Endosome fusion induced by diphtheria toxin translocation domain

3 downloads 217470 Views 1MB Size Report
Jun 10, 2008 - Once in the cytosol, the free A chain refolds into its biologically ..... 100 control E349K P308S DT. DTs. CRM197 diameter < 0.5µm diameter 0.5- ...
Endosome fusion induced by diphtheria toxin translocation domain Antonella Antignani and Richard J. Youle* Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive MSC 3704, Bethesda, MD 20892 Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved April 7, 2008 (received for review December 12, 2007)

Endosomal cargo travels through a dynamic vesicle network en route to degradation by lysosomes or recycling through the Golgi apparatus back to the cell surface. Rab5 is a key determinant of the early endosomes by organizing effector proteins in specific subdomains and mediating early endosome fusion. We find that early endosome morphogenesis and maturation is disrupted by diphtheria toxin (DT). Rab5 bound endosomes increase in size and in Rab5 content due to luminal toxin exposure, whereas Rab7 positive endosomes are not detectably altered. These changes appear to be caused by an activity of the toxin entry domain (T domain) as mutations inactivating either the receptor binding (CRM107) or ADP-ribosyl transferase (CRM197) activities do not inhibit the effect of DT on endosome morphogenesis. In contrast, mutations in the T domain or diminishing the endosomal pH gradient, which prevents T domain membrane insertion, inhibit these endosome changes. The change in size appears to be due to changing the early endosome fission-fusion equilibrium. The Rab5 membrane exchange rate, assessed with photoactivatable GFP-Rab5, decreases in the presence of DT. These changes to endosomes may reflect activities of the T domain that mediate toxin entry to the cytosol. The nontoxic mutant DT, CRM197, yields a new tool to manipulate endosome dynamics in living cells. early endosomes 兩 Rab5 兩 Rab7

M

any toxic proteins of bacterial and plant origin enzymatically modify substrates within the cytosol of mammalian cells (1). These toxins translocate certain protein domains across the plasma membrane or, after endocytosis, across the membrane of intracellular organelles by a number of different mechanisms. Diphtheria toxin is synthesized and secreted by Corynebacterium diphtheriae as a single chain polypeptide containing three distinctly different functional domains: the aminoterminal enzymatic ‘‘A chain,’’ the translocation or ‘‘T domain,’’ (residues 202–378), and the carboxyl-terminal receptor binding domain (residues 386–535). Diphtheria toxin (DT) binds to a cell surface receptor and becomes endocytosed into clathrin-coated vesicles. After vesicle uncoating, DT is delivered to early endosomes and is proteolytically cleaved by furin to generate a disulphide-linked, heterodimeric A chain-B chain form of the toxin. The low pH in endosomes generated by a proton-pumping vacuolar ATPase (V-ATPase) induces all three toxin domains to form partially unfolded molten globule-like structures, thereby exposing hydrophobic helices in the T domain that insert into the endosomal membrane and mediate the translocation of the partially unfolded A chain across the endosomal membrane. Once in the cytosol, the free A chain refolds into its biologically active conformation and inhibits protein synthesis by the ADPribosylation of EF-2, leading to the death of the cell (2, 3). How the A chain crosses the endosomal membrane into the cytosol remains unclear. The structure of native DT shows that the T domain consists of a bundle of 10 ␣-helices (4). Six of these helices fold similarly to helices of the Bcl-2 family members (5) and the channel-forming domains of certain colicins (6). The two long hydrophobic helices (TH8 and TH9) buried within the native T domain are thought to insert into the membrane bilayer 8020 – 8025 兩 PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23

