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Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4[sub +] T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of... [ABSTRACT FROM AUTHOR]

Nuclear-encoded proteins destined for insertion into the mitochondrial outer membrane, follow the same general pathway for import as proteins that are translocated to interior compartments within the organelle. This observation is true both for β-barrel-type proteins and for proteins that contain hydrophobic α-helical transmembrane segments. In this review, we describe what is known about the various steps leading to protein insertion into the outer membrane, and discuss the energetics that favor vectorial translocation into and across this membrane. The selection of the outer membrane during import may involve a lateral release of the translocating polypeptide from the import machinery so that the appropriate domains of the protein become embedded in the lipid bilayer. One type of topogenic domain that can guarantee such selection of the outer membrane is a signal-anchor sequence of the type characterized for the bitopic protein Mas70p. It is suggested that a signal-anchor sequence selective for the mitochondrial outer membrane causes abrogation of polypeptide translocation and triggers the release of the transmembrane segment into the surrounding lipid bilayer, prior to any possibility for the commitment of translocation to the interior of the organelle. Specific structural features of the signal-anchor sequence specify its orientation in the membrane, and can confer on this sequence the ability to form homo-oligomers and hetero-oligomers. Strategies other than a signal-anchor sequence may be employed by other classes of proteins for selection of the outer-membrane. Of note is the ability of the outer-membrane import machinery to catalyze integration of the correct set of proteins into the outer-membrane bilayer, while allowing proteins that are destined for integration into the bilayer of the inner membrane to pass through unimpeded. Again, however, different proteins may employ different strategies. One model proposes that this can be accomplished by a combination of a matrix-targeting signal and a distal stop-transfer sequence. In this model, the formation of contact sites, which is triggered when the matrix-targeting signal engages the import machinery of the inner membrane, may prevent the outer-membrane translocon from recognizing and responding to the downstream stop-transfer domain. This allows the transmembrane segment to pass across the outer-membrane, and subsequently integrate into the inner membrane. [ABSTRACT FROM AUTHOR]