Biomedical subjects

Dina Raveh

Publications and source records attributed to Dina Raveh.

8 recordsLinked to original sources

PubMed2006

Nuclear import of ho endonuclease utilizes two nuclear localization signals and four importins of the ribosomal import system.

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.

Active Transport, Cell Nucleus↗

PubMed2006

Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 complexes.

SCF complexes are E3 ubiquitin-protein ligases that mediate degradation of regulatory and signaling proteins and control G1/S cell cycle progression by degradation of G1 cyclins and the cyclin-dependent kinase inhibitor, Sic1. Interchangeable F-box proteins bind the core SCF components; each recruits a specific subset of substrates for ubiquitylation. The F-box proteins themselves are rapidly turned over by autoubiquitylation, allowing rapid recycling of SCF complexes. Here we report a role for the UbL-UbA protein Ddi1 in the turnover of the F-box protein, Ufo1. Ufo1 is unique among F-box proteins in having a domain comprising multiple ubiquitin-interacting motifs (UIMs) that mediate its turnover. Deleting the UIMs leads to stabilization of Ufo1 and to cell cycle arrest at G1/S of cells with long buds resembling skp1 mutants. Cells accumulate substrates of other F-box proteins, indicating that the SCF pathway of substrate ubiquitylation is inhibited. Ufo1 interacts with Ddi1 via its UIMs, and Deltaddi1 cells arrest when full-length UFO1 is overexpressed. These results imply a role for the UIMs in turnover of SCF(Ufo1) complexes that is dependent on Ddi1, a novel activity for an UbL-UbA protein.

Amino Acid Motifs↗

PubMed2006

The F-box protein, Ufo1, maintains genome stability by recruiting the yeast mating switch endonuclease, Ho, for rapid proteasome degradation.

We describe a unique E3, the F-box protein, Ufo1, of yeast. Ufo1 recruits the mating switch endonuclease, Ho, to the SCF complex for ubiquitylation. In addition to the F-box and WD40 protein-protein interaction domains found in all F-box proteins, Ufo1 has a unique domain comprising multiple copies of the ubiquitin-interacting motif. Ufo1 interacts with the UbL-UbA protein, Ddi1, via its UIMs, and this is required for turnover of SCFUfo1 complexes. This is a novel function for an UbL-UbA protein. Deletion of the genomic UFO1UIMs is lethal and our data indicate that Ufo1deltaUIM acts as a dominant negative leading to inhibition of the SCF pathway of substrate degradation and to cell cycle arrest. Furthermore, we found that Ddi1 is required for the final stages of degradation of Ho endonuclease. In the absence of Ddi1, Ho does not form a complex with the 19S RP and is stabilized. Stabilization of Ho leads to perturbation of the cell cycle and to the formation of multi-budded cells. Our experiments uncover a novel role for the ubiquitin-proteasome system in maintenance of genome stability.

Cell Cycle↗

PubMed2005

Proteasome plasticity.

The 26S proteasome is responsible for regulated proteolysis of most intracellular proteins yet the focus of intense regulatory action itself. Proteasome abundance is responsive to cell needs or stress conditions, and dynamically localized to concentrations of substrates. Proteasomes are continually assembled and disassembled, and their subunits subject to a variety of posttranslational modifications. Furthermore, as robust and multi-tasking as this complex is, it does not function alone. A spattering of closely associating proteins enhances complex stability, fine-tunes activity, assists in substrate-binding, recycling of ubiquitin, and more. HEAT repeat caps activate proteasomes, yet share remarkable features with nuclear importins. Fascinating cross talk even occurs with ribosomes through common maturation factors. The dynamics of proteasome configurations and how they relate to diverse activities is the topic of this review.

Animals↗

PubMed2005

The DNA damage-inducible UbL-UbA protein Ddi1 participates in Mec1-mediated degradation of Ho endonuclease.

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 Deltaufo1 mutants that do not support its degradation. Ddi1 binds the proteasome via its N-terminal ubiquitin-like 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.

Cytoplasm↗

PubMed2004

Homology modeling and mutational analysis of Ho endonuclease of yeast.

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.

Amino Acid Sequence↗

PubMed2003

DNA damage response-mediated degradation of Ho endonuclease via the ubiquitin system involves its nuclear export.

Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1. A PEST sequence marks Ho for degradation. Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation. Mutation of PEST residue threonine 225 stabilizes Ho, yet HoT225A still binds Ufo1 in vitro. Stable HoT225A accumulates within the nucleus, whereas HoT225E is degraded. Deletion of the nuclear exportin Msn5 traps native Ho in the nucleus and extends its half-life. These experiments suggest that Ho is degraded in the cytoplasm. In mec1 mutants stable Ho accumulates within the nucleus; Ho produced in mec1 cells does not bind Ufo1. Thus the MEC1 pathway has functions both in phosphorylation of Thr-225 for nuclear export and in additional phosphorylations for binding Ufo1. Cells with HO under its genomic promoter, but stabilized by deletion of the Msn5 exportin, proliferate, but are multibudded. These experiments elucidate some of the links between the DNA damage response and degradation of Ho by the ubiquitin system.

Base Sequence↗

PubMed2002

Stable chloroplast transformation of the unicellular red alga Porphyridium species.

Red algae are extremely attractive for biotechnology because they synthesize accessory photosynthetic pigments (phycobilins and carotenoids), unsaturated fatty acids, and unique cell wall sulfated polysaccharides. We report a high-efficiency chloroplast transformation system for the unicellular red microalga Porphyridium sp. This is the first genetic transformation system for Rhodophytes and is based on use of a mutant form of the gene encoding acetohydroxyacid synthase [AHAS(W492S)] as a dominant selectable marker. AHAS is the target enzyme of the herbicide sulfometuron methyl, which effectively inhibits growth of bacteria, fungi, plants, and algae. Biolistic transformation of synchronized Porphyridium sp. cells with the mutant AHAS(W492S) gene that confers herbicide resistance gave a high frequency of sulfomethuron methyl-resistant colonies. The mutant AHAS gene integrated into the chloroplast genome by homologous recombination. This system paves the way for expression of foreign genes in red algae and has important biotechnological implications.

Acetolactate Synthase↗