upon exposure to the low pH of endosomes (7–9), and several other helices adhere along the luminal side of the membrane (10). Studies in planar lipid bilayers have shown that under low pH conditions the T domain is capable of translocating the A chain across the bilayer in the absence of other proteins (11). However, Ratts et al. (12) provide evidence that the translocation of DT A chain out of purified early endosomes is not autonomous as it requires the addition of a cytosolic translocation factor complex composed in part by Hsp90 and thioredoxin reductase. Studying the kinetics of toxin entry, Hudson and Neville (13) conclude that the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the entry of DT into the cytosol. They propose a model in which DT enters the cytosol in a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. The size of a single bolus of DT calculated from data over the range of 10⫺11 to 10⫺9 M DT was found to remain constant at ⬇20 molecules. They 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 the simultaneous release of all of the toxin molecules in an endosome. The process by which a destabilized vesicle releases its toxin content to the cytosol compartment may be thought of as a functional lysis of the membrane vesicle barrier, but the actual physical process of toxin release from endosomes is unknown. Intracellular membrane traffic transfers materials within vesicular compartments to other membrane-bounded compartments through membrane destabilizing reactions that permit fusion of vesicles with the target cellular compartments. Rab proteins, which constitute the largest family of monomeric GTPases and their effectors coordinate consecutive stages of membrane transport, such as vesicle formation, vesicle and organelle motility, and tethering of vesicles with target membranes during the endocytic and exocytic pathways. These proteins are highly compartmentalized, making them excellent markers of vesicle subtype (14). Rab5 is localized on the membrane of early endosomes, where it regulates clathrincoated vesicle-mediated transport from the plasma membrane to the early endosomes and homotypic early endosome fusion through the recruitment of a large number of effector proteins (15, 16). As early endosomes become acidified, they form the first cellular environment suitable for DT translocation to the cytosol. Here, we report the real-time visualization of endosomes during DT entry and the consequences of DT translocation on the status of the endosomal membranes. By analysis of the dynamics of endosomes, using Rab5 as a marker, we identify Author contributions: A.A. and R.J.Y. designed research; A.A. performed research; A.A. and R.J.Y. analyzed data; and A.A. and R.J.Y. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. *To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0711707105/DCSupplemental.

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0711707105

500

control 400 300 200 100 0

500

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

DT 400

count

B

DT

300 200 100 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 500

C CRM197

CRM197

count

400 300 200 100 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

diameter (µm) Fig. 1. Endosome changes in the presence of DT. (A–C) HeLa cells, transfected with a plasmid encoding EYFP-Rab5 for 12–15 h, were incubated in the absence (A) or presence (B) of 0.5 nM DT (A and B) or CRM197 (C) for 30 min at 37°C. The cells were examined by confocal microscopy and the calculation of the diameter of Rab5a positive endosomes was performed by using Volocity software. (Scale bars: 10 ␮m.) (D) The histogram represents the distribution of the calculated diameters of Rab5 positive endosomes (n ⫽ 2,000) chosen randomly in control and toxin treated cells, and the black curves represent the Gaussian distribution of the histogram data.

dramatic consequences on endosome size due to the DT translocation domain insertion into membranes. Results Early Endosome Morphology in the Presence of DT. Subsequent to HeLa cell surface receptor binding, DT, at a concentration (1 nM) that causes a 90% reduction in protein synthesis after 4 h (17), enters transferrin (Tf) positive endosomal compartments within 5 min and is progressively concentrated over 30 min into vesicles with characteristics of the degradative pathway (18). To probe potential alterations in the endosomal compartment during the DT membrane translocation process, we visualized HeLa cells transiently expressing EYFP-Rab5 after exposure to DT. Substantial overexpression of Rab5 was shown to cause pronounced enlargement of the early endosomes (19, 20). In our experiments, EYFP-Rab5 was expressed at sufficiently low levels that the enlargement was not observed (Fig. 1A). Under these conditions, we found that cells incubated with 0.5 nM DT for 30 min display enlarged Rab5 positive vesicles (Fig. 1B) compared with untreated cells (Fig. 1 A). A mutation of glycine 52 to glutamate in the DT catalytic domain (CRM197) causes the loss of the ADP ribosylation activity and cytotoxic activity (21). We found that CRM197 also induces the formation of enlarged Rab5 positive vesicles (Fig. 1C), showing that the effect on endosomes is not a consequence of DT inhibition of protein synthesis. When we analyzed the distribution of the diameters of Rab5 positive endosomes (n ⫽ 2,000) chosen randomly in control and toxin treated cells, we found that the major fraction of endosomes Antignani and Youle

