Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells.
ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant 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 found in uninfected cells. Infected Sf9 cells incorporated [3H]myristate 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 chromatography and identified by microsequencing of HPLC-purified tryptic peptides. Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation. The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP. Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2.
Kunz
B C
BC
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Muczynski
K A
KA
Welsh
C F
CF
Stanley
S J
SJ
Tsai
S C
SC
Adamik
R
R
Chang
P P
PP
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006160:Guanosine Triphosphate / Q000378:metabolism; D006882:Hydroxyapatites; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D006882:Hydroxyapatites; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D017886:Durapatite; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors