Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1821 - 6 Synthesis of alpha-D-glucosylglycerol by alpha-glucosidase and some of its characteristics; Takenaka F et al.; It has been found that alpha-D-glucosylglycerol (GG) is contained in such traditional Japanese foods brewed by using koji as sake, miso and mirin, and that GG is formed by transglucosylation to glycerol that is produced by yeast with alpha-glucosidase (EC 3.2.1.20) from koji in the sake mash . GG has also been found to consist of three components, 2-O-alpha-D-glucosylglycerol (GG-II), (2R)-1-O-alpha-D-glucosylglycerol (R-GG-I) and (2S)-1-O-alpha-D-glucosylglycerol (S-GG-I) . GG was synthesized from a mixture of maltose and glycerol by the batch method, using alpha-glucosidase (transglucosidase L-AMANO) . alpha-Glucosidase seemed to be so stable that the amount of GG increased about 5-fold compared with that in the first reaction by the daily addition of maltose for 10 d . Syrupy GG obtained was found to have the following characteristics: about 0.55-fold sweetness compared with sucrose, high thermo-stability, low heat-colorability, low Maillard reactivity, low hygroscopicity, high water-holding capacity, non-cariogenicity and low digestibility. Yeast, 2000 Nov, 16(15), 1437 - 43 Disruption and functional analysis of six ORFs on chromosome IV: YDL053c, YDL072c, YDL073w, YDL076c, YDL077c and YDL080c; Blasco A et al.; Six open reading frames (ORFs) from chromosome IV, YDL053c, YDL072c, YDL073w, YDL076c, YDL077c and YDL080c, were disrupted using the long flanking homology technique (LFH) to replace each target locus with the KanMX4 selection marker . We have also constructed plasmids containing replacement cassettes (pYORC) and the cognate clones (pYCG) for each ORF . Disruption of five of the ORFs-YDL053c, YDL072c, YDL073w, YDL076c and YDL080c (THI3)-resulted in no distinctive phenotype with respect to temperature or nutritional requirements . However, disruption of YDL077c (also known as VAM6) exhibited an slow growth phenotype in minimal media and also in rich media containing glycerol as a carbon source . The homozygous disruptant diploid corresponding to this gene also failed to sporulate . Yeast, 2000 Nov, 16(15), 1359 - 63 Progression of cell cycle monitored by dielectric spectroscopy and flow-cytometric analysis of DNA content; Asami K et al.; A dielectric method has already been developed for the real-time monitoring of cell cycle progression in synchronized cell culture (Asami et al., 1999) . This method, in combination with DNA content analysis by fluorescence-activated cell sorting (FACS), was applied to the synchronized cell culture of a CDC28-13th mutant (Saccharomyces cerevisiae) . In synchronous cell growth, relative permittivity epsilon (or dielectric constant) for the culture broth showed cyclic changes at low frequencies below 0.5 MHz, being correlated to phases in the cell cycle that were simultaneously determined by FACS . The epsilon increased in the period from S phase to G(2) phase and decreased between M and G(1) phases . Peaks in these cyclic changes of epsilon indicated the time when daughter cells segregated from mother cells . J Mol Biol, 2000 Nov 3, 303(4), 479 - 88 The mitochondrial import receptor Tom70: identification of a 25 kDa core domain with a specific binding site for preproteins; Brix J et al.; The mitochondrial import receptor of 70 kDa, Tom70, preferentially recognizes precursors of membrane proteins with internal targeting signals . We report the identification of a stably folded 25 kDa core domain located in the middle portion of Tom70 that contains two of the seven tetratricopeptide repeat motifs of the receptor . The core domain binds non-cleavable and cleavable preproteins carrying internal targeting signals with a specificity indistinguishable from the full-length receptor . Competition studies indicate that both types of preproteins interact with overlapping binding sites of the core domain and that at least one additional interaction site is present in the full-length receptor . We suggest a model of Tom70 function in import of membrane proteins whereby a hydrophobic preprotein concomitantly interacts with several binding sites of the receptor . J Biol Chem, 2001 Jan 26, 276(4), 2608 - 15 Epub 2000 Oct 27. A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin, ouabain, and SCH 28080; Farley RA et al.; Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase . Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase . The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080 . A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae . Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions . The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080 . The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase . In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080 . The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080. Eur J Biochem, 2000 Nov, 267(22), 6699 - 707 The expression of Cox17p in rodent tissues and cells; Kako K et al.; Previous works have reported the isolation of a novel polypeptide from porcine heart . Structural analysis has shown that it is a mammalian homologue of Cox17p, believed essential for the assembly of functional cytochrome c oxidase and delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast . Although the human, mouse and porcine homologs of this small protein have already been cloned or purified, the function of Cox17p in the mammalian system has not yet been elucidated . To investigate the physiological function of Cox17p in mammals, we performed Northern blot analysis using probes containing the mouse and rat sequences obtained by RT-PCR . The hybridization signals were detected in all mouse tissues, but notably intense signals were observed in heart, brain and kidney RNA samples . Some of the neuroendocrine and endocrine cell lines showed higher expression levels than fibroblasts . The highest expression level of Cox17p mRNA in mouse brain was observed in the pituitary sample . While in rat heart, Cox17p mRNA expression was detected from early development, in rat brain, embryonic and postnatal changes in the expression were observed . Immunocytochemical analysis showed that Cox17p immunoreactivity was strong in the pituitary cell line, AtT-20 . These findings suggested that Cox17p is not only part of the respiratory chain but also involved in brain and endocrine functions. Exp Gerontol, 2000 Sep, 35(6-7), 879 - 96 The network and the remodeling theories of aging: historical background and new perspectives; Franceschi C et al.; Two general theories, i.e . "the network theory of aging" (1989) and "the remodeling theory of aging" (1995), as well as their implications, new developments, and perspectives are reviewed and discussed . Particular attention has been paid to illustrate: (i) how the network theory of aging fits with recent data on aging and longevity in unicellular organisms (yeast), multicellular organisms (worms), and mammals (mice and humans); (ii) the evolutionary and experimental basis of the remodeling theory of aging (immunological, genetic, and metabolic data in healthy centenarians, and studies on the evolution of the immune response, stress and inflammation) and its recent development (the concepts of "immunological space" and "inflamm-aging"); (iii) the profound relationship between these two theories and the data which suggest that aging and longevity are related, in a complex way, to the capability to cope with a variety of stressors. Exp Gerontol, 2000 Sep, 35(6-7), 695 - 702 Werner syndrome protein: biochemical properties and functional interactions; Bohr VA et al.; Werner syndrome is a premature aging syndrome displaying numerous signs and symptoms found in normal aging . The disease is associated with a mutation in the WRN gene . We have purified the Werner protein (WRN) and studied its biochemical activities and its protein interactions . WRN is a helicase and an exonuclease and also has an associated ATPase activity . WRN interacts physically and functionally with replication protein A (RPA), which stimulates its helicase activity . We have studied the WRN exonuclease activity and found that it can be blocked by certain DNA lesions and not by others . Thus, while WRN does not bind to DNA damage, it may have properties that allow it to sense the presence of damage in DNA . More recently we have found other protein interactions that involve physical and functional interactions with WRN, which could suggest a role for WRN in DNA repair. J Exp Bot, 2000 Oct, 51(351), 1763 - 4 Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C; Vranova E et al.; A protein phosphatase 2C (PP2C)-homologous cDNA was isolated from Nicotiana tabacum (NtPP2C1) . The deduced protein sequence of 416 amino acids showed the highest degree of similarity to the PP2C of Arabidopsis thaliana (AtPP2CA) implicated in abscisic acid signalling . The expression of NtPP2C1 was strongly induced by drought, but repressed by oxidative stress and heat shock . It is suggested that NtPP2C1 operates at the junction of drought, heat shock and oxidative stress. Science, 2000 Oct 27, 290(5492), 806 - 9 Direct coupling between meiotic DNA replication and recombination initiation; Borde V et al.; During meiosis in Saccharomyces cerevisiae, DNA replication occurs 1 . 5 to 2 hours before recombination initiates by DNA double-strand break formation . We show that replication and recombination initiation are directly linked . Blocking meiotic replication prevented double-strand break formation in a replication-checkpoint-independent manner, and delaying replication of a chromosome segment specifically delayed break formation in that segment . Consequently, the time between replication and break formation was held constant in all regions . We suggest that double-strand break formation occurs as part of a process initiated by DNA replication, which thus determines when meiotic recombination initiates on a regional rather than a cell-wide basis. J Struct Biol, 2000 Sep, 131(3), 240 - 7 Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors; Bayliss R et al.; NTF2 and importin-beta are transport factors that mediate nuclear protein import and which interact with nuclear pore proteins (nucleoporins) during translocation from the cytoplasm to the nucleus through nuclear pore complexes . We employed a native gel electrophoresis method to assess the interaction of nucleoporin constructs that contain FxFG sequence repeats with NTF2 and truncation mutants of importin-beta to determine suitable fragments for crystallization . Based on these data, we obtained crystals of complexes between yeast NTF2 and a construct containing five FxFG nucleoporin repeats from the yeast nucleoporin Nsp1p and between a construct containing residues 1-442 of human importin-beta and the same nucleoporin construct . The yeast NTF2-nucleoporin crystals have trigonal symmetry and diffract past 2.8 A resolution using synchrotron radiation, whereas the importin-beta-nucleoporin complex crystals have P2(1)2(1)2 orthorhombic symmetry and diffract past 3.2 A resolution . Acta Biochim Pol, 2000, 47(2), 269 - 79 Aging and longevity genes; Jazwinski SM; The genetics of aging has made substantial strides in the past decade . This progress has been confined primarily to model organisms, such as filamentous fungi, yeast, nematodes, fruit flies, and mice, in which some thirty-five genes that determine life span have been cloned . These genes encode a wide array of cellular functions, indicating that there must be multiple mechanisms of aging . Nevertheless, some generalizations are already beginning to emerge . It is now clear that there are at least four broad physiological processes that play a role in aging: metabolic control, resistance to stress, gene dysregulation, and genetic stability . The first two of these at least are common themes that connect aging in yeast, nematodes, and fruit flies, and this convergence extends to caloric restriction, which postpones senescence and increases life span in rodents . Many of the human homologs of the longevity genes found in model organisms have been identified . This will lead to their use as candidate human longevity genes in population genetic studies . The urgency for such studies is great: The population is graying, and this research holds the promise of improvement in the quality of the later years of life. Endocrine, 2000 Aug, 13(1), 55 - 62 c-Jun targets amino terminus of androgen receptor in regulating androgen-responsive transcription; Bubulya A et al.; The human androgen receptor (hAR) is a member of the nuclear receptor superfamily and functions as a ligand-inducible transcription factor . We have previously proposed that c-Jun mediates the transcriptional activity of this receptor . The modular nature of hAR was used in this study to generate several fusions with the heterologous DNA-binding domain of the yeast transcription factor GAL4 in an attempt to identify the c-Jun-responsive domains within the receptor . Our results suggest that the target of c-Jun action is the amino terminus (AB region) of the receptor and that hAR amino acids 502-521 are critical for the c-Jun response . Additionally, amino acids 503-555 were shown to harbor an autonomous transactivation that is stimulated by c-Jun . Furthermore, we demonstrated that transcription intermediary factor-2 (TIF-2), a coactivator that acts on the activation function-2, stimulates the full-length hAR . These results suggest that c-Jun and TIF-2 can work together as coactivators on the hAR by targeting distinct portions of the receptor. J Biol Chem, 2001 Jan 19, 276(3), 2132 - 8 Epub 2000 Oct 26. Intracellular calcium mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in immortalized hippocampal cells; Chung KC et al.; Regulation of intracellular calcium levels plays a central role in cell survival, proliferation, and differentiation . A cell-permeable, tumor-promoting thapsigargin elevates the intracellular calcium levels by inhibiting endoplasmic reticulum Ca(2+)-ATPase . The Src-tyrosine kinase family is involved in a broad range of cellular responses ranging from cell growth and cytoskeletal rearrangement to differentiation . The immediate early gene pip92 is induced in neuronal cell death as well as cell growth and differentiation . To resolve the molecular mechanism of cell growth by intracellular calcium mobilization, we have examined the effect of thapsigargin and subsequent intracellular calcium influx on pip92 expression in immortalized rat hippocampal H19-7 cells . An increase of intracellular calcium ion levels induced by thapsigargin stimulated the expression of pip92 in H19-7 cells . Transient transfection of the cells with kinase-inactive mitogen-activated protein kinase kinase (MEK) and Src kinase or pretreatment with the chemical MEK inhibitor PD98059 significantly inhibited pip92 expression induced by thapsigargin . When constitutively active v-Src or MEK was overexpressed, the transcriptional activity of the pip92 gene was markedly increased . Dominant inhibitory Raf-1 blocked the transcriptional activity of pip92 induced by thapsigargin . The transcription factor Elk1 is activated during thapsigargin-induced pip92 expression . Taken together, these results suggest that an increase of intracellular calcium ion levels by thapsigargin stimulates the pip92 expression via Raf-MEK-extracellular signal-regulated protein kinase- as well as Src kinase-dependent signaling pathways. J Biol Chem, 2001 Feb 16, 276(7), 4901 - 8 Epub 2000 Oct 25. Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair; Qiu J et al.; Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease . It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability . Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization . In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage . Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability . Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast . Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei . Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature. Curr Biol, 2000 Oct 5, 10(19), R708 - 11 Gene silencing: two faces of SIR2; Gottschling DE; There are still many mysteries surrounding how silenced regions of the eukaryotic genome are created and maintained . But recent discoveries about the most evolutionarily conserved silencing protein, Sir2p, have provided new mechanistic insights into these processes. Curr Biol, 2000 Oct 5, 10(19), 1221 - 4 Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality; Esposito G et al.; Polymerase zeta (Pol zeta) is an error-prone DNA polymerase {1}, which in yeast is involved in trans-lesion synthesis (TLS) and is responsible for most of the ultraviolet (UV) radiation-induced and spontaneous mutagenesis {2-4} . Pol zeta consists of three subunits: REV1, a deoxycytidyl-transferase {5}; REV7, of unclear function {6}; and REV3, the catalytic subunit . REV3 alone is sufficient to carry out TLS, but association with REV1 and REV7 enhances its activity {5, 7} . Experiments using human cells treated with UV radiation indicate also that mammalian Pol zeta is involved in TLS {7} . The peculiar mutagenic activity of Pol zeta {4,7,8} suggests a possible role in somatic hypermutation of immunoglobulin (Ig) genes {9} . Here, we report that, unlike in yeast where the REV3 gene is not essential for life {4}, disruption of the mouse homologue (Rev3l) resulted in early embryonic lethality . In Rev3l(-/-) embryos, no haematopoietic cells other than erythrocytes could be identified in the yolk sac . Rev3l(-/-) haematopoietic precursors were unable to expand in vitro and no haematopoietic cells could be derived from the intraembryonic haematogenic compartment (splanchnopleura) . Fibroblasts could not be derived from the Rev3l(-/-) embryos, and Rev3l(-/-) embryonic stem (ES) cells could not be obtained . This is the first evidence that an enzyme involved in TLS is critical for mammalian development. Curr Biol, 2000 Oct 5, 10(19), 1217 - 20 Disruption of the developmentally regulated Rev3l gene causes embryonic lethality; Wittschieben J et al.; The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions {1} . Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis {2} . Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines {3} . To study the function of mammalian Rev31, we inactivated the gene in mice . Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter . Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc) . Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development . Tissues in many areas were disorganised, with significantly reduced cell density . Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes . Embryonic death coincided with the period of more widely distributed Rev3l expression . The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals. Curr Biol, 2000 Oct 5, 10(19), 1213 - 6 Disruption of mouse polymerase zeta (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro; Bemark M et al.; Multiple DNA polymerases exist in eukaryotes . Polymerases alpha, delta and epsilon are mainly responsible for chromosomal DNA replication in the nucleus and are required for proliferation . In contrast, the repair polymerases beta and eta are not essential for cellular proliferation in yeast or mice, but a lack of either polymerase can lead, respectively, to defects in base excision repair or the ability