Knocking out ubiquitin proteasome system function in vivo and in vitro with genetically encodable tandem ubiquitin.

At present, the 26S proteasome-specific inhibitor is not available. We constructed polyubiquitin derivatives that contained a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. When these artificial polyubiquitins (tUbs, tandem ubiquitins) were overproduced in the wild-type yeast strain, growth was strongly inhibited, probably because of inhibition of the 26S proteasome. We also found that several substrates of the ubiquitin-proteasome pathway were stabilized by expressing tUbs in vivo. tUbs containing four units or more of the ubiquitin monomer were found to form a complex with the 26S proteasome. We showed that tUb bound to the 26S proteasome inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 (six-mer) messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.

Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity.

For analysis of the mechanism of the 26S proteasome-mediated protein degradation in vitro, the preparation of well-defined substrate, the ubiquitinated proteins, of the 26S proteasome is inevitable. However, no method has been available to ubiquitinate a given protein. Here, we propose a relatively simple method for preparation of the ubiquitinated substrates using HECT-type ubiquitin ligase Rsp5, termed the PY motif-insertion method. The principle of this method is that the PY motif, known as the Rsp5-binding motif, is inserted into protein to be ubiquitinated by Rsp5. In this communication, we describe that Sic1 was successfully ubiquitinated by the PY motif-insertion method and demonstrate that Sic1 thus ubiquitinated was degraded by the purified yeast 26S proteasome.

A novel factor required for the SUMO1/Smt3 conjugation of yeast septins.

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to be conjugated to other proteins and modulate their functions in various important processes. Similar to the ubiquitin system, SUMO1/Smt3 is activated in an ATP-dependent reaction by thioester bond formation with E1 (activating enzyme), transferred to E2 (conjugating enzyme), and passed to a substrate lysine. It remained unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in the final transfer of this modifier. Here we report a novel factor Siz1 (YDR409w) required for septin-sumoylation of budding yeast, possibly acting as E3. Siz1 is a member of a new family (Miz1, PIAS3, etc.) containing a conserved domain with a similarity to a zinc-binding RING-domain, often found in ubiquitin ligases. In the siz1 mutant septin-sumoylation was completely abolished. A conserved cysteine residue in the domain was essential for this conjugation. Furthermore, Siz1 was localized at the mother-bud neck in the M-phase and physically bound to both E2 and the target proteins.

Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates.

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RING-like domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1.Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.

Regulation of the localization of Dbf2 and mob1 during cell division of saccharomyces cerevisiae.

The mitotic exit network (MEN) governs Cdk inactivation. In budding yeast, MEN consists of the protein phosphatase Cdc14, the ras-like GTPase Tem1, protein kinases Cdc15, Cdc5, Dbf2 and Dbf2-binding protein Mob1. Tem1, Dbf2, Cdc5 and Cdc15 have been reported to be localized at the spindle pole body (SPB). Here we report changes of the localization of Dbf2 and Mob1 during cell division. Dbf2 and Mob1 localize to the SPBs in anaphase and then moves to the bud neck, just prior to actin ring assembly, consistent with their role in cytokinesis. The neck localization, but not SPB localization, of Dbf2 was inhibited by the Bub2 spindle checkpoint. Cdc14 is the downstream target of Dbf2 in Cdk inactivation, but we found that the neck localization of DbP2 and Mob1 was dependent on the Cdc14 activity, suggesting that Dbf2 and Mob1 function in cytokinesis at the end of the mitotic signaling cascade.

Genetic dissection of the yeast 26S proteasome: cell cycle defects caused by the Deltarpn9 mutation.

Rpn9 is one of the subunits of the regulatory particle of the yeast 26S proteasome and is needed for stability or efficient assembly of the 26S proteasome. As anticipated from the fact that the rpn9 disruptant grew at 25 degrees C but arrested in G2/M phase at 37 degrees C, the CDK inhibitor Sic1p was found to be degraded at the G1/S boundary in the Deltarpn9 cells. The degradation of the anaphase inhibitor Pds1p was delayed in the Deltarpn9 cells. Clb2p in M phase, as well as that ectopically expressed in G1 and S phases, was degraded more slowly in the Deltarpn9 cells than in the wild type cells, indicating that the 26S proteasome lacking Rpn9 uses Sic1p as a better substrate than Pds1p and Clb2p. These results, in addition to the fact that multiubiquitinated proteins were accumulated in the Deltarpn9 cells incubated at 37 degrees C, strongly suggest that Rpn9 is involved in the proteolysis of a subset of the substrates degraded by the 26S proteasome. The Deltarpn9 Deltapds1 double mutant was unable to elongate spindle at a restrictive temperature, suggesting that some protein(s) other than Scc1 (cohesin) should be degraded during progression of anaphase.

Pho85 kinase, a yeast cyclin-dependent kinase, regulates the expression of UGP1 encoding UDP-glucose pyrophosphorylase.

The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85) highly homologous to the Cdc28 kinase (Cdc28). Ten cyclin-like proteins are known to interact with Pho85, and combination with different cyclins is believed to be responsible for distinct Pho85 functions, including phosphate metabolism, carbon source utilization and cell cycle regulation. However, only a limited number of substrates of Pho85 kinase, including Pho4, Gsy2 and Sicl, have so far been identified. To search for more targets of Pho85 and to clarify the genetic control mechanisms by Pho85 kinase in these cellular functions, we carried out a genome-wide analysis of the effect of a pho85Delta mutation on gene expression. We found that expression of various genes involved in carbon metabolism are affected by the mutation and that among them, UGP1 promoter activity was increased in the absence of Pho85 kinase. This increase in the promoter activity was not observed in a pho4Delta mutant or with a mutant UGP1 promoter that is devoid of putative Pho4 and Bas2 binding sites, suggesting that UGP1 expression is modulated by Pho85 through Pho4. We also found that expression of several Pho85-cyclin genes were altered by the carbon source, the growth phase and Pho85 kinase itself.

Nis1 encoded by YNL078W: a new neck protein of Saccharomyces cerevisiae.

An uncharacterized gene, YNL078W, was isolated by the two-hybrid screening method using SHS1 (one of the septin genes) as bait and designated NIS1 (Neck protein Interacting with Septins). Nis1 interacts with all septins in the two-hybrid assay system. Physical interaction between Nis1 and Shs1 in vivo was confirmed by a co-immunoprecipitation experiment. Neither disruption nor overexpression of NIS1 caused a prominent phenotypic change. NAP1 was isolated by two-hybrid screening using NIS1 as bait. We detected physical interaction between Nis1 and Nap1 in vivo by a co-immunoprecipitation experiment. Nis1 was found to bind Gin4 and Kcc4 in the two-hybrid assay. Thus, a number of the proteins interacting with Nis1 are members of the mitotic signaling network. The stable maintenance of Nis1 was dependent on Nap1. Nis1 was phosphorylated throughout the cell cycle and was less abundant in G2/M phase. GFP-Nis1 is localized in the nucleus throughout the cell cycle and in the bud neck at G2/M phase in a septin-dependent manner. Altogether, the findings suggest that Nis1 may play a non-essential role in the mitotic signaling network.

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae.

Las21/Gpi7 contains a heavy-metal-associated motif at its N-terminus. When this motif was disrupted by amino acid substitution, the cells acquired weak copper-resistance. We found that the previously isolated las21 mutants were strongly resistant to copper. Metallothionein is necessary for the expression of the copper-resistance of the las21 mutants. However, hyper-production of metallothionein is unlikely to be the cause of copper-resistance of the las21 mutants. Copper-sensitive mutants (collectively called Cus mutants) were isolated from the las21delta and characterized. One of the Cus genes was found to be PBS2, which encodes Hog1 MAP kinase kinase, indicating that the Hog1 MAP kinase pathway is needed for the expression of copper-resistance of the las21 mutants. As expected, the las21delta hog1delta strain was no longer copper-resistant. We found that Hog1 was constitutively activated in las21delta cells and in ssk1delta las21delta cells but not in sho1delta las21delta cells. Inactivation of either FSR2/MCD4 or MPC1/GPI13, both of which are involved in GPI anchor synthesis, like LAS21, caused a similar level of constitutive activation of Hog1 kinase and copper-resistance as found in the las21delta strain. The constitutive activation was canceled by introducing the sskl mutation, but not the sho1 mutation, in each GPI anchor mutant tested, suggesting that the defect in GPI anchor synthesis specifically affects the Slnl branch of the MAP kinase pathway. Since the wild-type cells grown in YPD containing 0.5 M NaCl do not show copper-resistance, mere activation of Hog1 is not sufficient for expression of copper-resistance. We propose that a defect in GPI anchor synthesis has multiple consequences, including activation of the Hog1 MAP kinase cascade and conferring copper-resistance.

