Reference: Dasilva LF, et al. (2013) The C-terminal Residues of Saccharomyces cerevisiae Mec1 Are Required for Its Localization, Stability and Function. G3 (Bethesda)

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Abstract

Mec1, a member of the PIKK (phosphoinositide three-kinase-related kinase) family of proteins, is involved in the response to replicative stress and DNA damage, and in telomere maintenance. An essential 30-35 residue FATC domain is found at the C-terminus of all PIKK family members. To investigate the roles of the C-terminal residues of Mec1, we characterized alleles of Saccharomyces cerevisiae mec1 that alter the FATC domain. A change of the terminal tryptophan to alanine resulted in temperature sensitive growth, sensitivity to hydroxyurea, and diminished kinase activity in vitro. Addition of a terminal glycine or deletion of one, two or three residues resulted in loss of cell viability and kinase function. Each of these Mec1 derivatives was less stable than wild-type Mec1, eluted abnormally from a size exclusion column, and showed reduced nuclear localization. We identified rpn3-L140P, which encodes a component of the 19S proteasomal regulatory particle of the 26S proteasome, as a suppressor of the temperature-sensitive growth caused by mec1-W2368A. The rpn3-L140P allele acted in a partially dominant fashion. It was neither able to suppress the inviability of the C-terminal truncations or additions, nor the hydroxyurea sensitivity of mec1-W2368A. The rpn3-L140P allele restored Mec1-W2368A to near wild-type protein levels at 37 degrees , an effect partially mimicked by the proteasome inhibitor MG-132. Our study supports a role for the C-terminus in Mec1 folding and stability, and suggests a role for the proteasome in regulating Mec1 levels.

Reference Type
Journal Article
Authors
Dasilva LF, Pillon S, Genereaux J, Davey MJ, Gloor GB, Karagiannis J, Brandl CJ
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