CHARACTERIZATION OF RECOMBINANT AND ENDOGENOUS ADP-RIBOSYLATION FACTORS SYNTHESIZED IN SF9 INSECT CELLS

ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-k Da guanine nucleotide-binding proteins that participate in protein tra fficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 f rom bovine retinal cDNA was expressed in Sf9 insect cells using recomb inant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E. coli rARF 2 ). The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were fo und in uninfected cells. Infected Sf9 cells incorporated [H-3]myristat e only into the 20-kDa protein. Sf9 cell recombinant ARF 2 (Sf9 rARF 2 ) and Sf9 ARF were separated by isoelectric focusing or ion-exchange c hromatography and identified by microsequencing of HPLC-purified trypt ic peptides. Sf9 ARF displayed considerable sequence identity to mamma lian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dep endent manner cholera toxin-catalyzed ADP-ribosylation. The K(a) for G TP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the a bility of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the K(a) for GTP. Despite the sequence identities and the fact that b oth were synthesized in insect cells, the endogenous Sf9 ARF was funct ionally different from Sf9 rARF 2.