from DT and CRM197 cells had diameters in the interval of 0.7–1.2 ␮m, whereas, in control cells, most endosomes had a diameter between 0.4 and 0.6 ␮m. We measured also a clear increase in the proportion of endosomes with a diameter ⬎1 ␮m in DT and CRM197 treated cells compared with untreated cells (Fig. 1D). This DT-induced alteration in morphology of the vesicles does not require Rab5 overexpression because we detected enlarged endosomes in untransfected cells treated with DT and immunostained with anti-Rab5 antibody (data not shown). These enlarged Rab5 positive vesicles stain positive for another marker, EEA1, confirming that they represent early endosomes. The quantification of the colocalization of Rab5 with EEA1, represented as a Pearson’s correlation coefficient, indicates less association between Rab5 and EEA1 in control cells compared with that in the early endosomes in DT and CRM 197 treated cells [supporting information (SI) Fig. S1]. This is likely due to the higher number of enlarged endosomes in DT treated cells compared with the control cells. We compared the percentage of Rab5 fluorescent volumes distributed in three diameter ranges in control and toxin treated cells (Fig. S2). We considered the fluorescent volumes with a diameter ⬍0.5 ␮m as one group that would include the coated and uncoated vesicle populations (22), those vesicles with a diameter between 0.5 and 1 ␮m and those vesicles with a diameter ⬎1 ␮m. We found that in cells treated with wild-type DT or CRM197, almost 60% of EYFP-Rab5 positive endosomes have a diameter ⬎1 ␮m compared with 30% in control cells (Fig. S2). The toxin ricin, that transits early endosomes after caveolae mediated endocytosis before translocation to the cytosol from within the ER, did not affect vesicle diameter (Fig. S2). We also analyzed the populations of Rab5 positive vesicles in cells incubated with a chemical conjugate of human transferrin (Tf) and a diphtheria toxin mutant (CRM107) with a 10,000-fold reduced affinity for the DT receptor (Tf-CRM107) (23). This immunotoxin binds and enters cells via the transferrin receptor before translocation across membranes via the DT T domain. Like DT and CRM197, this toxin increases the population of Rab5 positive vesicles with diameters ⬎1 ␮m in diameter, indicating that the effect does not depend on DT receptor binding (Fig. S2). Thus, DT mutants either in the receptor binding domain or in the enzymatic domain retain endosome enlargement activity. Mutations E349K and P308S in the T domain inhibit DT membrane translocation (23–25). We individually inserted these two mutations in an inactive version of the DT with a point mutation into the catalytic domain (E148S) (26, 27). We expressed and purified DT-E148S (DTs), DTsE349K, and DTsP308S and examined their effect on the early endosomes. The percentage of Rab5 fluorescent volumes distributed in three diameter ranges in control and cells treated with each of the three recombinant toxins was compared with that of wild-type DT and CRM107. Recombinant DTs is able to induce endosome enlargement similar to wild-type DT and CRM197 whereas the two mutants, DTsE349K and DTsP308S, were largely deficient in this activity supporting the model that the DT T domain is responsible for endosome enlargement (Fig. 2 A and B). Low pH in endosomes is required for the translocation of DT by triggering a conformational change in the T domain that exposes hydrophobic domains and initiates toxin membrane insertion (28, 29). Lysosomotropic agents, such as NH4⫹Cl⫺, that raise the endosomal pH by reducing the proton gradient across the membrane block DT toxicity (30). To test whether the endosomal enlargement induced by DT depends on the insertion of the T domain into the endosomal membrane we incubated HeLa cells with 1 mM NH4⫹Cl⫺ in the presence of DT or CRM197 and determined the size of the Rab5 positive endosomes. In the presence of NH4⫹Cl⫺, ⬇70% of the endosomes in PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23 兩 8021

