In vivo hexamerisation and characterisation of the Arabidopsis thaliana AAA ATPase complex using FRET-FLIM and FCS.

J.C.M. Aker, R. Hesselink, R. Engel, J.W. Borst, A.J.W.G. Visser, S.C. de Vries

Research output: Contribution to journalArticleAcademicpeer-review

39 Citations (Scopus)

Abstract

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
Original languageEnglish
Pages (from-to)339-350
JournalPlant Physiology
Volume145
Issue number2
DOIs
Publication statusPublished - 2007

Keywords

  • conformational-changes
  • living cells
  • protein
  • p97
  • p97/vcp
  • receptor
  • membrane
  • cycle
  • phosphorylation
  • degradation

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