Nuclear Import of Ho Endonuclease Utilizes Two Nuclear Localization Signals and Four Importins of the Ribosomal Import System *

Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel.
Current Genetics (Impact Factor: 2.68). 10/2008; 54(5):271-81. DOI: 10.1007/s00294-008-0216-8
Source: PubMed

ABSTRACT 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.

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Available from: Dina Raveh, Sep 28, 2015
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    • "Xpo1p/Crm1p is also, at least indirectly, required for normal mRNA production and export (Feng et al. 1999; Strasser et al. 2000; Hammell et al. 2002; Dong et al. 2007). Msn5p has also been shown to act as a nuclear export factor, exporting phosphorylated nuclear transcription factors (Kaffman et al. 1998a; DeVit and Johnston 1999; Gorner et al. 2002; Queralt and Igual 2003; Durchschlag et al. 2004; Ueta et al. 2007), the HO endonuclease (Bakhrat et al. 2008), and Whi5p, the yeast ortholog of Rb (Taberner et al. 2009). A consensus nuclear export signal (NES) for Msn5p has been elusive, but its preference for phosphorylated proteins suggests a role for regulated export. "
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    • "We also carried out an in vitro binding experiment between GST-Msn5, His 6 -Swi5 and His 6 -Gsp1/Ran expressed in bacteria. No specific interaction between Msn5 and Swi5 could be detected, which raises the possibility that the in vivo interaction between Msn5 and Swi5 could involve either additional bridging protein(s) or, most probably, a post-translational modification of Swi5 as occurs with other Msn5 cargo proteins [36] [37] [38] [39] [42] [43]. Given that Msn5 is a karyopherin, its in vivo physical interaction with Swi5 raises the possibility that Msn5 may control the subcellular localization of the transcription factor. "
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