CELL BIOLOGY

control

count

A

D

control

DTs

80

- NH4Cl

+ NH4Cl

control

control

DT

DT

CRM197

CRM197

DTsP308S

diameter < 0.5µm diameter 0.5-1µm diameter > 1µm

60

B

40 20 0

7

19

M

CR

s DT

DT S 08 P3 K 49 E3 l ro nt co

+ NH4Cl

60 40 20

7

19

l

80

M

100

DT

0

CR

8022 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0711707105

80

diameter 1 µm

ro nt co

cells treated with DT and CRM197 had a diameter ⬍1 ␮m, similar to that of control untreated cells (Fig. 3 A and B). There is, however, a 10% increase in the population of vesicles with diameters ⬍1 ␮m (Fig. 3 A and B) due to NH4⫹Cl⫺ alone. This indicates that the increase in endosomal size occurs due to T domain insertion into early endosomes and not at later stages of the toxin trafficking pathway. When HeLa cells were treated with nocodazole to depolymerize microtubules and inhibit the transport of the endosomal carrier vesicles (ECV) to late endosomes (31), although the control endosomes were smaller, DT retained the capacity to enlarge them (data not shown). We considered the possibility that the endosome enlargement induced by the T domain reflects an activity associated with DT translocation. If T domains act cooperatively to mediate translocation, a prediction consistent with the bolus model, additional T domain activity supplied from inactive DT molecules, such as that in CRM197, may increase DT toxicity. Measuring protein synthesis inhibition in Vero cells by different DT concentrations in the presence or in the absence of CRM197 revealed that CRM197 at a concentration below that which competes for receptor binding increased cell sensitivity to DT 50-fold (Fig. 3C). This indicates that T domains function cooperatively, consistent with the model that the change in the endosome morphology is correlated to the delivery of the DT catalytic domain into the cytosol. A dominant negative Rab5 mutant, S34N, inhibits fusion

C

100

DT 7 19 M CR l ro nt co

Fig. 2. DT translocation and endosome morphology. (A) HeLa cells, transfected with a plasmid encoding EYFP-Rab5 for 12–15 h, were incubated in the absence (control) or presence of 0.5 nM DTs, DTsE349K, and DTsP308K for 30 min at 37°C. (Scale bars: 10 ␮m.) (B) Calculation of the fraction of different endosome diameters in 80% of the Rab5a positive HeLa cells per well (70 – 80 cells) treated without or with 0.5 nM wild-type DT, CRM197, DTs, DTsE349K, and DTsP308S. The cells were examined by confocal microscopy, and the calculation of the diameter of Rab5a positive endosomes was performed by using Volocity software. (Scale bars: 10 ␮m.)

% of total fluorescent volumes

% of total voxel count

B 100

A

DTsE349K

Protein synthesis (% of control)

A

DT DT+CRM197

60 40 20 0

10-14 10-13 10-12 10-11 10-10 10-9 10-8 Concentration [M]

Fig. 3. Endosome perturbation alters toxin activity. (A and B) HeLa cells, transfected with the plasmid encoding EYFP-Rab5 for 12–15 h, were incubated without or with 0.5 nM diphtheria toxin or CRM197 for 30 min at 37°C (A) in the presence or absence of 5 mM NH4Cl (A and B). (Scale bars: 10 ␮m.) The calculation of the diameters of Rab5 positive endosomes was performed considering 80% of all transfected cells per well for each conditions using Volocity software (B). (C) Vero cells (1 ⫻ 104cells) were incubated with different concentrations of DT in the absence (open squares) or in the presence (filled squares) of 1 ⫻ 10⫺12 M CRM197. Cells were incubated with the toxins for 3 h, and the protein synthesis inhibition was measured as described in ref. 24.

between early endosomes and, when overexpressed, induces the accumulation of very small endocytic vesicles (20). We compared the effect of DT and CRM197 on endosomes in cells expressing Rab5 (S34N) relative to the effect in control cells. Rab5 (S34N) expression reduced the dimensions of early endosomes and prevented any increase in vesicle diameter induced by DT or CRM197 (Fig. 4A). These results were confirmed by the Antignani and Youle

% of total fluorescent volumes

B

control

control

DT

DT

CRM197

CRM197

120

diameter 1µm

Rab5

Rab5 S34N

80 60 40 20 0

control

DT

CRM 197 control DT

CRM 197

Fig. 4. Role of Rab5 in endosome fusion. (A) HeLa cells, transfected with the plasmid encoding EYFP-Rab5S34N for 12–15 h, were incubated with 0.5 nM diphtheria toxin or CRM197 for 30 min at 37°C. (B) The calculation of the diameters of Rab5 positive endosomes was performed considering 80% of transfected cells for each conditions, using Volocity software. (Scale bars: 10 ␮m.)

analysis of the diameters of ⬇30,000 fluorescent endosomes considering 80% of the total transfected cells per experiment (Fig. 4B). Thus, the effect of DT on endosome size requires Rab5 activity. Dynamics of Rab5 Association with Single Endosomes in Living Cells.

