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

Abstract

Activity of Ho, the yeast mating switch endonuclease, is restricted to a narrow time window of the cell cycle. Ho is unstable and despite being a nuclear protein is exported to the cytoplasm for proteasomal degradation. We report here the molecular basis for the highly efficient nuclear import of Ho and the relation between its short half-life and passage through the nucleus. The Ho nuclear import machinery is functionally redundant, being based on two bipartite nuclear localization signals, recognized by four importins of the ribosomal import system. Ho degradation is regulated by the DNA damage response and Ho retained in the cytoplasm is stabilized, implying that Ho acquires its crucial degradation signals in the nucleus. Ho arose by domestication of a fungal VMA1 intein. A comparison of the primary sequences of Ho and fungal VMA1 inteins shows that the Ho nuclear localization signals are highly conserved in all Ho proteins, but are absent from VMA1 inteins. Thus adoption of a highly efficient import strategy occurred very early in the evolution of Ho. This may have been a crucial factor in establishment of homothallism in yeast, and a key event in the rise of the Saccharomyces sensu stricto.Ho endonuclease initiates a mating type switch in Saccharomyces cerevisiae and related yeasts by making a site-specific double strand break in a 24-bp cognate site in the mating type gene, MAT. Repair of the double strand break is by gene conversion using one of the silent cassettes of mating type information (HML␣ or HMRa) as a template. Repair occurs before replication of the MAT locus and each daughter cell has the new mating type with a regenerated Ho cognate site (1). Ho activity is tightly regulated: HO is transcribed briefly at the end of G 1 , its transcription is restricted to haploid mother cells, i.e. cells that have divided at least once (2), and the protein is rapidly degraded by the ubiquitin-26 S proteasome system (3). Cells in which Ho is retained in the nucleus beyond its normal time window of activity show perturbation of the cell cycle (4). Ho is marked for degradation by functions of the DNA damage response (DDR), 7 specifically the MEC1, RAD9, and CHK1 pathway (5). Despite being a nuclear protein, Ho must exit the nucleus to be degraded in the proteasomes. The DDR functions are important for Ho phosphorylation: phosphorylation of threonine 225 is crucial for Ho nuclear export and additional phosphorylations are required for recruitment of Ho for ubiquitylation. Ho is ubiquitylated by the SCF (Skp1-Cdc53-F-box protein) E3 ubiquitin ligase complex, to which it is recruited by the F-box protein Ufo1 (6). In mec1 mutants Ho is stabilized and accumulates in the nucleus; conversely trapping Ho in the nucleus by deletion of its nuclear exportin, Msn5, leads to stabilization of the protein (4). Ddi1 binds ubiquitylated Ho and is required for interaction of Ho with the proteasome; in its absence Ho is stabilized. The finding that Ho is not degraded within the nucleus, but in the cytoplasm, is further strengthe...

The nuclear-encoded inteins of fungi

“…These studies indicate that HO is a domesticated form of VMA intein; HO sequences from diVerent saccharomycetes form a clade nested within the VDEs, suggesting a single origin with no evidence of horizontal transmission (Koufopanou and Burt, 2005;Bakhrat et al, 2006). HO is present in a large clade of saccharomycetes; some of the members of this clade do not have VDE.…”

“…NLS-like regions were not detected in the endonuclease domains of other fungal inteins such as those of the PRP8 proteins (Bakhrat et al, 2006).…”

Nuclear import by karyopherin-βs: Recognition and inhibition

“…First, Kap120p binds and imports the Ho endonuclease through a 13-residue basic NLS in its C-terminal region that is distinct from a second NLS, which is recognized by Kap121p and Kap123p [125]. Another Kap120p cargo Rpf1p is mislocalized only when all three Kap120p, Kap119p and Kap114p are mutated [116].…”

PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae

“…The importin-β pathway (Kap95 in yeast) recognizes the classical NLS through the adaptor protein importin-α (Kap60 in yeast). The β-karyopherins Kap108, Kap120, Kap123, and Kap114 are functionally redundant and act as importins or exportins [24][25][26]. Not only the β-karyopherins can recognize more than one cargo and potentially also more than one NLS, but also one cargo can be recognized by more than one β-karyopherin [27,28].…”