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The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb₃ in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb₃ clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb₃ lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb₃ (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb₃ (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb₃, STxB-Gb₃ complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.