Rab5 levels dynamically fluctuate on individual early endosomes during fusion and fission events that eventually, over time, link all endosomes into a network (32). The early endosomes, containing cargo destined for lysosomes, through repetitive fusion events, accumulate this cargo into fewer and larger endosomes, which are rapidly removed from the early-endosomal network via the loss of Rab5 and the concomitant acquisition of Rab7 (32). We asked whether DT can alter the dynamic network of this endocytotic pathway and whether there is a change in Rab5 dynamics along with toxin induced enlargement of Rab5 positive endosomes. We used Rab5 fused to a photoactivable form of GFP (PAGFP), a mutant of the Aequorea victoria GFP (33), that makes it possible to select proteins in specific regions of interest and analyze the kinetics of their localization within cells. We tracked 50 photoactivated endosomes (1–2 endosomes per transfected cell) from cells incubated with and without DT and measured the fluorescence intensity of each endosome every 20 s. We confirmed that photoactivated endosomes in control cells expressing PAGFP-Rab5 were the same size as those in EGFP-Rab5 expressing cells, whereas photoactivated endosomes in DT treated cells were larger and displayed a higher level Antignani and Youle

of PAGFP-Rab5 fluorescence (Fig. 5 A–C). We found that in control cells the amount of Rab5 associated with a given endosome fluctuated considerably over 5 min, as shown by a significant decrease in GFP-Rab5 fluorescence and by the termination of almost half of the analyzed traces before 5 min (Fig. 5C). As seen in the study in ref. 32, fission of Rab5 positive tubules below levels of detection and release of Rab5 from the membrane could explain the decrease. PA-GFP-Rab5 displayed a higher level of fluorescence intensity on the large endosomes in DT treated cells and was more stable than the PAGFP-Rab5 on control endosomes (Fig. 5 B and C). Comparing the average fluorescence intensity with time after photoactivation of Rab5 on endosomes (Fig. 5D) revealed that the fluorescence of Rab5 on the DT and CRM197 treated endosomes diminished more slowly over time than Rab5 on the control endosomes. The Rab5 fluorescence dropped off almost completely after 5 min in control cells, whereas endosomes from DT and CRM197 retained 40% of the initial fluorescence value. A high level of Rab5 loss causing track terminations resulted in the larger standard deviation values in the control endosomes. The track terminations occurred less frequently in DT treated cells. Because Rab5 positive endosomes are brighter and bigger, it was possible track them during the entire time series (5 min). Interestingly, from the analysis of the traces over 5 min, we found that, in contrast to the differential loss of Rab5 fluorescence, the variation in the endosome diameter is similar in DT and CRM197 containing vesicles relative to the control (Fig. S3). Thus, the greater endosome size in DT treated cells appears to reflect the greater retention of Rab5 on DT endosomes. This could be due to less fission of tubules or less Rab5 release that maintains greater fusion activity. Rab7, another member of the Rab GTPase protein family, has been localized to late endosomes and plays a key role downstream of Rab5 in regulating membrane transport leading from early to late endosomes (34, 35). Therefore, we examined Rab7 to see whether it is present in these enlarged endosomes in toxin treated cells and found no difference in the diameter or morphology of Rab7 positive endosomes between control cells and cells treated with DT or CRM197 (data not shown). Thus, DT appears to affect early endosomes upstream of Rab7 by inhibiting the exchange of Rab5 with Rab7. Discussion A remaining gap in understanding DT activity is the process of translocation of the A chain across membranes to the cytosol. Early endosomes are the first sorting station from which endocytosed materials are directed to various cellular destinations and they represent the crucial compartment along the endocytic pathway from which the DT catalytic domain productively crosses into the cytosol. Using fluorescently labeled Rab5, a regulatory GTPase that mediates maturation of endosomes, we have shown that DT induces an enlargement in the diameter of the early endosomes and this enlargement is correlated to its cytotoxic activity. The enlargement of the endosomes depends on the DT T domain, because mutations in the translocation domain abolish this alteration in the endosomal morphology, whereas mutations either in the receptor binding domain or in the catalytic domain do not inhibit this activity of DT. Raising the pH in the endosomal compartment blocks the DT-induced enlargement of endosomes, indicating that acidity-driven conformational change of the T domain in the endosome lumen is required for this activity. Vesicle swelling may be induced by DT translocation due to a flux of low molecular weight molecules from the cytosol. However, because Rab5 inhibition eliminates the early endosome enlargement, we suggest that DT perturbs the morphology of endosomes by either increasing the rate of early endosome fusion or decreasing the rate of fission. PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23 兩 8023

