Wortmannin Treatment Induces Changes in Arabidopsis Root Proteome and Post-Golgi Compartments

Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacký University , Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic.
Journal of Proteome Research (Impact Factor: 4.25). 04/2012; 11(6). DOI: 10.1021/pr201111n
Source: PubMed


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.

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    • "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 [37]. However, the relocalization of RabA1b to plasma membrane upon wortmannin treatment [37] differs from the accumulation, clustering, fusion or swelling of TGN and MVB compartments, observed by fluorescently tagged RabA1d, RabA4b, RabA1e, VTI12, FYVE and RabF2a [52]. Under wortmannin treatment, RabA1d is relocated into wortmannin-induced multivesicular compartments and is downregulated [52]. "
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    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.
    BMC Plant Biology 09/2014; 14(1):252. DOI:10.1186/s12870-014-0252-0 · 3.81 Impact Factor
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    ABSTRACT: The growing interest in the investigation of endocytosis, vesicular transport routes, and corresponding regulatory mechanisms resulted in the exploitation of cell biological, genetic, biochemical, and proteomic approaches. Methods and techniques such as site-directed and T-DNA insertional mutagenesis, RNAi, classical inhibitor treatments, and recombinant GFP technology combined with confocal laser scanning microscopy (CLSM) and electron and immune-electron microscopy were routinely employed for investigation of endocytosis in plant cells. However, new approaches such as high-throughput confocal microscopy screens on mutants and proteomic analyses on isolated vesicular compartments and root cells treated with vesicular trafficking inhibitors (both focused on the identification of new endosomal proteins), together with chemical genomics and advanced microscopy approaches such as Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), light sheet-based fluorescence microscopy, and super-resolution microscopy provided a significant amount of new data and these new methods appear as extremely promising tools in this field.
    Endocytosis in plants, Edited by Jozef Šamaj, 01/2012: pages 1-36; Springer., ISBN: ISBN 978-3-642-32462-8
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