Overexpression of Arabidopsis AGD7 Causes Relocation of Golgi-Localized Proteins to the Endoplasmic Reticulum and Inhibits Protein Trafficking in Plant Cells1(C)(OA) (original) (raw)
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Plant Physiology, 2002
ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play important roles in intracellular trafficking in animal and yeast cells. Here, we investigated the roles of two Arf homologs, Arf1 and Arf3 of Arabidopsis, in intracellular trafficking in plant cells. We generated dominant negative mutant forms of Arf 1 and Arf3 and examined their effect on trafficking of reporter proteins in protoplasts. Arf1[T31N] inhibited trafficking of H+-ATPase:green fluorescent protein (GFP) and sialyltransferase (ST):GFP to the plasma membrane and the Golgi apparatus. In addition, Arf1[T31N] caused relocalization of the Golgi reporter protein ST:GFP to the endoplasmic reticulum (ER). In protoplasts expressing Arf1[T31N], ST:red fluorescent protein remained in the ER, whereas H+-ATPase:GFP was mistargeted to another organelle. Also, expression of Arf1[T31N] in protoplasts resulted in profound changes in the morphology of the ER. The treatment of protoplasts with brefeldin A had exactl...
Plant Science, 2006
ADP-ribosylation factor (ARF) and ARF-like (ARL) proteins are small GTPases that undergo a GDP/GTP nucleotide exchange cycle. ARF proteins are important regulators of cellular trafficking. Knowledge of plant ARF proteins and their regulators is emerging from genetic and cellular studies. The Arabidopsis BIG2 ARF-guanine nucleotide exchange factor (GEF) gene is expressed in several tissues and the encoded protein is a typical Arabidopsis ARF-GEF. We address here the specificity of BIG2 for different Arabidopsis ARF proteins. The in vitro effect of the catalytic Sec7 domain of BIG2 on the guanine nucleotide exchange rate of five ARF and ARL proteins was measured using real time fluorescence spectroscopy. The Sec7 domain catalyzed nucleotide exchange on ARF1, but had essentially no effect on the exchange rate of ARF8, ARF9, ARL1, and ARL8a. In Western blots with an anti-BIG2 polyclonal antibody, a BIG2 fraction was detected in membranes, especially those deriving from the Golgi apparatus. The activity of the BIG2 Sec7 domain was unaffected by the Sec7 inhibitor brefeldin A, suggesting that BIG2 is a BFAinsensitive GEF for ARF1 or a close homolog. These studies contribute to our understanding of the biochemical and physiological specificity of ARF and ARF-GEF interactions in plants.
PLANT PHYSIOLOGY, 2007
Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes. In addition, although ARF1 appears to be central to the recruitment of both COPI components and the golgin, we have established a different subcellular distribution of these ARF1 effectors. Live cell imaging demonstrates that GDAP1 and COPI are distributed on Golgi membranes. However, GDAP1 is also found on ARF1-labeled structures that lack coatomer, suggesting that the membrane environment, rather than ARF1 alone,...
The Plant Cell, 2014
GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/ EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants.
Correct Targeting of Plant ARF GTPases Relies on Distinct Protein Domains
Traffic, 2008
Indispensable membrane trafficking events depend on the activity of conserved small guanosine triphosphatases (GTPases), anchored to individual organelle membranes. In plant cells, it is currently unknown how these proteins reach their correct target membranes and interact with their effectors. To address these important biological questions, we studied two members of the ADP ribosylation factor (ARF) GTPase family, ARF1 and ARFB, which are membrane anchored through the same N-terminal myristoyl group but to different target membranes. Specifically, we investigated how ARF1 is targeted to the Golgi and post-Golgi structures, whereas ARFB accumulates at the plasma membrane. While the subcellular localization of ARFB appears to depend on multiple domains including the C-terminal half of the GTPase, the correct targeting of ARF1 is dependent on two domains: an N-terminal ARF1 domain that is necessary for the targeting of the GTPase to membranes and a core domain carrying a conserved MxxE motif that influences the relative distribution of ARF1 between the Golgi and post-Golgi compartments. We also established that the N-terminal ARF1 domain alone was insufficient to maintain an interaction with membranes and that correct targeting is a protein-specific property that depends on the status of the GTP switch. Finally, an ARF1-ARFB chimera containing only the first 18 amino acids from ARF1 was shown to compete with ARF1 membrane binding loci. Although this chimera exhibited GTPase activity in vitro, it was unable to recruit coatomer, a known ARF1 effector, onto Golgi membranes. Our results suggest that the targeting of ARF GTPases to the correct membranes may not only depend on interactions with effectors but also relies on distinct protein domains and further binding partners on the Golgi surface.
AGD5 is a GTPase‐activating protein at the trans‐Golgi network
2010
Summary 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.
Nonselective chemical inhibition of Sec7 domain-containing ARF GEFs in Arabidopsis
The Plant cell, 2018
Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF GEF-deficient mutants. Drug affinity-responsive targe...
Post-Golgi protein traffic in the plant secretory pathway
Plant Cell Reports, 2007
The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441-447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate. In the post-Golgi direction, these include vacuole and plasma membrane, and specialized transport routes to each are required to prevent mislocalization. Recent evidence in plant cells points to the existence of post-Golgi organelles that function as intermediate stations for efficient protein traffic, as well as to the influence of small GTPases such as Rabs and ARFs on post-Golgi trafficking. This review focuses on the latest findings on post-Golgi trafficking routes and on the involvement of GTPases and their effectors on the trafficking of proteins in the plant secretory pathway.