Prolonged display or rapid internalization of the IgG-binding protein ZZ anchored to the surface of cells using the diphtheria toxin T domain

We have shown previously that the diphtheria toxin transmembrane domain (T) may function as a membrane anchor for soluble proteins fused at its C-term inus. Binding to membranes is triggered by acidic pH. Here, we further char acterized this anchoring device. Soluble proteins may be fused at the N-ter minus of the T domain or at both extremities, without modifying its membran e binding properties. This allows one to choose the orientation of the prot ein to be attached to the membrane. Maximum binding to the cell surface is reached within 1 h. Anchoring occurs on cells previously treated with prote inase K, suggesting that T interacts with the lipid phase of the membrane w ithout the help of cell surface proteins. Binding does not permeabilize cel ls or affect cell viability, despite the fact that it permeabilizes liposom es and alters their structure. When attached to L929 fibroblasts, the prote ins are not internalized and remain displayed at their surface for more tha n 24 It. When bound to K562 myeloid cells, the molecules are internalized a nd degraded. Thus, depending on the cell type, soluble proteins may be anch ored to the surface of cells by the T domain for an extended time or direct ed towards an internalization pathway.