Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.
ABSTRACT. In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membraneassociated proteins called SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors). Sequence analysis identified a total of 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8 QcSNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. Almost all of them were ubiquitously expressed through out all tissues examined. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of the SNARE proteins were located on specific intracellular compartments: 6 in the endoplasmic reticulum, 9 in the Golgi apparatus, 4 in the trans-Golgi network (TGN), 2 in endosomes, 17 on the plasma membrane, 7 in both the prevacuolar compartment (PVC) and vacuoles, 2 in TGN/PVC/vacuoles, and 1 in TGN/PVC/plasma membrane. Some SNARE proteins showed multiple localization patterns in two or more different organelles, suggesting that these SNAREs shuttle between the organelles. Furthermore, the SYP41/SYP61-residing compartment, which was defined as the TGN, was not always located along with the Golgi apparatus, suggesting that this compartment is an independent organelle distinct from the Golgi apparatus. We propose possible combinations of SNARE proteins on all subcellular compartments, and suggest the complexity of the post-Golgi membrane traffic in higher plant cells.
BACKGROUND Patients with peripheral artery disease have an increased risk of cardiovascular morbidity and mortality. Antiplatelet agents are widely used to reduce these complications. METHODS This was a multicentre, double-blind, randomised placebo-controlled trial for which patients were recruited at 602 hospitals, clinics, or community practices from 33 countries across six continents. Eligible patients had a history of peripheral artery disease of the lower extremities (previous peripheral bypass surgery or angioplasty, limb or foot amputation, intermittent claudication with objective evidence of peripheral artery disease), of the carotid arteries (previous carotid artery revascularisation or asymptomatic carotid artery stenosis of at least 50%), or coronary artery disease with an ankle-brachial index of less than 0·90. After a 30-day run-in period, patients were randomly assigned (1:1:1) to receive oral rivaroxaban (2·5 mg twice a day) plus aspirin (100 mg once a day), rivaroxaban twice a day (5 mg with aspirin placebo once a day), or to aspirin once a day (100 mg and rivaroxaban placebo twice a day). Randomisation was computer generated. Each treatment group was double dummy, and the patient, investigators, and central study staff were masked to treatment allocation. The primary outcome was cardiovascular death, myocardial infarction or stroke; the primary peripheral artery disease outcome was major adverse limb events including major amputation. This trial is registered with ClinicalTrials.gov, number NCT01776424, and is closed to new participants. FINDINGS Between March 12, 2013, and May 10, 2016, we ; HR 0·67, 95% CI 0·45-1·00, p=0·05). The median duration of treatment was 21 months. The use of the rivaroxaban plus aspirin combination increased major bleeding compared with the aspirin alone group (77 [3%] of 2492 vs 48 [2%] of 2504; HR 1·61, 95% CI 1·12-2·31, p=0·0089), which was mainly gastrointestinal. Similarly, major bleeding occurred in 79 (3%) of 2474 patients with rivaroxaban 5 mg, and in 48 (2%) of 2504 in the aspirin alone group (HR 1·68, 95% CI 1·17-2·40; p=0·0043). INTERPRETATION Low-dose rivaroxaban taken twice a day plus aspirin once a day reduced major adverse cardiovascular and limb events when compared with aspirin alone. Although major bleeding was increased, fatal or critical organ bleeding was not. This combination therapy represents an important advance in the management of patients with peripheral artery disease. Rivaroxaban alone did not significantly reduce major adverse cardiovascular events compared with asprin alone, but reduced major adverse limb events and increased major bleeding. FUNDING Bayer AG. Methods This was a multicentre, double-blind, randomised placebo-controlled trial for which patients were recruited at 602 hospitals, clinics, or community practices from 33 countries across six continents. Eligible patients had a history of peripheral artery disease of the lower extremities (previous peripheral bypass surgery or angioplasty, limb or foot amputation, i...
For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.
Ara6 of Arabidopsis thaliana is a novel member of the Rab/Ypt GTPase family with unique structural features. It resembles Rab5 GTPases best, but lacks a large part of the C-terminal hypervariable region and the cysteine motif, and instead harbors an extra stretch of amino acid residues containing myristoylation and palmitoylation sites at the N-terminus. Ara6 is tightly associated with membranes and is expressed constitutively. In contrast, the conventional Rab5 ortholog, Ara7, is highly expressed only in actively dividing cells. Examination of green fluorescent protein (GFP)-tagged proteins indicates that both Ara6 and Ara7 are distributed on a subpopulation of endosomes and suggests their roles in endosomal fusion. The endosomal localization of Ara6 requires N-terminal fatty acylation, nucleotide binding and the C-terminal amino acid sequence coordinately. Proteins similar to Ara6 are found only in higher plants and thus represent a novel class of Rab GTPases regulating endocytic function in a plant- specific manner.
Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.
Endosomal trafficking plays an integral role in various eukaryotic cell activities and serves as a basis for higher-order functions in multicellular organisms. An understanding of the importance of endosomal trafficking in plants is rapidly developing, but its molecular mechanism is mostly unknown. Several key regulators of endosomal trafficking, including RAB5, which regulates diverse endocytic events in animal cells, are highly conserved. However, the identification of lineage-specific regulators in eukaryotes indicates that endosomal trafficking is diversified according to distinct body plans and lifestyles. In addition to orthologues of metazoan RAB5, land plants possess a unique RAB5 molecule, which is one of the most prominent features of plant RAB GTPase organization. Plants have also evolved a unique repertoire of SNAREs, the most distinctive of which are diverse VAMP7-related longins, including plant-unique VAMP72 derivatives. Here, we demonstrate that a plant-unique RAB5 protein, ARA6, acts in an endosomal trafficking pathway in Arabidopsis thaliana. ARA6 modulates the assembly of a distinct SNARE complex from conventional RAB5, and has a functional role in the salinity stress response. Our results indicate that plants possess a unique endosomal trafficking network and provide the first indication of a functional link between a specific RAB and a specific SNARE complex in plants.
There is a debate over how protein trafficking is performed through the Golgi apparatus. In the secretory pathway, secretory proteins that are synthesized in the endoplasmic reticulum enter the early compartment of the Golgi apparatus called cis cisternae, undergo various modifications and processing, and then leave for the plasma membrane from the late (trans) cisternae. The cargo proteins must traverse the Golgi apparatus in the cis-to-trans direction. Two typical models propose either vesicular transport or cisternal progression and maturation for this process. The vesicular transport model predicts that Golgi cisternae are distinct stable compartments connected by vesicular traffic, whereas the cisternal maturation model predicts that cisternae are transient structures that form de novo, mature from cis to trans, and then dissipate. Technical progress in live-cell imaging has long been awaited to address this problem. Here we show, by the use of high-speed three-dimensional confocal microscopy, that yeast Golgi cisternae do change the distribution of resident membrane proteins from the cis nature to the trans over time, as proposed by the maturation model, in a very dynamic way.
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