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Promoting the degradation of Hsp90 client proteins by inhibiting Hsp90, an important protein chaperone, has been shown to be a promising new anticancer strategy. In this study, we show that an oxazoline analogue of apratoxin A (oz-apraA), a cyclodepsipeptide isolated from a marine cyanobacterium, promotes the degradation of Hsp90 clients through chaperone-mediated autophagy (CMA). We identify a KFERQ-like motif as a conserved pentapeptide sequence in the kinase domain of epidermal growth factor receptor (EGFR) necessary for recognition as a CMA substrate. Mutation of this motif prevents EGFR degradation by CMA and promotes the degradation of EGFR through the proteasomal pathway in oz-apraA--treated cells. Oz-apraA binds to Hsc70/Hsp70. We propose that apratoxin A inhibits Hsp90 function by stabilizing the interaction of Hsp90 client proteins with Hsc70/Hsp70 and thus prevents their interactions with Hsp90. Our study provides the first examples for the ability of CMA to mediate degradation of membrane receptors and cross talks of CMA and proteasomal degradation mechanisms.

Ligand--receptor complexes are internalized by a variety of endocytic mechanisms. Some are initiated within clathrin-coated membranes, whereas others involve lipid microdomains of the plasma membrane. In neurons, where alternative targeting to short- or long-range trafficking routes underpins the differential processing of synaptic vesicle components and neurotrophin receptors, the mechanism giving access to the axonal retrograde pathway remains unknown. To investigate this sorting process, we examined the internalization of a tetanus neurotoxin fragment (TENT [H.sub.c]), which shares axonal carriers with neurotrophins and their receptors. Previous studies have shown that the TeNT [H.sub.c] receptor, which comprises polysialogangliosides, resides in lipid microdomains. We demonstrate that TeNT [H.sub.c] internalization also relies on a specialized clathrin-mediated pathway, which is independent of synaptic vesicle recycling. Moreover, unlike transferrin uptake, this AP-2--dependent process is independent of epsin1. These findings identify a pathway for TENT, beginning with the binding to a lipid raft component (GD1b) and followed by dissociation from GD1b as the toxin internalizes via a clathrin-mediated mechanism using a specific subset of adaptor proteins.

It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >19(super 5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices. Key words: Aerolysin, cholesterol, microdomains, glycosyl phosphatidylinositol-anchored protein, oligomerization

An examination of the acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) found that it behaves like the cyclic pentameric complexes of cholera toxis and Eschrichia coli enterotoxin B-rings. Electron microscopy revealed that flat oligomers adsorb to mica in orientations that correspond to hexameric surfaces. Results indicate that VacA activation is characterized by its disassembly before contact or entry into target cells.

Shiga toxin is a bacterial toxin elucidated by Salmonella dysenteriae. Immunofluorescence studies, confocal microscopy, recycling experiments and immunoelectron microscopy were undertaken to determine the transport of the B fragment of this toxin in HeLa cells. Results reveal that the B fragment of the toxin is transported to the Golgi apparatus directly from early/recycling endosomes.