Ho endonuclease initiates a mating type switch by making a double-strand break at the mating type locus, MAT. Ho is marked by phosphorylation for rapid destruction by functions of the DNA damage response, MEC1, RAD9, and CHK1. Phosphorylated Ho is recruited for ubiquitylation via the SCF ubiquitin ligase complex by the F-box protein, Ufo1. Here we identify a further DNA damage-inducible protein, the UbL-UbA protein Ddi1, specifically required for Ho degradation. Ho interacts only with Ddi1; it does not interact with the other UbL-UbA proteins, Rad23 or Dsk2. Ho must be ubiquitylated to interact with Ddi1, and there is no interaction when Ho is produced in mec1 or ⌬ufo1 mutants that do not support its degradation. Ddi1 binds the proteasome via its N-terminal ubiquitinlike domain (UbL) and interacts with ubiquitylated Ho via its ubiquitin-associated domain (UbA); both domains of Ddi1 are required for association of ubiquitylated Ho with the proteasome. Despite being a nuclear protein, Ho is exported to the cytoplasm for degradation. In the absence of Ddi1, ubiquitylated Ho is stabilized and accumulates in the cytoplasm. These results establish a role for Ddi1 in the degradation of a natural ubiquitylated substrate. The specific interaction between Ho and Ddi1 identifies an additional function associated with DNA damage involved in its degradation.

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

The F-box protein, Ufo1, recruits Ho endonuclease to the SCFUfo1 complex for ubiquitylation. Both ubiquitylated Ho and Ufo1 are transferred by the UbL-UbA protein, Ddi1, to the 19S Regulatory Particle (RP) of the proteasome for degradation. The Ddi1-UbL domain binds Rpn1 of the 19S RP, the Ddi1-UbA domain binds ubiquitin chains on the degradation substrate. Here we used complex reconstitution in vitro to identify stages in the transfer of Ho and Ufo1 from the SCFUfo1 complex to the proteasome. We report SCFUfo1 complex at the proteasome formed in the presence of Ho. Subsequently Ddi1 is recruited to this complex by interaction between the Ddi1-UbL domain and Ufo1. The core of Ddi1 binds both Ufo1 and Rpn1; this interaction confers specificity of SCFUfo1 for Ddi1. The substrate-shield model predicts that Ho would protect Ufo1 from degradation and we find that Ddi1 binds Ho, Ufo1, and Rpn1 simultaneously forming a complex for transfer of Ho to the 19S RP. In contrast, in the absence of Ho, Rpn1 displaces Ufo1 from Ddi1 indicating a higher affinity of the Ddi1-UbL for the 19S RP. However, at high Rpn1 levels there is synergistic binding of Ufo1 to Ddi1 that is dependent on the Ddi1-UbA domain. Our interpretation is that in the absence of substrate, the Ddi1-UbL binds Rpn1 while the Ddi1-UbA binds ubiquitin chains on Ufo1. This would promote degradation of Ufo1 and disassembly of SCFUfo1 complexes.

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...

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.