Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors.
The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of approximately 200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are approximately 20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5'-[gamma-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5'-[gamma-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.
Sata
M
M
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Donaldson
J G
JG
Moss
J
J
Vaughan
M
M
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D020126:Brefeldin A; D002352:Carrier Proteins / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology*; D017931:DNA Primers; D005656:Fungal Proteins / Q000187:drug effects / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D016324:Sequence Tagged Sites
D002352:Carrier Proteins; D003517:Cyclopentanes; D017931:DNA Primers; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; C066755:Sec7 guanine nucleotide exchange factors; D009243:NAD; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors