Diphtheria Toxin Translocation across Endosome Membranes

Abstract

By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADPribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.Translocation of proteins across membranes is an important process in living cells. In eukaryotic cells proteins located in plasma membrane, lysosomes, and the Golgi complex are all synthesized on ribosomes attached to the endoplasmic reticulum, co-translationally translocated into the lumen of endoplasmic reticulum, and then transferred to appropriate destinations. Secreted proteins are also translocated into the endoplasmic reticulum, followed by exocytosis. Proteins localized in mitochondria, peroxisomes, chloroplasts, and nuclei are synthesized in the cytosol and translocated post-translationally into those organelles. The mechanisms of membrane penetration by these newly synthesized proteins and the machinery needed for their translocation have been studied in great detail.Translocation of proteins from outside the cell to inside also occurs. A number of protein toxins exert their toxic effects in mammalian cells by enzymatically modifying an intracellular target. At least a portion of the protein must be therefore be translocated across the cell membrane. Cholera toxin, Escherichia coli heat-labile toxin, and pertussis toxin ADP-ribosylate subunits of trimeric G proteins and cause constitutive activation of adenylate cyclases (1-3). Diphtheria toxin (DT) 1 and Pseudomonas aeruginosa exotoxin A inactivate elongation factor 2 and inhibit protein synthesis (4, 5). Several plant toxins as well as the Shiga toxin inactivate ribosomes by hydrolyzing specific sites in ribosome RNA (6 -8). Tetanus and botulinum neurotoxins cleave proteins that are part of the machinery for exocytosis in neurons (9). Other toxins, including Clostridium difficile toxin (10) and anthrax toxin (11), also affect intracellular targets.The mechanisms...

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

“…In fact, the time course of bafilomycin A " -induced GLUT4 translocation from intracellular stores to plasma membranes observed in the present study (Figure 4) corresponds well with the reported rapid effect of this agent in increasing endosomal pH. Similarly, the maximally activating concentration of bafilomycin A " with regard to GLUT4 translocation is similar to the bafilomycin A " concentration found to be effective in blocking endosomal acidification in 3T3-L1 adipocytes ( Figure 3) and in other living cells of several types [21,[24][25][26]45]. Together, these results are consistent with the notion that the altered glucose transporter membrane dynamics result from the specific effect of bafilomycin A " on V-ATPase.…”

“…Alkalinization of the endocytic structure is accompanied by disappearance of the orange fluorescence [25,26]. In agreement with these findings, staining of control 3T3-L1 adipocytes with the dye for 10 min resulted in a granular appearance of the orange fluorescence associated with the acidified endosomes\lysosomes, whereas cytosol and nuclei showed green fluorescence [24][25][26] (Figure 3 ; and results not shown). Treatment of 3T3-L1 adipocytes with 20-800 nM bafilomycin A " for 30 min at 37 mC prior to Acridine Orange staining resulted in a dose-dependent quenching of the granular orange fluorescence (Figure 3), whereas the green fluorescence remained practically unaltered.…”

“…Following serum starvation and treatment with bafilomycin A " for 30 min at 37 mC at the concentrations indicated in the Figure legends, the living cells were stained with Acridine Orange using previously described protocols [24][25][26]. Briefly, treated cells were further incubated at 37 mC for 10 min with 5 µg\ml Acridine Orange in intracellular buffer.…”

Membrane Protein Insertion Regulated by Bringing Electrostatic and Hydrophobic Interactions into Play

“…Acid residues located in the loop linking TH8 to TH9 are good candidates for this regulation. There is much evidence indicating that residues Glu-349 and Asp-352 are responsible for the pH dependence of the insertion of TH8 and TH9 in the membrane (55)(56)(57). Nevertheless, it has been reported that other residues may also contribute to the pH-dependent membrane insertion of TH8-TH9 (34).…”

Vacuolar‐type H+‐ATPase in mouse bladder epithelium is responsible for urinary acidification

“…Briefly, acridine orange (6 uM in PBS) was injected into the bladder lumen via the urethra, and incubated for a further 10 min. Under this condition, acridine orange penetrates through the cell membrane, and accumulates in acidic compartments [18,19]. Then, mice were killed and the bladder was isolated.…”