A NosA-specific bacteriophage can be used to select denitrification-defective mutants of Pseudomonas stutzeri

phi PS5, a double-stranded DNA bacteriophage of Pseudomonas stutzeri JM604 that adsorbs specifically to the outer-membrane protein NosA, was isolated from stagnant irrigation ditch water. Mutant strains that do not produce NosA are resistant to phi PS5 and cannot grow anaerobically with N2O as the sole electron acceptor. phi PS5 did not adsorb to nosA mutants and adsorption to the wild-type strain was reduced when cells were grown with a high concentration of copper, a condition that represses the synthesis of NosA. The isolation of spontaneous phi PS5-resistant mutants yielded strains that were clearly defective in growth on N2O at about a 10% incidence. About half of these strains could respire N2O when supplied with a high concentration of exogenous copper.     

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.     

Requirements for Cu(A) and Cu-S center assembly of nitrous oxide reductase deduced from complete periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida

Bacterial nitrous oxide (N(2)O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N(2)O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu(A) site in the periplasm. There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco. We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida. An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N(2)O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N(2)O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu(A) metallation. Both of these were found to be dispensable elements for N(2)O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N(2)O reductase maturation.     

Budding yeast Dsk2 protein forms a homodimer via its C-terminal UBA domain

Budding yeast Dsk2 is a family of UbL-UBA proteins that can interact with both polyubiquitin and the proteasome, and is thereby thought to function as a shuttle protein in the ubiquitin-proteasome pathway. Here we show that Dsk2 can homodimerize via its C-terminal UBA domain in the absence of ubiquitin. Dsk2 mutants defective in the UBA domain do not dimerize and do not bind polyubiquitin. The expression of Dsk2 UBA mutants fails to restore the growth defect caused by DSK2 disruption although that of wild-type Dsk2 can restore the defect. These results suggest that Dsk2 homodimerization via the UBA domain plays a role in regulating polyubiquitin binding in the ubiquitin-proteasome pathway.     

Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.     

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 ubiquitin-like genes are involved in duplication of the microtubule organizing center

KAR1 is required for duplication of the Saccharomyces cerevisiae microtubule organizing center, the spindle pole body (SPB) (Rose, M.D., and G.R. Fink, 1987. Cell. 48:1047-1060). Suppressors of a kar1 allele defective for SPB duplication were isolated in two genes, CDC31 and DSK2 (Vallen, E.A., W.H., M. Winey, and M.D. Rose. 1994. Genetics. 137:407-422). To elucidate the role of DSK2 in SPB duplication, we cloned the gene and found it encodes a novel ubiquitin-like protein containing an NH2 terminus 36% identical to ubiquitin. The only other known yeast ubiquitin-like protein is encoded by the nucleotide excision repair gene RAD23 (Watkins, J.F.,P. Sung, L. Prakash, and S. Prakash. 1993. Mol. Cell. Bio. 13:7757-7765). Unlike ubiquitin, the NH2- terminal domain of Dsk2p is not cleaved from the protein, indicating that Dsk2p is not conjugated to other proteins. Although the DSK2-1 mutation alters a conserved residue in the Dsk2p ubiquitin-like domain, we detect no differences in Dsk2p or Cdc31p stability. Therefore, DSK2 does not act by interfering with ubiquitin-dependent protein degradation of these proteins. Although DSK2 is not essential, a strain deleted for both DSK2 and RAD23 is temperature sensitive for growth due to a block in SPB duplication. In addition, overexpression of DSK2 is toxic, and the DSK2-1 allele causes a block in SPB duplication. Therefore, DSK2 dosage is critical for SPB duplication. We determined that CDC31 gene function is downstream of DSK2 and KAR1. Dsk2p is a nuclear-enriched protein, and we propose that Dsk2p assists in Cdc31 assembly into the new SPB.     

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 Ca(2+)-induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division.     

Interacting factors and cellular localization of SR protein-specific kinase Dsk1

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A)(+) RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G(2) phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.     

