Nuclear export of Ho endonuclease of yeast via Msn5.
Exportin-5, an evolutionarily conserved nuclear export factor of the beta-karyopherin family, exports phosphorylated proteins and small noncoding RNAs. Msn5, the yeast ortholog, exports primarily phosphorylated cargoes including Ho endonuclease and a number of transcription factors and regulatory proteins. The Msn5-mediated nuclear export of Ho is dependent on phosphorylation of Thr225 by kinases of the DNA damage response pathway. Although Msn5 has been the object of many studies, no NES sequence capable of binding the exportin and/or of leading to Msn5-dependent export of a heterologous protein has been identified. Here we report identification of a 13-residue Ho sequence that interacts with Msn5 in vitro and directs Msn5-dependent nuclear export of GFP in vivo. A single point mutation in this 13-mer Ho NES abrogates both interaction with Msn5 and nuclear export of Ho and of GFP. However, this mutation, or of T225A, both of which abrogate nuclear export of Ho, does not interfere with its interaction with Msn5 implying that the exportin makes multiple contacts with its cargo. This can explain the lack of a conserved NES in Msn5 cargoes. Our results identify essential criteria for Msn5-mediated nuclear export of Ho: phosphorylation on HoT225, and interaction with the 13-mer Ho NES sequence.
Bakhrat
Anya
A
Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel.
Baranes-Bachar
Keren
K
Reshef
Dan
D
Voloshin
Olga
O
Krichevsky
Oleg
O
Raveh
Dina
D
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D017124:Conserved Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D028884:Karyopherins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C109731:ASF1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D028884:Karyopherins; C401306:MSN5 protein, S cerevisiae; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; C530845:HO protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific