The C-terminal residues of Saccharomyces cerevisiae Mec1 are required for its localization, stability, and function.
Mec1, a member of the phosphoinositide three-kinase-related kinase (PIKK) family of proteins, is involved in the response to replicative stress and DNA damage and in telomere maintenance. An essential 30 to 35 residue, the 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 not able to suppress the inviability of the C-terminal truncations or additions or the hydroxyurea sensitivity of mec1-W2368A. The rpn3-L140P allele restored Mec1-W2368A to nearly wild-type protein levels at 37°, 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.
Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada N6A5C1.
Lance F
LF
DaSilva
Samantha
S
Pillon
Julie
J
Genereaux
Megan J
MJ
Davey
Gregory B
GB
Gloor
Jim
J
Karagiannis
Christopher J
CJ
Brandl
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D004795:Enzyme Stability; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D047908:Intracellular Signaling Peptides and Proteins; C403291:RPN3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex
FATC domain; Mec1; PIKK proteins; Rpn3