Translocation from endosomes

Translocation of the catalytic A fragment of DT from acidified endosomes depends crucially on the low pH environment in this organelle (Sandvig and Olsnes, 1980; Moskaug et al., 1988). At endosomal pH, both DTA and DTB fragments undergo conformational change (Blewitt et al., 1985). A major consequence of this in terms of the B fragment is that two hydrophobic helices that are normally buried in the T domain become exposed. Exposure of these two helices is followed by their insertion into the endosomal membrane, possibly accompanied by other regions of the DTB fragment, in particular other helices of the T domain (Silverman et al., 1994). In this way the DTB fragment forms, or forms part of, a proteinaceous translocation pore through which the DTA fragment is able to pass (Montecucco and Papini, 1995). At endosomal pH, DTA partially unfolds to facilitate the membrane translocation step. When this partial unfolding is constrained by introducing disulfide bonds into DTA, the cytotoxicity of DT is significantly reduced (Falnes et al., 1994). After translocation the reducing nature of the cytosol breaks the disulfide bond joining DTA to the membrane-associated DTB, and the higher pH in the cytosol causes DTA to refold into its biologically active conformation. This model for DT membrane translocation is based on considerable experimental evidence, including directly assessing the effect of low pH on DT conformation by a variety of techniques including circular dichroism (Blewitt et al., 1985), and demonstrating that at low pH DTB inserts into membranes and DTA traverses them (Eriksen et al., 1994). Phase partitioning experiments using Triton X-114 solutions showed that at higher pH values DT enters the aqueous phase, whereas at pH values of 5.5 or less, it partitions into the detergent phase (Sandvig and Moskaug, 1987). In other words, exposure to low pH converts the behaviour of DTB from that characteristic of a soluble protein to that of a membrane protein. The DTB T domain consists of a series of stacked helices, a structural feature shared with other proteins capable of forming pores in membranes such as colicin and insecticidal S-endotoxin (Parker and Pattus, 1993). Because DTA translocation into the cytosol requires low pH, increasing endosomal pH by treating cells with reagents such as ammonium chloride protects against DT. Conversely DT bound to the cell surface can directly cross the plasma membrane when the extracellular pH is lowered to or below that found in endosomes (Stenmark et al., 1988).

DT is therefore a protein capable of effecting its own membrane translocation in response to appropriate environmental conditions, that is, low pH. Under these conditions, translocation is possible because the T domain of DTB contributes to the formation of a proteinaceous membrane channel through which DTA is able to pass. Other toxins such as PE, ST and ricin do not translocate from endosomes, apparently because they do not contain regions or domains capable of forming, or contributing to the formation of, translocation channels. Because of this, these toxins must be transported to intracellular locations where pre-existing translocation channels exist. To reach such locations, the toxins must be transported beyond endosomes.

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