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After 30 min, samples were analyzed in a 1- 0

After 30 min, samples were analyzed in a 1- 0.5-cm quartz cuvette using an ISS (Champaign, Illinois) fluorometer at an excitation wavelength of 341 nm. a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry. for 10 min, and the supernatant was incubated at 4C for 1 h with rabbit anti-PA antibody (1:500 dilution) and then with protein A-Sepharose beads for an additional hour. The beads were then washed and suspended in a gel sample buffer made up of 2% SDS. Samples were resolved on 3-8% Tris-acetate or 8% Tris-glycine gels and transferred to polyvinylidene difluoride and probed with anti-PA goat serum or an anti-EGFP mouse monoclonal antibody. Pyrene Fluorescence. N306C PA63 was produced and labeled SIRT6 with pyrene as described in ref. 17. Labeled (PA63)7 prepore was mixed with soluble CMG2 or soluble TEM8 as described in ref. 17, except that 1 mM MgCl2 was included in the mixture. pH was adjusted to the indicated values by using 0.1 equivalents of either 1 M Tris, pH 8; 1 M HEPES, pH 7; 1 M BisTris, pH6; or 1 M sodium acetate, pH 5. After 30 min, samples were analyzed in a 1- 0.5-cm quartz cuvette using an ISS (Champaign, Illinois) fluorometer at an excitation wavelength of 341 nm. Emission was measured from (Rac)-PT2399 360 to 600 nm and normalized against the maximal fluorescence at 384 nm. Intoxication Assays. For LFN-DTA intoxication and MEK1 cleavage assays, cells were pretreated with or without 30 mM NH4Cl in supplemented F12 medium for 1 h at 37C. In the LFN-DTA experiments, cells were incubated with the indicated concentrations of PA83 and 10-9 MLFN-DTA for 2 h at 37C. The medium was then removed and replaced with fresh medium, and the cells were incubated for an additional 2 h at 37C. The medium was then replaced with DMEM without glutamine, leucine, and sodium pyruvate (MP Biomedicals, Irvine, CA) supplemented with 2 mM l-glutamine (Rac)-PT2399 (Invitrogen), 0.45 mM sodium pyruvate (Sigma), and 1 Ci/ml, 173 Ci/mmol [3H]leucine (1 Ci = 37 GBq; PerkinElmer). Cells were incubated at 37C for a further 2 h, washed three times with cold PBS, resuspended in EcoLume scintillation fluid (MP Biomedicals), and assayed with a Top-Count NXT microplate scintillation counter (Packard). In the MEK1 cleavage assays cells were incubated with 2.5 10-8 M PA83 and 5 10-9 M LF for 2 h at 4C, washed, and shifted to 37C for the indicated times. In the case of the 4-h time point, the medium was exchanged with fresh NH4Cl-containing medium after 2 h had elapsed. Cells were lysed in TBS-containing 1% Nonidet P-40 as described above, and 12-mg protein samples were resolved by 8% acrylamide Tris-glycine SDS/PAGE, transferred to a polyvinylidene difluoride membrane, and detected with anti-MEK1 N-terminal and C-terminal antibodies, followed by anti-rabbit-horseradish peroxidase secondary antibodies and SuperSignal femto Western detection reagent (Pierce). Results To determine whether the TEM8 and CMG2 receptors influence the triggering mechanism that leads to anthrax toxin pore formation, we investigated the pH thresholds for this process in PA receptor-deficient CHO-R1.1 cells (2) engineered to express either receptor (Fig. 5, which is published as supporting information on the PNAS web site). PA83 (Rac)-PT2399 and LF were added to the cells in the presence of ammonium chloride, a lysosomotropic agent that elevates the pH of acidic endosomes to approximately pH 6.5 or higher (29, 30). Pore formation was monitored by the conversion of PA to an SDS-resistant oligomeric form (7, 16) (Fig. 1 and in conditions under which the toxin subunit was fully occupied by soluble receptors, as judged by native PAGE (Fig. 6, which is published as supporting information on the PNAS web site). The pH was adjusted to various values, and the resulting excimer fluorescence was monitored. When bound to TEM8, (PA63)7 pore formation occurred at pH values ranging from pH 6.8 to 7.1, but, when bound to.