Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains.
ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.
Cohen
Lee Ann
LA
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, and Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Honda
Akira
A
Varnai
Peter
P
Brown
Fraser D
FD
Balla
Tamas
T
Donaldson
Julie G
JG
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D010716:Phosphatidylinositols; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C102474:cytohesin-2; C040985:phosphatidylinositol receptors; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human