Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Δssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Δssa1, Δssa3, or Δssa4 strains. Likewise, in vitro import was impaired for the Δssa2 strain, but not for the other Δssa strains. The cytosol was identified as the site of the Δssa2 defect; Δssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Δssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Δssa2 Vid vesicles, providing Δssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.

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Fluorescence resonance energy transfer (FRET) was used to monitor pH-dependent structural changes in the cholera toxin B subunit (CTB) and the membranes with which CTB associates. The distance separating the single tryptophan (Trp88) of each CTB monomer and a pyrene probe linked to the membrane-imbedded tail of ganglioside GM1 is not influenced by pH in a range from 3.5 to 7.5, consistent with the position of Trp88 in the GM1 binding site of CTB. In contrast, the distance between the pyrene probe on GM1 and coumarin, stilbene, or fluorescein probes covalently linked to specific sites on CTB appears to increase significantly as the pH is lowered to 5.0 or less. This conformational change is not accompanied by detectable changes in the distance between Trp88 and these extrinsic probe positions in the presence of nonfluorescent GM1. However, when the distance from Trp88 to the extrinsic probes is monitored as a function of pH in the absence of GM1, a conformational change is seen which indicates that receptor binding influences the character of pH-dependent conformational changes that occur within CTB. Interestingly, the observed change in CTB conformation is accompanied by a change in the relative position of GM1 within the membrane as judged by FRET from the pyrene probe on GM1 to a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) probe linked to the polar head group of phosphatidylethanolamine and positioned at the membrane surface. Taken together, the data imply that low endosomal pH is capable of inducing structural changes in CTB, which, in turn, exert effects on the structure of the membrane to which CTB is bound. These phenomena may have a role in (1) processing of cholera toxin within the endosomal compartments of some target cell types, (2) determining the lag time between cholera toxin binding and the target cell response to cholera intoxication, or (3) the efficiency of CTB and cholera toxin as mucosal adjuvants.

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