title: A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export.
abstract: The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.
authors.affiliation: Department of Biological Sciences, Colgate University, Hamilton, NY 13346, USA. efinn87@gmail.com
authors.forename: Erin M
authors.initials: EM
authors.lastname: Finn
authors.forename: Elise P
authors.initials: EP
authors.lastname: DeRoo
authors.forename: George W
authors.initials: GW
authors.lastname: Clement
authors.forename: Sheila
authors.initials: S
authors.lastname: Rao
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Kruse
authors.forename: Kate M
authors.initials: KM
authors.lastname: Kokanovich
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Belanger
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D022022:Nuclear Pore / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C109868:CRZ1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D028884:Karyopherins; C401306:MSN5 protein, S cerevisiae; C062748:NSP1 protein, S cerevisiae; C077731:NUP100 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010649:Phenylalanine; D005998:Glycine