I read a paper. The authors performed in vivo coimmunoprecipitation to investigate the interaction between rsl1 (e3 ligase) and ABA receptor (putative substrate protein). ABA receptor band can be enriched by rsl1 only when MG132 is added during coip. If MG132 is not added, there is no band. The authors did not add ATP and ubiquitin to the reaction sample, so the ubiquitylation and degradation can only be dependent on the endogenous ATP and Ub. Why can MG132 affect interaction between ABA receptor and rsl1? How can it enhance the interaction between them? And why is there no band when the authors did not add MG132? Is the e3-substrate complex less stable when ubiquitylation can occur fast enough?
MG132 is almost certainly not enhancing the interaction of Rsl1 and the ABA receptor per se, but maintaining the levels of the ABA receptor by preventing degradation by the proteasome during the IP, and consequently increasing signal. Eukaryotic cell lysates have plenty of ubiquitin and ATP, the E3 ligases and proteasomes may very well function in promoting turnover if not inhibited. Quenching ATP may also have worked to inhibit the proteasome, but MG132 has great specificity, and MG132-dependent stabilization indicates that the fate of the ubiquitinated protein is proteasomal degradation.
According to my experience working with E3-ligase (SKP2) and myc degradation.Both MG115 and 132 inhibits the 20S proteasome activity and this stabilizes the substrate even though it is ubiquitylated. The substrate in your question seems has very short half-life and therefore it is not detectable during CoIp without proteasome inhibitors. Your question about the MG132 affect complexes between E3 ligase and the substrate is very relevant. According to our results; this would not affect the interaction directly it just stabilize the substrate and make it detectible but MG132 treatment of the cells longer time will induce the cells into the cell arrest and then the transcription of the substrate affected or signal mechanisms(such as kinase activation) for increased interaction between the E3 ligase and the substrate.
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