TY - JOUR

T1 - Coordinated activation of ARF1 GTPases by ARF-GEF GNOM dimers is essential for vesicle trafficking in arabidopsis

AU - Brumm, Sabine

AU - Singh, Manoj K.

AU - Nielsen, Mads Eggert

AU - Richter, Sandra

AU - Beckmann, Hauke

AU - Stierhof, York Dieter

AU - Fischer, Angela Melanie

AU - Kumaran, Mande

AU - Sundaresan, Venkatesan

AU - Jürgens, Gerd

N1 - Publisher Copyright: ã 2020 ASPB.

PY - 2020

Y1 - 2020

N2 - Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1cGTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1cGTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

AB - Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1cGTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1cGTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

U2 - 10.1105/tpc.20.00240

DO - 10.1105/tpc.20.00240

M3 - Journal article

C2 - 32487565

AN - SCOPUS:85089129827

VL - 32

SP - 2491

EP - 2507

JO - The Plant Cell

JF - The Plant Cell

SN - 1040-4651

IS - 8

ER -