Wortmannin is a widely used pharmaceutical compound which is employed to define vesicular trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinases in a dose-dependent manner leading to the inhibition of protein vacuolar sorting and endocytosis. Combined proteomics and cell biological approaches have been used in this study to explore the effects of wortmannin on Arabidopsis root cells, especially on proteome and endomembrane trafficking. On the subcellular level, wortmannin caused clustering, fusion, and swelling of trans-Golgi network (TGN) vesicles and multivesicular bodies (MVBs) leading to the formation of wortmannin-induced multivesicular compartments. Appearance of wortmannin-induced compartments was associated with depletion of TGN as revealed by electron microscopy. On the proteome level, wortmannin induced massive changes in protein abundance profiles. Wortmannin-sensitive proteins belonged to various functional classes. An inhibition of vacuolar trafficking by wortmannin was related to the downregulation of proteins targeted to the vacuole, as showed for vacuolar proteases. A small GTPase, RabA1d, which regulates vesicular trafficking at TGN, was identified as a new protein negatively affected by wortmannin. In addition, Sec14 was upregulated and PLD1 alpha was downregulated by wortmannin.
"RabA1b co-localize with VAMP721/722, R-SNARE proteins that operate in the secretory pathway and only partially and unstably associate with TGN, Golgi and endosomes . However, the relocalization of RabA1b to plasma membrane upon wortmannin treatment  differs from the accumulation, clustering, fusion or swelling of TGN and MVB compartments, observed by fluorescently tagged RabA1d, RabA4b, RabA1e, VTI12, FYVE and RabF2a . Under wortmannin treatment, RabA1d is relocated into wortmannin-induced multivesicular compartments and is downregulated . "
[Show abstract][Hide abstract] ABSTRACT: Background
Small Rab GTPases are important regulators of vesicular trafficking in plants. AtRabA1d, a member of the RabA1 subfamily of small GTPases, was previously found in the vesicle-rich apical dome of growing root hairs suggesting a role during tip growth; however, its specific intracellular localization and role in plants has not been well described.ResultsThe transient expression of 35S::GFP:RabA1d construct in Allium porrum and Nicotiana benthamiana revealed vesicular structures, which were further corroborated in stable transformed Arabidopsis thaliana plants. GFP-RabA1d colocalized with the trans-Golgi network marker mCherry-VTI12 and with early FM4-64-labeled endosomal comparments. Late endosomes and endoplasmic reticulum labeled with FYVE-DsRed and ER-DsRed, respectively, were devoid of GFP-RabA1d. The accumulation of GFP-RabA1d in the core of brefeldin A (BFA)-induced-compartments and the quantitative upregulation of RabA1d protein levels after BFA treatment confirmed the association of RabA1d with early endosomes/TGN and its role in vesicle trafficking. Light-sheet microscopy revealed involvement of RabA1d in root development. In root cells, GFP-RabA1d followed cell plate expansion consistently with cytokinesis-related vesicular trafficking and membrane recycling. GFP-RabA1d accumulated in disc-like structures of nascent cell plates, which progressively evolved to marginal ring-like structures of the growing cell plates. During root hair growth and development, GFP-RabA1d was enriched at root hair bulges and at the apical dome of vigorously elongating root hairs. Importantly, GFP-RabA1d signal intensity exhibited an oscillatory behavior in-phase with tip growth. Progressively, this tip localization dissapeared in mature root hairs suggesting a link between tip localization of RabA1d and root hair elongation. Our results support a RabA1d role in events that require vigorous membrane trafficking.Conclusions
RabA1d is located in early endosomes/TGN and is involved in vesicle trafficking. RabA1d participates in both cell plate formation and root hair oscillatory tip growth. The specific GFP-RabA1d subcellular localization confirms a correlation between its specific spatio-temporal accumulation and local vesicle trafficking requirements during cell plate and root hair formation.
[Show abstract][Hide abstract] ABSTRACT: Each membrane trafficking pathway involves several evolutionarily conserved key molecules, including RAB GTPases and SNARE proteins. Distinct sets of RAB and SNARE molecules regulate tethering and fusion of the carrier membrane to the target membrane for different trafficking pathways. These proteins are thought to control the specificity of directional targeting and membrane fusion to the correct target organelles. These molecules also exhibit distinctive subcellular localizations and are, therefore, regarded as earmarks for organelles. Several subgroups of RAB and SNARE are widely conserved among eukaryotic lineages, indicating ancient origins and conserved functions. In contrast, recent comparative genomics indicated RAB and SNARE members have expanded in a lineage-specific manner. This finding suggests novel trafficking routes, which are unique to each lineage, developed during evolution. Plant-unique sets of RAB and SNARE proteins have been identified and characterized in recent years. In this chapter, we summarize the conserved and unique features of plant RAB and SNARE proteins with a special focus on post-Golgi trafficking pathways, including the endocytic pathway.
Endocytosis in Plants, 01/2012: pages 201-216; , ISBN: 978-3-642-32462-8
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