Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein.
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.
Shah
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Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen, The Netherlands.
Gadella
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TW
van Erp
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H
Hecht
V
V
de Vries
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SC
D020816:Amino Acid Motifs; D000818:Animals; D017360:Arabidopsis / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004735:Energy Transfer; D005453:Fluorescence; D006031:Glycosylation; D016350:Leucine Zippers; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018411:Spodoptera; D014415:Tunicamycin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D008565:Membrane Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D020929:Mitogen-Activated Protein Kinase Kinases