Modifications to the C-terminus of Arf1 alter cell functions and protein interactions.
Arf family proteins are approximately 21-kDa GTP-binding proteins that are critical regulators of membrane traffic and the actin cytoskeleton. Studies examining the complex signaling pathways underlying Arf action have relied on recombinant proteins comprised of Arf fused to epitope tags or proteins, such as glutathione S-transferase or green fluorescent protein, for both cell-based mammalian cell studies and bacterially expressed recombinant proteins for biochemical assays. However, the effects of such protein fusions on the biochemical properties relevant to the cellular function have been only incompletely studied at best. Here, we have characterized the effect of C-terminal tagging of Arf1 on (i) function in Saccharomyces cerevisiae, (ii) in vitro nucleotide exchange and (iii) interaction with guanine nucleotide exchange factors and GTPase-activating proteins. We found that the tagged Arfs were substantially impaired or altered in each assay, compared with the wild-type protein, and these changes are certain to alter actions in cells. We discuss the results related to the interpretation of experiments using these reagents and we propose that authors and editors consistently adopt a few simple rules for describing and discussing results obtained with Arf family members that can be readily applied to other proteins.
Jian
Xiaoying
X
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bldg 37 Room 2042, Bethesda, MD 20892, USA.
Cavenagh
Margaret
M
Gruschus
James M
JM
Randazzo
Paul A
PA
Kahn
Richard A
RA
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry*; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry; D020690:GTPase-Activating Proteins / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D000939:Epitopes; D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1