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Published on August 10, 2007; 10.1104/pp.107.103986

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Received June 26, 2007
Accepted August 3, 2007

In vivo Hexamerization and Characterization of the Arabidopsis AAA ATPase CDC48A-complex using FRET-FLIM and FCS

José Aker , Renske Hesselink , Ruchira Engel , Rumyana Karlova , Jan Willem Borst , Antonie J.W.G. Visser , and Sacco C. de Vries *

Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands; Microspectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

* Corresponding author; email: Sacco.deVries{at}wur.nl.

The Arabidopsis thaliana AAA ATPase CDC48A was fused to Cerulean Fluorescent Protein (CrFP) and Yellow Fluorescent Protein (YFP). AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer (FRET) -based Fluorescence Lifetime Imaging Microscopy (FLIM) using CDC48A-CrFP and -YFP showed interaction between two adjacent protomers, demonstrating homo-oligomerization to occur in living plant cells. Interaction between CDC48A and the 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. FRET-FLIM was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing Fluorescence Correlation Spectroscopy (FCS), it was shown that CDC48A hexamers are part of larger complexes.




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