title: AGD5 is a GTPase-activating protein at the trans-Golgi network.
abstract: 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.
authors.affiliation: Department of Plant, Soil and Environmental Science, viale delle Idee, University of Florence, Sesto Fiorentino, FI 50019, Italy. gstefano@msu.edu
authors.forename: Giovanni
authors.initials: G
authors.lastname: Stefano
authors.forename: Luciana
authors.initials: L
authors.lastname: Renna
authors.forename: Marika
authors.initials: M
authors.lastname: Rossi
authors.forename: Elisa
authors.initials: E
authors.lastname: Azzarello
authors.forename: Susanna
authors.initials: S
authors.lastname: Pollastri
authors.forename: Federica
authors.initials: F
authors.lastname: Brandizzi
authors.forename: Frantisek
authors.initials: F
authors.lastname: Baluska
authors.forename: Stefano
authors.initials: S
authors.lastname: Mancuso
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: C503841:ARF1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D014157:Transcription Factors