The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

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Hydrophobins self assemble into amphipathic films at hydrophobic-hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the ␣-helical state that is formed upon binding to a hydrophobic solid, and the ␤-sheet state, which is formed at the air-water interface. Here, we show that the formation of the ␤-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the ␣-helical state of SC3. The second intermediate is rich in ␤-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.

Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probe the structural basis of annexin IV-induced membrane aggregation and the inhibition of this property by protein kinase C phosphorylation. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr6Ala, T6A) phosphorylation of threonine 6 were produced for these studies and compared with wild-type annexin IV. In vitro assays showed that unmodified wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild-type or the T6D mutant, promote vesicle aggregation. Electron crystallographic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopic images of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated a separation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In this orientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergo face-to-face self-association with the annexin layer of a second vesicle. The 2.0-A resolution crystal structure of the T6D mutant showed that the mutation causes release of the N-terminal tail from the protein core. This change would preclude the face-to-face annexin self-association required to aggregate vesicles. The data suggest that reversible complex formation through phosphorylation and dephosphorylation could occur in vivo and play a role in the regulation of vesicle trafficking following changes in physiological states.