Reciprocal regulation of nuclear import of the yeast MutSalpha DNA mismatch repair proteins Msh2 and Msh6.
DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSalpha (Msh2/Msh6) and MutSbeta (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.
Hayes
Alicia P
AP
Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, United States.
Sevi
Leah A
LA
Feldt
Megan C
MC
Rose
Mark D
MD
Gammie
Alison E
AE
D021581:Active Transport, Cell Nucleus / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D051718:MutS Homolog 2 Protein / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C098790:MSH6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C489751:MSH2 protein, S cerevisiae; D051718:MutS Homolog 2 Protein