Nuclear export of Ho endonuclease of yeast via Msn5.

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


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.

Download full-text


Available from: Dina Raveh,
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell's genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or "Nups"), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC's role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.
    Genetics 03/2012; 190(3):855-83. DOI:10.1534/genetics.111.127803 · 5.96 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Inactivation of S. cerevisiae β-karyopherin Msn5 causes hypersensitivity to the overexpression of mitotic cyclin Clb2 and aggravates growth defects of many mutant strains in mitotic exit, suggesting a connection between Msn5 and mitotic exit. We determined that Msn5 controlled subcellular localization of the mitotic exit transcription factor Swi5, since it was required for Swi5 nuclear export. Msn5 physically interacted with the N-terminal end of Swi5. Inactivation of Msn5 caused a severe reduction in cellular levels of Swi5 protein. This effect occurred by a post-transcriptional mechanism, since SWI5 mRNA levels were not affected. The reduced amount of Swi5 in msn5 mutant cells was not due to an increased protein degradation rate, but to a defect in Swi5 synthesis. Despite the change in localization and protein level, Swi5-regulated transcription was not defective in the msn5 mutant strain. However, a high level of Swi5 was toxic in the absence of Msn5. This deleterious effect was eliminated when Swi5 nuclear import was abrogated, suggesting that nuclear export by Msn5 is important for cell physiology, because it prevents toxic Swi5 nuclear accumulation.
    Biochimica et Biophysica Acta 02/2012; 1823(4):959-70. DOI:10.1016/j.bbamcr.2012.02.009 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Saccharomyces cerevisiae Start repressor Whi5, the functional analogue of mammalian pRB, shuttles between the nucleus and the cytoplasm throughout the cell cycle: enters into the nucleus at the end of mitosis and remains nuclear until Start. We studied the mechanisms involved in this spatial regulation. The nuclear import depends on the beta-karyopherins of the classical import pathway Kap95 and Cse1. Whi5 contains a monopartite and a bipartite classical NLS localized in its N-terminal region which are functionally redundant. A fragment of Whi5 containing these NLSs is able to constitutively accumulate a GFP(4) protein inside the nucleus throughout the cell cycle, which suggests that the Whi5 nuclear import is not cell cycle-regulated. The nuclear export of Whi5 is assisted by beta-karyopherin Msn5. A two-hybrid assay indicates a physical interaction between Whi5 and Msn5. We identified a fragment of Whi5 with export activity from amino acids 51 to 167. Interestingly, this region drives the export of a chimeric nuclear protein in a cell cycle-regulated pattern similarly to that observed for Whi5. Moreover, the nuclear export driven by Whi5(51-167) depends on the phosphorylation of specific Ser residues. Finally, we identified Cdc14 as the phosphatase required for the nuclear accumulation of Whi5.
    Cell cycle (Georgetown, Tex.) 10/2009; 8(18):3010-8. DOI:10.4161/cc.8.18.9621 · 4.57 Impact Factor
Show more