CELL BIOLOGY

Rab5 S34N

Rab5

A

A pre

control

B

+ CRM197 pre

postact.

postact. post

post +1min

+1min

+3 min

+3 min

80 70 60 50 40 30 20 10 0

control

0

60

120

180

240

400 350 300 250 200 150 100 50 300 0

DT

D

Rab5-PAGFP fluorescence (%)

Rab5-PAGFP fluorescence

C

0

60

120 180 time (sec)

240

300

cont rol

100

DT

CRM 197

80 60 40 20 0 0

60

120

180

240

300

time (sec)

Fig. 5. Dynamics of the early endosomes in the presence of DT. (A and B) HeLa cells were transfected with PAGFP-Rab5 for 24 –28 h and incubated in the absence (A) or presence (B) of 0.5 nM CRM197 for 30 min at 37°C. To photoactivate PAGFP, ROIs marked with yellow, red, and white arrows were irradiated with a 405-nm laser. PAGFP-Rab5 was imaged by using 488-nm laser excitation before (pre) and 30 s after (post) 405-nm light photoactivation. Images of two photoactivated endosomes (marked with the yellow and red arrows) were captured every 20 s for 5 min as series of z sections covering the entire thickness of the cell (A and B), showing the endosomes labeled with the red arrows (B Center), yellow arrows (B Right), and white arrows (data not shown). (C and D) Changes in the fluorescence intensities within photoactivated endosomes were measured over the time (C), and percentages of initial PAGFP-Rab5 fluorescence were plotted versus time (D). The data represent the mean ⫾ SD of 50 –55 endosomes during time-lapse measurements from control cells and cells incubated with DT or CRM197 (D). (Scale bars: 10 ␮m.)

Endosomes receive incoming vesicles from the plasma membrane. Recycling cargo exits in Rab4-positive tubules, which frequently grow out and detach from Rab5-positive cores through constant nucleation of Rab4 domains. Cargo destined for late endosomes, such as DT, is retained in individual endosomes. Thus, by parallel sorting activities and interconnectivity of endosomes, degradative cargo gradually accumulates in fewer and bigger endosomes. Rab5 vesicles convert to Rab7 vesicles, removing the Rab7 vesicles from the early-endosomal network and rendering them fusion-competent with late endosomes, although homotypic fusion events can temporarily restore Rab5 levels. We have shown that DT is able to both increase the level of excessively fused endosomes and maintain endosomes in a Rab5-rich state consistent with the promotion of homotypic early endosome fusion. Using photoactivable GFP-Rab5, we found that DT slows down the rate of extraction of Rab5 from endosomal membranes. The increased fusion and the inhibition of the subsequent maturation of the early endosomes by DT may allow the accumulation of the toxin in the early endosome until it reaches the ‘‘quantum’’ (13) for release into the cytosol. The Bcl-2 family of proteins, important regulators of programmed cell death in multicellular organisms (36), have two central hydrophobic ␣-helices surrounded by four amphipatic ␣-helices, an ␣-helix organization similar to that of the DT T domain (5). As with the structurally related helices 8 and 9 of DT that insert into membranes at low pH, helices 5 and 6 of Bcl-xL and Bax are hypothesized to insert into membrane bilayers during apoptosis (37). Interestingly, recent results show that Bax (38) and Bcl-xL (39) can mediate fusion of mitochondria. Thus, the membrane inserting T domain of DT may share an activity to activate membrane fusion processes with Bcl-2 family members albeit acting differentially on endosomes and mitochondria, respectively. How endosome fusion may participate in the native toxicity of DT remains unclear. We have analyzed the protein synthesis inhibition activity of DT in HeLa cells stably expressing GFP-Rab5 and GFP-Rab5S34N and found that Rab5 transfected cells were 8024 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0711707105