A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation

We developed a growth test to screen for yeast mutants defective in endoplasmic reticulum (ER) quality control and associated protein degradation (ERAD) using the membrane protein CTL*, a chimeric derivative of the classical ER degradation substrate CPY*. In a genomic screen of approximately 5,000 viable yeast deletion mutants, we identified genes necessary for ER quality control and degradation. Among the new gene products, we identified Dsk2p and Rad23p. We show that these two proteins are probably delivery factors for ubiquitinated ER substrates to the proteasome, following their removal from the membrane via the Cdc48-Ufd1-Npl4p complex. In contrast to the ERAD substrate CTG*, proteasomal degradation of a cytosolic CPY*-GFP fusion is not dependent on Dsk2p and Rad23p, indicating pathway specificity for both proteins. We propose that, in certain degradation pathways, Dsk2p, Rad23p and the trimeric Cdc48 complex function together in the delivery of ubiquitinated proteins to the proteasome, avoiding malfolded protein aggregates in the cytoplasm.     

Identification of two isoforms of Dsk2-related protein XDRP1 in Xenopus eggs

The budding yeast UbL-UBA protein Dsk2 has a UbL domain at its N-terminus and a UBA domain at its C-terminus, and thus functions as a shuttle protein in the ubiquitin-proteasome pathway. In this report we describe two isoforms of Xenopus Dsk2-related protein, XDRP1L and XDRP1S. Difference of the two proteins in sequence was that the UbL domain of XDRP1S lacks 15 residues in the middle part of that of XDRP1L. Both XDRP1L and XDRP1S were expressed in Xenopus eggs. XDRP1L and XDRP1S bound to polyubiquitinated proteins via their UBA domains. XDRP1L also bound to the proteasome via its UbL domain, whereas the XDRP1S UbL domain was less likely to bind to the proteasome. Instead, XDRP1S not XDRP1L bound to monomeric cyclin A and prevented its degradation. The existence of such Dsk2-isoforms in Xenopus eggs suggests that the shuttling function via the UbL-UBA protein Dsk2 is evolutionally conserved across species.     

Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23.

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.     

Solution structure of family 21 carbohydrate-binding module from Rhizopus oryzae glucoamylase

Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1(+): Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.     

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.     

A Cdc2-sensitive interaction of the UbL domain of XDRP1S with cyclin B mediates the degradation of cyclin B in Xenopus egg extracts

The yeast UbL-UBA protein Dsk2 is thought to act as a shuttle protein that delivers polyubiquitinated proteins to the proteasome. Previously, we identified Xenopus Dsk2-related protein, XDRP1, as a cyclin A-interacting protein. Using Xenopus egg extracts, we further characterized its two isoforms, XDRP1L and XDRP1S, with respect to cyclin binding and its degradation. Polyubiquitinated cyclins bound to the UBA domain of XDRP1L and XDRP1S, whereas monomeric cyclins A and B bound to the UbL domain of XDRP1S but not to XDRP1L. Binding of XDRP1S with monomeric cyclins was affected by a Cdc2-mediated phosphorylation of either the XDRP1S UbL domain or cyclins. Degradation of cyclin B was also prevented by XDRP1S in a Cdc2-sensitive manner. Loss of the XDRP1S-cyclin interaction allowed cyclins to be degraded in calcium-treated CSF extracts. These results suggest that the shuttling pathway via the UbL-UBA protein XDRP1 participates in degradation of mitotic cyclins in Xenopus eggs.     

Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit

Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with ΔRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Δp54 proteasomes, but the interaction can be fully restored by complementing the Δp54 deletion with Flag-p54.     

Structure of the UBA domain of Dsk2p in complex with ubiquitin molecular determinants for ubiquitin recognition

The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containing protein from Saccharomyces cerevisiae, is involved in the ubiquitin-proteasome proteolytic pathway and has been implicated in spindle pole duplication. Here we present the solution structure of the UBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structure reveals that the UBA domain uses a mode of ubiquitin recognition that is similar to that of the CUE domain, another ubiquitin binding motif that shares low sequence homology but high structural similarity with UBA domains. These two domains, as well as the structurally unrelated ubiquitin binding motif UIM, provide a common, crucial recognition site for ubiquitin, comprising a hydrogen-bonding acceptor for the amide group of Gly-47, and a methyl group that packs against the hydrophobic pocket of ubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.