Minoru Funakoshi

Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome

Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2. K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene. These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.

Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability.

Degradation of polyubiquitinated proteins by the proteasome often requires accessory factors; these include receptor proteins that bind both polyubiquitin chains and the regulatory particle of the proteasome. Overproduction of one such factor, Dsk2, is lethal in Saccharomyces cerevisiae and we show here that this lethality can be suppressed by mutations in SEM1, a gene previously recognized as an ortholog of the human gene encoding DSS1, which binds the BRCA2 DNA repair protein. Yeast sem1 mutants accumulate polyubiquitinated proteins, are defective for proteasome-mediated degradation and cannot grow under various stress conditions. Moreover, sem1 is synthetically lethal with mutations in proteasome subunits. We show that Sem1 is a component of the regulatory particle of the proteasome, specifically the lid subcomplex. Loss of Sem1 impairs the stability of the 26S proteasome and sem1Δ defects are greatly enhanced by simultaneous deletion of RPN10. The Rpn10 proteasome subunit appears to function with Sem1 in maintaining the association of the lid and base subcomplexes of the regulatory particle. Our data suggest a potential mechanism for this protein-protein stabilization and also suggest that an intact proteasomal regulatory particle is required for responses to DNA damage.

Identification of XDRP1; a Xenopus protein related to yeast Dsk2p binds to the N-terminus of cyclin A and inhibits its degradation

Using the N‐terminus of cyclin A1 in a two‐hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin‐like domain in its N‐terminus and shows significant homology in its C‐terminal 50 residues to Saccharomyces cerevisiae Dsk2 and Schizosaccharomyces pombe dph1. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A‐dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B‐type cyclins. The N‐terminal ubiquitin‐like domain of XDRP1, but not the C‐terminal Dsk2‐like domain, is required for interaction with cyclin A. XDRP1 requires residues 130–160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A. The addition of bacterially expressed XDRP1 protein to frog egg extract inhibited the Ca2+‐induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division.

Yeast Pth2 is a UBL domain‐binding protein that participates in the ubiquitin–proteasome pathway

Ubiquitin‐like (UBL)‐ubiquitin‐associated (UBA) proteins such as Rad23 and Dsk2 mediate the delivery of polyubiquitinated proteins to the proteasome in the ubiquitin–proteasome pathway. We show here that budding yeast peptidyl‐tRNA hydrolase 2 (Pth2), which was previously recognized as a peptidyl‐tRNA hydrolase, is a UBL domain‐binding protein that participates in the ubiquitin–proteasome pathway. Pth2 bound to the UBL domain of both Rad23 and Dsk2. Pth2 also interacted with polyubiquitinated proteins through the UBA domains of Rad23 and Dsk2. Pth2 overexpression caused an accumulation of polyubiquitinated proteins and inhibited the growth of yeast. Ubiquitin‐dependent degradation was accelerated in the pth2Δ mutant and was retarded by overexpression of Pth2. Pth2 inhibited the interaction of Rad23 and Dsk2 with the polyubiquitin receptors Rpn1 and Rpn10 on the proteasome. Furthermore, Pth2 function involving UBL‐UBA proteins was independent of its peptidyl‐tRNA hydrolase activity. These results suggest that Pth2 negatively regulates the UBL‐UBA protein‐mediated shuttling pathway in the ubiquitin–proteasome system.

Xenopus cyclin A1 can associate with Cdc28 in budding yeast, causing cell‐cycle arrest with an abnormal distribution of nuclear DNA

Cyclins play a regulatory role in cell cycle progression, associated with cyclin‐dependent kinases. We have investigated the structure–function relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae.

Amino acid residues required for Gtr1p-Gtr2p complex formation and its interactions with the Ego1p-Ego3p complex and TORC1 components in yeast

The yeast Ras‐like GTPases Gtr1p and Gtr2p form a heterodimer, are implicated in the regulation of TOR complex 1 (TORC1) and play pivotal roles in cell growth. Gtr1p and Gtr2p bind Ego1p and Ego3p, which are tethered to the endosomal and vacuolar membranes where TORC1 functions are regulated through a relay of amino acid signaling interactions. The mechanisms by which Gtr1p and Gtr2p activate TORC1 remain obscure. We probed the interactions of the Gtr1p‐Gtr2p complex with the Ego1p‐Ego3p complex and TORC1 subunits. Mutations in the region (179–220 a.a.) following the nucleotide‐binding region of Gtr1p and Gtr2p abrogated their mutual interaction and resulted in a loss in function, suggesting that complex formation between Gtr1p and Gtr2p was indispensable for TORC1 function. A modified yeast two‐hybrid assay showed that Gtr1p‐Gtr2p complex formation is important for its interaction with the Ego1p‐Ego3p complex. GTP‐bound Gtr1p interacted with the region containing the HEAT repeats of Kog1p and the C‐terminal region of Tco89p. The GTP‐bound Gtr2p suppressed a Kog1p mutation. Our findings indicate that the interactions of the Gtr1p‐Gtr2p complex with the Ego1p‐Ego3p complex and TORC1 components Kog1p and Tco89p play a role in TORC1 function.

Isolation and characterisation of a mutation in the PMR1 gene encoding a Golgi membrane ATPase, which causes hypersensitivity to over-expression of Clb3 in Saccharomyces cerevisiae

Abstract We screened for mutant strains of Saccharomyces cerevisiae that are sensitive to overexpression of specific cyclins, and identified mutations in two genes that caused growth inhibition in response to mild over-expression of Clb3. One was the ANP1 gene, which encodes a glycosyltransferase previously identified by a similar strategy using Clb2 instead of Clb3. This paper describes the second strain of S. cerevisiae that is hypersensitive to Clb3 expression. The gene mutated in this strain was identified as PMR1, which encodes a Ca2+-ATPase located in the Golgi membrane. The protein product of pmr1-1 was truncated at residue 409 and thus lacked the C-terminal ATPase domain. The pmr1-1 strain was hypersensitive to over-expression of Clb3, but not Cln2, Clb5 or Clb2. The lethality due to Clb3 expression in pmr1-1 could be suppressed by adding Ca2+ ions to the medium. The pmr1-1 strain proved to be defective in glycosylation, and the defects in glycosylation were exacerbated by high levels of Clb3. On induction of Clb3 expression in the pmr1-1 strain, the cells arrested at anaphase with an elongated daughter bud. We discuss possible interpretations of this synthetic lethal phenotype.

Ubiquitin chains in the Dsk2 UBL domain mediate Dsk2 stability and protein degradation in yeast

Ubiquitin-like (UBL)-ubiquitin-associated (UBA) proteins, including Dsk2 and Rad23, act as delivery factors that target polyubiquitinated substrates to the proteasome. We report here that the Dsk2 UBL domain is ubiquitinated in yeast cells and that Dsk2 ubiquitination of the UBL domain is involved in Dsk2 stability, depending on the Dsk2 UBA domain. Also, Dsk2 lacking ubiquitin chains impaired ubiquitin-dependent protein degradation and decreased the interaction of Dsk2 with polyubiquitinated proteins in cells. Moreover, Dsk2 ubiquitination affected ability to restore the temperature-sensitive growth defect of dsk2Δ. These results indicate that ubiquitination in the UBL domain of Dsk2 has in vivo functions in the ubiquitin-proteasome pathway in yeast.

Yeast Irc22 Is a Novel Dsk2-Interacting Protein that Is Involved in Salt Tolerance.

The yeast ubiquitin-like and ubiquitin-associated protein Dsk2 is one of the ubiquitin receptors that function in the ubiquitin-proteasome pathway. We screened the Dsk2-interacting proteins in Saccharomyces cerevisiae by a two-hybrid assay and identified a novel Dsk2-interacting protein, Irc22, the gene locus of which has previously been described as YEL001C, but the function of which is unknown. IRC22/YEL001C encodes 225 amino acid residues with a calculated molecular weight of 25 kDa. The Irc22 protein was detected in yeast cells. IRC22 was a nonessential gene for yeast growth, and its homologs were found among ascomycetous yeasts. Irc22 interacted with Dsk2 in yeast cells, but not with Rad23 and Ddi1. Ubiquitin-dependent degradation was impaired mildly by over-expression or disruption of IRC22. Compared with the wild-type strain, dsk2Δ exhibited salt sensitivity while irc22Δ exhibited salt tolerance at high temperatures. The salt-tolerant phenotype that was observed in irc22Δ disappeared in the dsk2Δirc22Δ double disruptant, indicating that DSK2 is positively and IRC22 is negatively involved in salt stress tolerance. IRC22 disruption did not affect any responses to DNA damage and oxidative stress when comparing the irc22Δ and wild-type strains. Collectively, these results suggest that Dsk2 and Irc22 are involved in salt stress tolerance in yeast.