ABSTRACT

II. BIOCHEMICAL CHARACTERIZATION OF ARFs 258 A. Purification and Characterization of Native ARF Proteins 258 B. Expression of Recombinant ARF Proteins 262

III. DISTRIBUTION OF ARF PROTEINS AND DEVELOPMENTAL EXPRESSION

IV. MOLECULAR CHARACTERIZATION OF ARF

V. ARF PROTEIN STRUCTURE

VI. CELLULAR FUNCTIONS OF ARFs

VII. SUMMARY

REFERENCES

I. CHOLERA TOXIN ACTIVATION OF ADENVLVL CYCLASE

Cholera toxin, a heterooligomeric protein secreted by Vibrio cholera, causes the pathogenesis of cholera due to its ability to activate adenylyl cyclase of intestinal mucosal cells and increase intracellular cAMP levels (Finkelstein, 1973; Kelly, 1986). Cholera toxin is composed of one A and five B subunits, the latter forming the cell membrane binding domain (Gill, 1976, 1977; for review see Moss and Vaughan, 1990). The A subunit is cleaved into two polypeptides, AI and A2 (Gill and Rappaport, 1979), and the active A 1 species is generated after reduction of a disulfide bond that links the two (Mekalanos, 1979b; Moss and Vaughan, 1990). The A1 protein is responsible for the enzymatic activities of cholera toxin (Moss et al., 1979). These include ( 1) NAD hydrolysis to ADP-ribose and nicotinamide (Moss et al., 1976); (2) transfer of ADP-ribose

to free arginine and simple guanidino compounds (Moss and Vaughan, 1977a; Mekalanos et al., 1979a); (3) ADP-ribosylation of nonspecific proteins, presumably due to available arginine residues that serve as toxin substrates (Moss and Vaughan, 1978); (4) auto-ADPribosylation ofCTA1 (Moss et al., 1980; Trepel et al., 1977); and (5) ADP-ribosylation of Gsa, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase (Cassel and Pfeuffer, 1978; Gill and Meren, 1978; Johnson et al., 1978; Northup etal., 1980).