The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.

The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.The adenylate cyclase toxin (ACT), 3 a multifunctional, single polypeptide toxin, is expressed by six of the eight members of the genus Bordetella and was discovered by detection of adenylate cyclase enzymatic activity in a commercial pertussis vaccine (1). ACT derives its cytotoxic effects from delivery of its 400-amino acid adenylate cyclase enzymatic domain into the cell cytoplasm, resulting in the unregulated, calmodulindependent conversion of ATP into cAMP (2-4). Translocation, the process by which the catalytic domain is delivered across the cytoplasmic membrane, is unaffected by cytochalasin D or ammonium chloride and is dependent on the interaction of the ϳ1000-amino acid cell-binding domain with the cell membrane (5).This cell-binding domain is homologous to the members of the RTX (repeat-in-toxin) family of pore-forming bacterial toxins, such as Escherichia coli hemolysin, HlyA, and several leukotoxins from organisms such as Pasteurella hemolytica and Actinobacillus actinomycetemcomitans (6, 7). The RTX region of ACT oligomerizes in the cell membrane independently of translocation, forming cation-selective pores and causing hemolysis of erythrocytes and nonapoptotic death of nucleated cells (8 -14). Membrane interaction and pore formation by ACT can occur in artificial lipid bilayers and liposomes and are influenced by lipid and glycolipid composition (11,(15)(16)(17)(18). Although ACT intoxicates a broad range of cells and is able to associate with artificial lipid membranes containing no proteins, the ␤ 2 -integrin, CD11b/CD18 (Mac-1), which is expressed on phagocytic leukocytes, has been shown to increase the potency of ACT by an order of magnitude and has been considered a receptor for ACT (19). Accordingly, most of the studies on the functional, cytotoxic effects of ACT have focused on CD11b/CD18-positive cells, beginning with the initial observation that the...

Simultaneous monitoring of ATP synthesis and K+ movements across pea mitochondrial membranes revealed information about the competition of the two processes for mitochondrial energy. In the presence of valinomycin and at low extramitochondrial K+ concentration, ADP could be phosphorylated rapidly. This occurred with a decrease in net potassium ion uptake. At higher external K+ concentrations respiratory energy was unavailable for ATP synthesis and only a portion of added ADP could be phosphorylated within a reasonable time. Magnesium ions were shown to have an inhibitor effect on the K+ uptake, and stimulated a greater rate of ATP synthesis. When valinomycin and ADP were added simultaneously so that phosphorylation of the ADP and enhancement of K+ uptake could complete for mitochondrial energy, K+ uptake was preferred over ATP synthesis.