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CELL BIOLOGY AND SIGNAL TRANSDUCTION

In Vivo Hexamerization and Characterization of the Arabidopsis AAA ATPase CDC48A Complex Using Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy^1,[W],[OA]

Laboratory of Biochemistry (J.A., R.H., R.E., R.K., J.W.B., A.J.W.G.V., S.C.d.V.) and Microspectroscopy Centre (R.E., J.W.B., A.J.W.G.V.), Wageningen University, 6703 HA Wageningen, The Netherlands

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sacco C. de Vries (sacco.devries{at}wur.nl).

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.103986

^* Corresponding author; e-mail sacco.devries{at}wur.nl.

Received June 26, 2007; accepted August 3, 2007; published August 10, 2007.

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