ; cules/cell volume (1 pl). The number is diminished at lower toxin concentrations (176 at 10"' M, 50 at lo-" M). The initial post-cycloheximide washout slopes or rate constants (and theircorresponding A-chain concentrations) represent in the quantal model a weighted average of rate constants Kl, K2, K3 . . . associated with fractions Fl, F,, F3 . . . corresponding to cell populations which have experienced 1, 2, 3, . . . quantal events during the cycloheximide incubation. The constant Kl is of interest because it represents the size e-' a" of one bolus or quantum. The Poisson formula Fn = n! ! ! indicates that thefraction of a population Fn experiencing n ! events is related to the average number of events a. When n ! = 0,Fn = e-". Fn is determined in Fig. 2 by they axis intercept ! ! at the inflection point of the two slopes, i.e. the fraction of ! ! cells protected from the rapid process whichreceived no ! ! quantal events. At lo-" and 10"' M DT, a equals 0.6 and 2.0, ! respectively. At loT9M DT, the inflection points is in doubt ! but is equal to or less than 2% and a is therefore 3.9 or greater. The size of the quantum is the weighted average divided by the average number of events a. The quantal size for lo-", M DT is 83, 88, and 77 (or less) molecules of and FIG. 5. Effects of NH&l on DT inhibition of protein syntheDT A-chain. The quantal size is constant over a range where sis after removal of cycloheximide. Vero cells in 96-well plates receptor occupancy changes by 50-fold (KA, for DT and Vero were incubated with cycloheximide for 30 min and toxin added to a cells is lo9 M - ~(4)).Over this same range the probability of final concentration of 0 (0)or 10"' M (0,0) as described in the entry (the second slopes in Fig. 2 or the slopes in Fig. 1) legend to Fig. 2. After 1.75 h incubation under culture conditions, the cells were washed and leucine-free medium alone (0)or containing change by 6-fold. This finding implies that there is a segre- 10 mM NH,CI (0)was added hack to the wells as described in the gating event operating between the receptors on the external legend to Fig. 2. Protein synthesiswas assayed at thetimes indicated membrane which are both filled and empty and the vesicles in Fig. 2. The I4C-labeledincorporation of no-toxin controls ranged from 1182 to 3212 cpm. from which DT is presumably released to thecytosol. + Tail"
T
m
........ . . . .
Diphtheria Toxin Entry slowly interconverted with time. The same conclusion can be drawn by observing kinetics of DT intoxication at subsaturatingconcentrations of DT. The time of onset of ADPribosylation for the entirepopulation of cells shown in Fig. 1 is dependent on the DTconcentration and thistime is much later than the onset of vesicle acidification. This dose-dependent lag is also pH dependent, and we observe that pH decreases of approximately 1 unit shortenthe lag. This iswhy in Fig. 1 we fail to observe a lag at lo-' M DT with RPMI medium which allows Vero cells to rapidly acidify the medium. With MEM bicarbonate medium and less acidification, a 10min lag is observed. A pH dependence of the dose-dependent lag period of ricin intoxication (in theopposite direction) has been observed and interpreted as reflecting an obligatory pH dependent first order processing step (23). The same may be true for DT. If this is the case there are likely two pH dependent steps for DT. An initial dose-dependent processing step which must precede an acid dependent insertion. The artificial bilayer systems may not require the first step for demonstration of phenomena associated with insertion and translocation. However, the insertion efficiency of the artificial system has not been compared to thatof the biological system and is probably low since the bilayers lack receptors. It is feasible that the processing step is obligatory when DT is inserted into the membrane from a receptor bound state. Considering the above information and the datapresented in thispaper we propose the followingmodel. DT binds receptors and enters endocytotic vesicles which are rapidly acidified. During the lag period an acid dependent processing step occurs with arate proportional tothe bound toxin concentration. Processed toxin is inserted intothe membrane and this step is also acid dependent. Either before or after insertion intothe membrane the DTis packaged into vesicles each vesicle containing the same amount of DT. The number of these vesicles made is related to receptor occupancy. The insertion of DT into thevesicular membrane destabilizes the membrane in some as yet undetermined manner, and this destabilization imparts to the vesicle a certain probability (probability constant) per unit timeof rapidly releasing all of its included toxin molecules to the cytosol. The probability that a cell willreceive a bolus of toxin is the probability constant times thenumber of vesicles containing a quantum of toxin which is proportional to receptor occupancy. When toxin release occurs all EF-2 is rapidly inactivated leading to a parallel rapid fall in protein synthesis in that cell. The first order rate of protein synthesis inactivationin a population of intoxicated cells is the result of a constant probability that a bolus of toxin will be released at random in any cell of the population. This probability defines the rate-limiting step in intoxication at all times after the processing step is largely completed. At any DT concentration the time required for any cell to experience on the average one bolus is the time (exclusive of lag) taken to reach 37% (e-') of control protein synthesis. Doubling this time gives the time required to experience on the average two such events. Because the rate of EF-2 inactivation is 5 to 12 times faster than the quantal entry (Figs. 2, 3, and 5), the results of second and third quantal entry events are not detectable and the rate-limiting process fits first order kinetics. The above modelis an attempt toincorporate our new data with previous knowledge on DT entry mechanisms. However, the data of this paper only requires a model having the
2679
following features: 1) DT A-chain entry to the cytosol compartment is rate-limiting for the intoxication process; 2) the rate-limiting entry is proportional to the receptor bound DT concentration; 3) entry is a concerted process delivering a bolus of toxin molecules to the cytosol of an individual cell; 4) the size of the bolus which is sufficient to rapidly inactivate all the EF-2 in a cell is constant over a wide range of receptor occupancy; 5) within a cell population the concerted entry event is random; and 6) steps preceding the entry event are acid-dependent but the entryitself is acid-independent. The process by which a destabilized vesicle releases its toxin content to thecytosol compartment may be thought of as a functional lysis of the membrane vesicle barrier, but the actual physical process is unknown. There aremany examples in biology wherematerials within vesicular compartments are transferred to other membrane bound compartments. The well-studied examples such as endocytosis, exocytosis, and the formation of secondary lysosomes are actually membrane fusion reactions. The stabilizing forces which keep vesicles intact as discreet entities and the local destabilizing forces which permit fusion reactions are poorly understood. However, these reactions can also be utilized by pathogens such as theenveloped RNA viruses to gain access to thecytosol of cells (24). Diphtheria toxin maybe another example of a pathogen exploiting a membrane destabilization reaction. Because the DT toxin entry is such a highly specific process which appears to involve quantal packaging of DT, we speculate that this process is physiological and is used by endogenous substances. Considering the many similarities of the protein toxins (25) we would not be surprised to find other examples of this phenomenon. Acknowledgment-We acknowledge the helpful suggestions and stimulating discussions of Dr. Richard Youle. REFERENCES 1. Pappenheimer, A. M., Jr. (1977) Annu. Reu. Biochem. 46,69-94 2. Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 265,10710-10716 3. Uchida, T., Pappenheimer, A. M., Jr., and Harper, A. A. (1973) J. Biol. Chem. 248,3845-3850 4. Middlebrook, J. L., Dorland, R. B., and Leppla, S. H.(1978) J. Biol. Chem. 263,7325-7330 5. Moynihan, M.R., and Pappenheimer, A. M., Jr. (1981) Infect. Immun. 32,575-582 6. Youle, R. J., and Neville, D. M., Jr. (1979) J. Biol. Chem. 2 5 4 , 11089-11096 7. Pappenheimer, A. M., Jr., and Gill, D. M. (1973) Science ( Wash. D. C.) 182,353-358 8. Yamaizumi, M., Uchida, T., Takamatsu, K., and Okada, Y.(1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 461-465 9. Draper,R. K., Chin, D., Eurey-Owens, O., Scheffler, I. E., and Simon, M. I. (1979) J. Cell Biol. 8 3 , 116-125 10. Marnell,M. H., Shia, S.-P., Stookey, M., and Draper,R. K. (1984) Infect. Immun. 4 4 , 145-150 11. Gill, D. M., Pappenheimer, A. M., Jr.,and Baseman, J. B. (1969) Cold Spring Harbor Symp. Quant. Biol. 34,595-602 12. Gale, E. F., Cundlittle, E., Reynolds, P. E., Richmond, M. H., and Waring,M. J. (1981) The Molecular Basis of Antibiotic Action pp. 402-547, John Wiley and Sons, New York 13. Eiklid, K., Olsnes, S., and Pihl, A. (1980) Exp. Cell Res. 126, 321-326 14. Honjo, T., Nishizuka, Y., and Hayaishi, 0. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 603-608 15. Draper, R. K., and Simon, M. I. (1980) J. Cell Biol. 87,849-854 16. Sandvig, K., and Olsnes, S. (1980) J. Cell Biol. 87,828-832
2680
Toxin
Diphtheria
17. Keen, J. H., Maxfield, F. R., Hardegree, M.C., and Habig, W.H. (1982)Proc. Natl. Acad.Sci. U.S. A. 79,2912-2916 22. 18. Galloway, C. J., Dean, G . E., Marsh, M., Rudnick, G., and
Entry 46,102-104 Donovan, J., Simon, M., and Montal, M.(1982)Biophys. J. 37, 256a
