title: Anthrax protective antigen: efficiency of translocation is independent of the number of ligands bound to the prepore.
abstract: Heptameric anthrax protective antigen (termed prepore), which assembles at the mammalian cell surface, competitively binds edema factor (EF) and/or lethal factor (LF). It then transports them to an acidic intracellular compartment and mediates their translocation across the membrane to the cytosol. Steric constraints limit to three the number of molecules of EF and/or LF that can bind simultaneously to prepore. To determine whether the number of ligand molecules bound per heptamer affects the efficiency of translocation, we measured the low-pH-triggered translocation of the radiolabeled protective antigen (PA(63))-binding domain of LF ((35)S-LF(N)) across the plasma membrane of CHO-K1 cells as a function of the degree of saturation of the prepore. The fraction translocated remained constant at approximately 0.4 as (35)S-LF(N) was varied from nil through saturating concentrations. The same constant value was observed when we held (35)S-LF(N) at a saturating concentration and varied the number of functional ligand sites per prepore by changing the ratio of wild-type PA to a ligand-binding mutant. Thus, prepore containing only a single ligand-binding site is capable of translocating its cargo as efficiently as one containing multiple binding sites. The results as a whole imply that heptamers with one, two, or three ligands bound translocate their ligands with the same efficiency, indicating that each ligand molecule is translocated independently from the others.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Sen
authors.initials: S
authors.lastname: Zhang
authors.forename: Kristina
authors.initials: K
authors.lastname: Cunningham
authors.forename: R J
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D008024:Ligands; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D008024:Ligands; D021122:Protein Subunits; C030325:anthrax toxin