ADP-ribosylation of membrane proteins catalyzed by cholera toxin: basis of the activation of adenylate cyclase.

Abstract

In the presence of ATP and a cytosolic factor, cholera toxin fragment A1 catalyzes the transfer of ADP-ribose from NAD to a number of soluble and membrane-bound proteins of the pigeon erythrocyte. Evidence is presented that suggests that the most readily modified membrane protein (M, 42,000 The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.] by cholera toxin involves the interaction of some part of the cyclase system exposed on the inner surface of the plasma membrane with NAD, a nucleoside triphosphate, and a cytoplasmic protein (1-4) and is conveniently studied in lysed pigeon erythrocytes. The likelihood that the NAD might function as an adenosine diphosphoribose donor in a toxincatalyzed reaction of the type NAD+ + protein T ADP-ribosyl protein + nicotinamide + H+ was increased by the demonstrations that the active A1 fragment of cholera toxin catalyzes the slow hydrolysis of NAD to ADP-ribose and nicotinamide (5) and the transfer of ADP-ribose to arginine and related compounds (6) and to itself (C. King, personal communication; ref. 7).If the postulated acceptor were adenylate cyclase or an associated membrane protein, it should be possible to demonstrate a toxin-dependent transfer of ADP-ribose from radioactive NAD to a small number of sites on pigeon erythrocyte ghosts. There is, however, a large toxin-independent incorporation of ADP-ribose which was difficult to decrease without losing the toxin response. We now report the resolution of this problem and the demonstration of several toxin-specific ADP-ribose acceptors, principally a 42,000 Mr membrane protein. METHODSThe medium used throughout consisted of 0.13 M NaCl, 0.01% sodium azide, Trasylol (aprotinin: FBA Pharmaceuticals) at 2 kallikrein inactivator units/ml, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), and NaOH to give pH 7.3 at 370. Washed and purified pigeon erythrocytes suspended in an equal volume of this medium were lysed by rapid freezing and then thawing. The lysate was centrifuged at 10,000 X g for 5 min to separate ghosts and cytosol. The cytosol was mixed with well-washed pig brain NAD glycohydrolase (Sigma) at a ratio of 1 ml/10 mg, agitated at 370 for 30 min, and then centrifuged to remove the enzyme. The ghosts were washed three times in 20 vol of medium. There are about 8 X 109 ghosts per ml (packed).Portions of lysate reconstituted from 1 part washed ghosts and 2 parts NAD-depleted cytosol fraction were incubated for 30 min at 250 or 370 with 10 mM thymidine, 5

The differential detergent solubilization of adenylate cyclase and polypeptides ADP‐ribosylated with cholera toxin suggests an excess of G/F protein relative to adenylate cyclase in rat pancreatic plasma membranes

“…Using similar conditions with [c~-32P] NAD +, we observed that cytosolic proteins were labelled in addition to plasma membrane proteins (not shown). This was in line with [6]; however, it was difficult to wash out pancreatic plasma membranes of all cytosolic components. Such difficulties have been avoided by replacing the whole erythrocyte cytosol either by a protein cofactor purified 1000-fold from this cytosol [18] or by an acetate or phosphate buffer used at high concentration [8].…”

“…The ADP-ribosylation of G/F proteins in membranes from erythrocytes [5,6,10] (but apparently not from hepatocytes [20]) requires the presence of erythrocyte cytosol in addition to cholera toxin. In [4] we used the whole erythrocyte cytosol and cholera toxin to activate adenylate cyclase in pancreatic plasma membranes.…”

“…Similarly, polypeptides 45 kM r and 55 kM r are the only one to be ADP-ribosylated by cholera toxin in G/F from rat liver membranes, a 130 kM r oligoprotein made of 3 polypeptides: 30 kMr, 45 kM r and 55 kM r [14]. In contrast, there is only one ADP-ribosylated polypeptide in pigeon [5,6] and human [8,9] erythrocyte ghosts and in thymus [12] …”

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

“…The cholera holotoxin molecule, a member of a family of bacterial heat-labile enterotoxins, is composed of a single A subunit (CT-A) and a pentamer of B subunits (CT-B) (Lospalluto and Finkelstein, 1972;Gill, 1976). The catalytic CT-A subunit disrupts regulation of ion channel function within the host intestinal epithelial cells causing inappropriate and substantial fluid efflux from the small intestine (Cassel and Pfeuffer, 1978;Gill and Meren, 1978). The CT-B pentamer required for secretion of the holotoxin outside the bacterial cell targets the CT-A subunit to the intestinal epithelium by interacting with host cell-surface GM 1 ganglioside (King and Van Heyningen, 1973).…”

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

“…In addition, both choleragen and LT require NAD for activation of adenylate cyclase in disrupted cells (9,11,12) and possess NAD glycohydrolase and ADPribosyltransferase activities (13)(14)(15). It has been proposed that both toxins exert their effects through the NAD-dependent ADP-ribosylation of either adenylate cyclase itself or of a protein critical to cyclase activation (9,11,(13)(14)(15)(16)(17). LT cross-reacts immunologically with antisera directed primarily against the B-subunits of choleragen (9,18,19).…”