AGD5 is a GTPase-activating protein at the trans-Golgi network.

ARF-GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which facilitate the activation or inactivation of ARF-GTPases, respectively. There are 15 predicted proteins that contain an ARF-GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF-GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF-GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans-Golgi network (TGN), where it co-localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post-Golgi structures of unknown nature. Taking advantage of the in vivo AGD5-ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF-GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post-Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN-localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane-localised ADP ribosylation factor B (ARFB), confirming that ARF-GAP specificity for ARF-GTPases within the cell environment may be spatially regulated.

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