Yeast Ulp1, an Smt3-specific protease, associates with nucleoporins.

Yeast Smt3 is a ubiquitin-like protein similar to the mammalian SUMO-1. Cdc3, a septin component, is known to be modified by Smt3. The level of this modification was affected by Smt3-specific protease mutation ulp1-ts or overexpression of ULP1. By two-hybrid screening, we isolated 5 UIP (Ulp1 interacting protein) genes. UIP1 was identical to NUP42 encoding a component of the nuclear pore complex (NPC). Gle1, another NPC-associating component, also interacted with Ulp1 in the two-hybrid system and co-immunoprecipitation experiment. Thus Ulp1 associates with nucleoporins and may interact with septin rings in the telophase.

Yeast Krr1p physically and functionally interacts with a novel essential Kri1p, and both proteins are required for 40S ribosome biogenesis in the nucleolus.

Using a two-hybrid screening with TOM1, a putative ubiquitin-ligase gene of Saccharomyces cerevisiae, we isolated KRR1, a homologue of human HRB2 (for human immunodeficiency virus type 1 Rev-binding protein 2). To characterize the gene function, we constructed temperature-sensitive krr1 mutants and isolated two multicopy suppressors. One suppressor is RPS14A, encoding a 40S ribosomal protein. The C-terminal-truncated rpS14p, which was reported to have diminished binding activity to 18S rRNA, failed to suppress the krr1 mutant. The other suppressor is a novel gene, KRI1 (for KRR1 interacting protein; YNL308c). KRI1 is essential for viability, and Kri1p is localized to the nucleolus. We constructed a galactose-dependent kri1 strain by placing KRI1 under control of the GAL1 promoter, so that expression of KRI1 was shut off when transferring the culture to glucose medium. Polysome and 40S ribosome fractions were severely decreased in the krr1 mutant and Kri1p-depleted cells. Pulse-chase analysis of newly synthesized rRNAs demonstrated that 18S rRNA is not produced in either mutant. However, wild-type levels of 25S rRNA are made in either mutant. Northern analysis revealed that the steady-state levels of 18S rRNA and 20S pre-rRNAs were reduced in both mutants. Precursors for 18S rRNA were detected but probably very unstable in both mutants. A myc-tagged Kri1p coimmunoprecipitated with a hemagglutinin-tagged Krr1p. Furthermore, the krr1 mutant protein was defective in its interaction with Kri1p. These data lead us to conclude that Krr1p physically and functionally interacts with Kri1p to form a complex which is required for 40S ribosome biogenesis in the nucleolus.

Yeast Pdr13p and Zuo1p molecular chaperones are new functional Hsp70 and Hsp40 partners.

The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae. The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses. We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant. ZUO1 encodes a DnaJ-related Hsp40. Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation. The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant. A zuo1 pdr13 double deletion mutant did not show additive phenotypes. Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p. Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones. In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p.

A network of proteins around Rvs167p and Rvs161p, two proteins related to the yeast actin cytoskeleton.

The Rvs161p and Rvs167p proteins of Saccharomyces cerevisiae, homologues of higher eukaryotes' amphiphysins, associate with actin and appear to be involved in several functions related to the actin cytoskeleton. In order to identify partners of the Rvsp proteins, yeast libraries constructed in two-hybrid vectors were screened using either Rvs167p or Rvs161p as a bait. The selected candidates, representing 34 ORFs, were then tested against both Rvsp proteins, as well as domains of Rvs167p or Rvs161p. Among the most significant ones, 24 ORFs were specific preys of Rvs167p only and two gave interactions with Rvs161p only. Interestingly, five ORFs were preys of both Rvs161p and Rvs167p (RVS167, LAS17, YNL094w, YMR192w and YPL249c). Analysis of putative functions of the candidates confirm involvement of the Rvsp in endocytosis/vesicle traffic, but also opens possible new fields, such as nuclear functions.

Ebp2p, yeast homologue of a human protein that interacts with Epstein-Barr virus nuclear antigen 1, is required for pre-rRNA processing and ribosomal subunit assembly.

BACKGROUND: A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response. RESULTS: EBP2, encoding the yeast homologue of a human protein that interacts with Epstein-Barr virus Nuclear Antigen 1, was cloned in a two-hybrid screen using RRS1 as a bait. The rrs1-1 mutation, which produces Rrs1p without the C-terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p-depleted cells, the rate of pre-rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild-type cells. The polysome pattern indicates that Ebp2p-depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half-mer polysomes. CONCLUSIONS: Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.

Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast.

A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.

Rapid isolation and characterization of the yeast proteasome regulatory complex.

The 26S proteasome, which catalyzes degradation of ubiquitinated proteins, is composed of the 20S proteasome and the 19S complex. Recently, it has been reported that the 26S complex can be dissociated into the lid complex and the 20S-proteasome-base complex in a mutant yeast and that the lid complex is required for ubiquitin-dependent proteolysis. In the present study, we established methods for rapid isolation of the 19S complex, the lid complex, and the base complex from wild-type yeast. The isolated 19S complex was capable of binding to the 20S proteasome to reconstitute the 26S proteasome. In contrast with the previously reported result showing that Rpn10, a multiubiquitin chain binding subunit, is a component of the base complex, we present evidence that the lid complex isolated from wild-type yeast contains Rpn10.

Nob1p, a new essential protein, associates with the 26S proteasome of growing saccharomyces cerevisiae cells.

Nob1p, which interacts with Nin1p/Rpn12, a subunit of the 19S regulatory particle (RP) of the yeast 26S proteasome, has been identified by two-hybrid screening. NOB1 was found to be an essential gene, encoding a protein of 459 amino acid residues. Nob1p was detected in growing cells but not in cells in the stationary phase. During the transition to the stationary phase, Nob1p was degraded, at least in part, by the 26S proteasome. Nob1p was found only in proteasomal fractions in a glycerol gradient centrifugation profile and immuno-coprecipitated with Rpt1, which is an ATPase component of the yeast proteasomes. These results suggest that association of Nob1p with the proteasomes is essential for the function of the proteasomes in growing cells.

Extragenic suppressors that rescue defects in the heat stress response of the budding yeast mutant tom1.

The TOM1 gene codes for a so-called HECT protein, a putative ubiquitin ligase, in Saccharomyces cerevisiae. Deletion of the entire gene (tom1-10) or the sequence encoding the HECT domain (tom1-2) causes temperature sensitivity for growth. Here we report the isolation of extragenic, recessive suppressors of tom1-2, which were designated tmr (for tom1 revertant) mutations. These were classified into eight complementation groups and six of the genes were identified: tmr1/cyr1, tmnr2/sch9, tmr3/zuo1, tmr4, tmr5/mot1, tmr6/sse1, tmr7 and tmr8/kre6. These results suggested that the tom1 phenotype can be rescued by down-regulating the cAMP/PKA pathway. It was found that the temperature sensitivity of the tom1-2 mutant is indeed suppressed by multiple copies of PDE2 or BCY1, which encode negative regulators of the cAMP/PKA pathway. The MSN2 gene, which encodes a zinc-finger transcription factor involved in the general stress response is also a multicopy suppressor of tom1. It was found that induction levels of both STRE-mediated (general stress response) and HSE-mediated gene expression (heat shock response) upon shift to high temperature are reduced by more than half in the tom1 mutant. Most of the isolated tmr mutations rescued one of the defects seen in both types of heat stress response in the tom1 mutant.

Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase.

Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.

Rpn9 is required for efficient assembly of the yeast 26S proteasome.

We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Deltarpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Deltarpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Deltarpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Deltarpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome.