Quantifying Degradation Rates of Transmembrane Receptor Kinases
Transmembrane receptor-kinases are widespread throughout eukaryotes and their activities are known to regulate all kinds of cellular responses in diverse organs and cell types. In order to guarantee the correct amplitude and duration of signals, receptor
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1. Introduction Regulated degradation of cytosolic proteins is achieved by the action of a plethora of E3 ubiquitin ligases which ubiquitinate the target protein, leading to its recruitment to the proteasome machinery (1). Degradation of transmembrane proteins, by contrast, occurs in specialized cellular compartments, termed lysosomes in
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_12, © Springer Science+Business Media, LLC 2011
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animal cells and vacuoles in yeast and plant cells. Therefore, recruitment of a plasma membrane protein for degradation will require its re-targeting to a different membrane compartment by means of successive membrane transport steps. After internalization from the plasma membrane, a protein is found in endosomes from which it can either be recruited into a recycling pathway back to the plasma membrane or be placed into an intraluminal vesicle of a maturing multivesicular body (2). Eventual fusion of such a multivesicular compartment with the vacuole will release the vesicle containing the transmembrane protein into the vacuolar lumen. This allows access of vacuolar proteases to both the cytosolic and extracellular/luminal part(s) of a transmembrane protein. The machinery that recruits the proteins into the multivesicular body consists of a series of so-called ESCRT complexes and all the evidence indicates that their function is conserved in all eukaryotic model organisms (3, 4). Different methods have been used for quantifying protein degradation rate. The easiest way probably consists in treating cells with cycloheximide (CHX) in order to block translation of new protein and measure the rate of disappearance of the pre-existing protein. However, this approach is based on the assumption that a generalized block of protein synthesis does not interfere with the effectiveness and functionality of the degradation machinery. This might apply to cytosolic proteins showing a high turn-over. However, it is much more problematic for transmembrane proteins, especially when their turn-over is not rapid, which would require prolonged treatment with CHX. Receptor turn-over has also been assessed by metabolic pulse-chase labeling with 35S-methionine and subsequent pull-downs (see ref. 5, for example). This technique is challenging, especially when used on whole organisms. It necessitates high doses of radioactivity, specific antibodies and cannot give any good temporal or spatial resolution of degradation rates. Therefore, we set out to develop an improved technique in order to measure the turn-over rate of the cell-surface receptor kinase Brassinosteroid-insensitive 1 (BRI1) in Arabidopsis. BRI1 is a receptor for Brassinosteroids – steroidal plant growth regulators, which are crucial for plant cell elongation (6). Instead of using metabolic pulse-chase labeling, we established a transgenic line that expresses an YFP-tagged BRI1 variant under the control of a heat-shock promoter
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