more susceptible to the action of the toxin than the Rab5S34N expressing cells (data not shown). However, for unknown reasons, both cells lines were more susceptible than control vector expressing lines. These initial results suggest that promotion of endosome fusion could be correlated with the cytotoxic effect of DT. Nevertheless, DT entry into cells increases Rab5 retention on endosomes and thereby causes their enlargement. This activity occurs after membrane insertion of the T domain and is correlated with membrane translocation process suggesting that DT crosses into the cytosol from these unusual endosomal structures. Materials and Methods Plasmids and Cell Culture. The EYFP-Rab5 plasmid was provided by H. McBride (University of Ottawa, Ottawa), and the EYFP-Rab5 S34N was provided by B. Ceresa (University of Oklahoma, Norman). The Rab5 cDNA, digested with KpnI and BamHI (New England Biolabs), was cloned into PAGFP-C1 vector, digested with KpnI and EcoRI. HeLa cells were grown in DMEM and 10% FBS. They were transfected by using the FuGENE (Roche) and treated as indicated in individual experiments 12–15 h after transfection with the EYFP constructs or 24 –28 h after transfection with PAGFP-Rab5. DT and CRM107 were purchased from List Biological Laboratories. Tf-CRM107 was synthesized as reported in ref. 23. Confocal Microscopy and Image Analysis. Cells were grown in two-well chambers for confocal microscopy. For immunofluorescence, cells were fixed for 30 min in 4% paraformaldehyde in PBS and were stained with anti-EEA1 or anti-Rab5 antibody (BD Biosciences) followed by staining with goat antimouse Alexa Fluor 568 antibody (Molecular Probes). Images were captured with a model LSM 510 microscope (Carl Zeiss MicroImaging), using a 63⫻ or 100⫻ Apochromat objective (Carl Zeiss MicroImaging). Projected series of z sections collected with intervals of 0.5– 0.75 ␮m between sections are shown. For photoactivation of PAGFP-Rab5, ROIs were selected as the smallest circle shape allowed by the image acquisition program. The same intervals between optical section were using for imaging. Postacquisition processing and analysis were performed with Volocity image analysis software (Improvision). For the analysis of PAGFP-Rab5, endosomal ROIs were selected in the first image collected after photoactivation (postactivation values). The same regions were transferred without change to the image obtained before photoactiva-

Antignani and Youle

ACKNOWLEDGMENTS. We thank Heidi McBride for the Rab plasmids and the helpful suggestions, Mariusz Karbowski and Carolyn L. Smith for assistance with the confocal microscopy, R. John Collier (Harvard Medical

School, Boston, MA) and Erwin London (State University of New York, Stony Brook, NY) for the DTE148S plasmids, G. H. Patterson (National Institute of Child Health and Human Development, Bethesda, MD) and J. Lippincott-Schwartz (National Institute of Child Health and Human Development, Bethesda, MD) for the PAGFP plasmid, and Brian Ceresa (University of Oklahoma, Norman, OK) for the Rab5S34N plasmid.

1. Sandvig K, van Deurs B (2005) Delivery into cells: Lessons learned from plant and bacterial toxins. Gene Ther 12:865– 872. 2. Collier RJ (2001) Understanding the mode of action of diphtheria toxin: A perspective on progress during the 20th century. Toxicon 39:1793–1803. 3. Lord JM, Smith DC, Roberts LM (1999) Toxin entry: How bacterial proteins get into mammalian cells. Cell Microbiol 1:85–91. 4. Choe S, et al. (1992) The crystal structure of diphtheria toxin. Nature 357:216 –222. 5. Muchmore SW, et al. (1996) X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death Nature 381:335–341. 6. Parker MW, Pattus F (1993) Rendering a membrane protein soluble in water: A common packing motif in bacterial protein toxins. Trends Biochem Sci 18:391–395. 7. Oh KJ, et al. (1996) Organization of diphtheria toxin T domain in bilayers: A sitedirected spin labeling study. Science 273:810 – 812. 8. Huynh PD, et al. (1997) Probing the structure of the diphtheria toxin channel. Reactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives. J Gen Physiol 110:229 –242. 9. Kachel K, Ren. J, Collier RJ, London E (1998) Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membraneinserted diphtheria toxin T domain. J Biol Chem 273:22950 –22956. 10. Wang J, Rosconi MP, London E (2006) Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether. Biochemistry 45:8124 – 8134. 11. Oh KJ, Senzel JL, Collier RJ, Finkelstein A (1999) Translocation of the catalytic domain of diphtheria toxin across planar phopholipid bilayers by its own T domain. Proc Natl Acad Sci USA 96:8467– 8470. 12. Ratts R, et al. (2003) The cytosolic entry of diphtheria toxin catalytic domain requires a host cell translocation factor complex. J Cell Biol 160:1139 –1150. 13. Hudson TH, Neville DM, Jr (1985) Quantal entry of diphtheria toxin to the cytosol. J Biol Chem 260:2675–2680. 14. Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117. 15. Christoforidis S, McBride HM, Burgoyne RD, Zerial M (1999) The Rab5 effector EEA1 is a core component of endosome docking. Nature 397:621– 625. 16. de Renzis S, Sonnichsen B, Zerial M (2002) Divalent Rab effectors regulate the subcompartmental organization and sorting of early endosomes. Nat Cell Biol 4:124 –133. 17. Youle RJ, Neville DM, Jr (1979) Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II. J Biol Chem 254:11089 –11096. 18. Lemichez E, et al. (1997) Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery Mol Microbiol 23:445– 457. 19. Bucci C, et al. (1992) The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway Cell 70:715–728.

