title: The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae.
abstract: Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.
authors.affiliation: Department of Biology, Colgate University, Hamilton, NY 13346, USA. kbelanger@colgate.edu
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Belanger
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Griffith
authors.forename: Heather L
authors.initials: HL
authors.lastname: Baker
authors.forename: Jeanne N
authors.initials: JN
authors.lastname: Hansen
authors.forename: Laura A Simmons
authors.initials: LA
authors.lastname: Kovacs
authors.forename: Justin S
authors.initials: JS
authors.lastname: Seconi
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Strine
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D028961:beta Karyopherins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; C082728:RFA1 protein, S cerevisiae; C082727:RFA2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins