In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.
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.
Aker
José
J
Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands.
Hesselink
Renske
R
Engel
Ruchira
R
Karlova
Rumyana
R
Borst
Jan Willem
JW
Visser
Antonie J W G
AJ
de Vries
Sacco C
SC
D000074183:ATPases Associated with Diverse Cellular Activities; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D018506:Gene Expression Regulation, Plant; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits; D021381:Protein Transport; D013050:Spectrometry, Fluorescence
D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D021122:Protein Subunits; D011494:Protein Kinases; C440035:SERK1 protein, Arabidopsis; D000074183:ATPases Associated with Diverse Cellular Activities; C525275:CDC48A protein, Arabidopsis
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