20. Stenmark H, et al. (1994) Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis EMBO J 13:1287–1296. 21. Uchida T, Pappenheimer AM, Harper AA (1972) Reconstitution of diphtheria toxin from two nontoxic cross-reacting mutant proteins. Science 175:901–903. 22. Gaidarov I, Santini F, Warren RA, Keen JH (1999) Spatial control of coated-pit dynamics in living cells. Nat Cell Biol 1:1–7. 23. Greenfield L, Johnson VG, Youle RJ (1987) Mutations in diphtheria toxin separate binding from entry and amplify immunotoxin selectivity. Science 238:536 –539. 24. Johnson VG, Youle RJ (1989) A point mutation of proline 308 in diphtheria toxin B chain inhibits membrane translocation of toxin conjugates. J Biol Chem 264:17739 –17744. 25. O’Keefe DO, Cabiaux V, Choe S, Eisenberg D, Collier RJ (1992) pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349 –Lys. Proc Natl Acad Sci USA 89:6202– 6206. 26. Barbieri JT, Collier RJ (1987) Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli. Infect Immun 55:1647–1651. 27. O’Keefe D, Collier RJ (1989) Cloned diphtheria toxin within the periplasm of Escherichia coli causes lethal membrane damage at low pH. Proc Natl Acad Sci USA 86:343–346. 28. Sandvig K, Olsnes S (1980) Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol 87:828 – 832. 29. Sandvig K, Olsnes S (1981) Rapid entry of nicked diphtheria toxin into cells at low pH. Characterization of the entry process and effects of low pH on the toxin molecule. J Biol Chem 256:9068 –9076. 30. Kim K, Groman NB (1965) In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J Bacteriol 90:1552–1556. 31. Bayer N, et al. (1998) Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: Implications for viral uncoating and infection. J Virol 72:9645–9655. 32. Rink J, Ghigo E, Kalaidzidis Y, Zerial M (2005) Rab conversion as a mechanism of progression from early to late endosomes. Cell 122:735–749. 33. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877. 34. Feng Y, PressB, Wandinger-Ness A (1995) Rab 7: An important regulator of late endocytic membrane traffic. J Cell Biol 131:1435–1452. 35. Meresse S, Gorvel JP, Chavrier P (1995) The rab7 GTPase resides on a vesicular compartment connected to lysosomes. J Cell Sci 108(Pt 11):3349 –3358. 36. Youle RJ, Strasser A (2008) The BCL-2 protein family: Opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59. 37. Antignani A, Youle RJ (2006) How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr Opin Cell Biol 18:685– 689. 38. Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ (2006) Role of Bax and Bak in mitochondrial morphogenesis Nature 443:658 – 662. 39. Delivani P, Adrain C, Taylor RC, Duriez PJ, Martin SJ (2006) Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics. Mol Cell 21:761–773.

Antignani and Youle

PNAS 兩 June 10, 2008 兩 vol. 105 兩 no. 23 兩 8025

CELL BIOLOGY

tion (preactivation values) or were corrected for the movement of the endosomes to the following postactivation images.