Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

Arabidopsis proteins were predicted which share an 80 residue zinc finger d omain known from ADP-ribosylation factor GTPase-activating proteins (ARF GA Ps). One of these is a 37 kDa protein, designated ZAC, which has a novel do main structure in which the N-terminal ARF GAP domain and a C-terminal C2 d omain are separated by a region without homology to other known proteins. Z ac promoter/beta -glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-det ected mainly in association with membranes and fractionated with Golgi and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and promp ted an analysis of the in vitro phospholipid-binding ability of ZAC. Phosph olipid dot-blot and liposome-binding assays indicated that fusion proteins containing the ZAC-C2 domain bind anionic phospholipids non-specifically, w ith some variance in Ca2+ and salt dependence. Similar assays demonstrated specific affinity of the ZAC N-terminal region (residues 1-174) for phospha tidylinositol 3-monophosphate (PI-3-P). Binding was dependent in part on an intact zinc finger motif, but proteins containing only the zinc finger dom ain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase- activating activity on Arabidopsis ARF proteins. These data identify a nove l PI-3-P-binding protein region and thereby provide evidence that this phos phoinositide is recognized as a signal in plants. A role for ZAC in the reg ulation of ARF-mediated vesicular transport in plants is discussed.