Study of the R38-K40 ribosome binding site in the Rack1 protein using the Morg1 protein model
Stress granules (SG) are the membraneless organelles which modulate the cells response to stress conditions. The transmission of protein Rack1 into the SG induces cancer cell resistance against some chemotherapeutics. The mechanism of its transfer is difficult to study because mammalian cell lines with mutated or depleted Rack1 are very unstable. Here we suggest using a similar protein Morg1 as a model to study Rack1 binding interactions, without altering the expression of Rack1 itself. We designed the protocol, which enables i) to create a stable MDCK cell line expressing mutated forms of Morg1, ii) to quantify the contribution of individual binding sites to the overall affinity of Rack1 to the ribosome, using fluorescent microscopy and polysome profile analysis.
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