Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein

J Mol Biol. 2001 Jun 8;309(3):641-55. doi: 10.1006/jmbi.2001.4706.

Abstract

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed by five leucine-rich repeats, a proline-rich region, a single transmembrane region and an intracellular kinase domain. The AtSERK1 cDNA was fused to two different variants of green fluorescent protein (GFP), a yellow-emitting GFP (YFP) and a cyan-emitting GFP (CFP), and transiently expressed in both plant protoplasts and insect cells. Using confocal laser scanning microscopy it was determined that the AtSERK1-YFP fusion protein is targeted to plasma membranes in both plant and animal cells. The extracellular leucine-rich repeats, and in particular the N-linked oligosaccharides that are present on them appear to be essential for correct localization of the AtSERK1-YFP protein. The potential for dimerization of the AtSERK1 protein was investigated by measuring the YFP/CFP fluorescence emission ratio using fluorescence spectral imaging microscopy. This ratio will increase due to fluorescence resonance energy transfer if the AtSERK1-CFP and AtSERK1-YFP fusion proteins interact. In 15 % of the cells the YFP/CFP emission ratio for plasma membrane localized AtSERK1 proteins was enhanced. Yeast-protein interaction experiments confirmed the possibility for AtSERK1 homodimerization. Elimination of the extracellular leucine zipper domain reduced the YFP/CFP emission ratio to control levels indicating that without the leucine zipper domain AtSERK1 is monomeric.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Arabidopsis / cytology*
  • Arabidopsis / drug effects
  • Arabidopsis / enzymology*
  • Arabidopsis / metabolism
  • Cell Line
  • Cell Membrane / metabolism
  • Dimerization
  • Energy Transfer
  • Fluorescence
  • Glycosylation
  • Leucine Zippers
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Mitogen-Activated Protein Kinase Kinases / chemistry*
  • Mitogen-Activated Protein Kinase Kinases / metabolism*
  • Plant Proteins / chemistry
  • Plant Proteins / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Transport
  • Protoplasts / cytology
  • Protoplasts / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Fluorescence
  • Spodoptera
  • Tunicamycin / pharmacology
  • Two-Hybrid System Techniques

Substances

  • Membrane Proteins
  • Plant Proteins
  • Recombinant Fusion Proteins
  • Tunicamycin
  • Mitogen-Activated Protein Kinase Kinases