to replicate past lesions induced by ultraviolet (UV) radiation {1-3} . Here, we have focused on polymerase zeta . This was first described as a non-essential product of the yeast REV3/REV7 genes involved in UV-induced mutagenesis, and was later implicated in trans-lesion synthesis {4,5} . Unlike in yeast, the mouse homologue (mRev3) was found to be essential for life . Homozygous mutant mice died in utero . Mutant embryos were considerably reduced in size at day 10.5 of development and usually aborted around day 12.5 . It is likely that this block reflects a need for mRev3 in proliferative clonal expansion (rather than in the production of a particular cell type) as mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture . Thus, mRev3 could be required to repair a form of externally induced DNA damage that otherwise accumulates during clonal expansion or, consistent with the high homology shared between its Rev7 partner and the mitotic checkpoint gene product Mad2 {6}, mRev3 might play a role in cell proliferation and genomic stability even in the absence of environmentally induced damage. Curr Biol, 2000 Oct 5, 10(19), 1182 - 90 Slk19p is necessary to prevent separation of sister chromatids in meiosis I; Kamieniecki RJ et al.; BACKGROUND: A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids remain joined, moving as a unit to one pole of the spindle rather than separating as they do in mitosis . It has long been known that the sustained linkage of sister chromatids through meiotic anaphase I is accomplished by association of the chromatids at the centromere region . The localization of the cohesin Rec8p to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, but the molecular basis for the regulation of Rec8p and sister kinetochores in meiosis remains a mystery . RESULTS: We show that the SLK19 gene product from Saccharomyces cerevisiae is essential for proper chromosome segregation during meiosis I . When slk19 mutants were induced to sporulate they completed events characteristic of meiotic prophase I, but at the first meiotic division they segregated their sister chromatids to opposite poles at high frequencies . The vast majority of these cells did not perform a second meiotic division and proceeded to form dyads (asci containing two spores) . Slk19p was found to localize to centromere regions of chromosomes during meiotic prophase where it remained until anaphase I . In the absence of Slk19p, Rec8p was not maintained at the centromere region through anaphase I as it is in wild-type cells . Finally, we demonstrate that Slk19p appears to function downstream of the meiosis-specific protein Spo13p in control of sister chromatid behavior during meiosis I . CONCLUSIONS: Our results suggest that Slk19p is essential at the centromere of meiotic chromosomes to prevent the premature separation of sister chromatids at meiosis I. Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12589 - 94 Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2; Lin CY et al.; Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells . Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis . In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein . GRASP65 appears to function in the postmitotic reassembly of Golgi stacks . In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase . Moreover, we show that Cdc2 can also phosphorylate GRASP65 . In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function . Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65 . These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division. Blood, 2000 Nov 1, 96(9), 3256 - 64 Human ABC7 transporter: gene structure and mutation causing X-linked sideroblastic anemia with ataxia with disruption of cytosolic iron-sulfur protein maturation; Bekri S et al.; The human protein ABC7 belongs to the adenosine triphosphate-binding cassette transporter superfamily, and its yeast orthologue, Atm1p, plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins . Previously, a missense mutation in the human ABC7 gene was shown to be the defect in members of a family affected with X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A) . Here, the promoter region and the intron/exon structure of the human ABC7 gene were characterized, and the function of wild-type and mutant ABC7 in cytosolic Fe/S protein maturation was analyzed . The gene contains 16 exons, all with intron/exon boundaries following the AG/GT rule . A single missense mutation was found in exon 10 of the ABC7 gene in 2 affected brothers with XLSA/A . The mutation was a G-to-A transition at nucleotide 1305 of the full-length cDNA, resulting in a charge inversion caused by the substitution of lysine for glutamate at residue 433 C-terminal to the putative sixth transmembrane domain of ABC7 . Expression of normal ABC7 almost fully complemented the defect in the maturation of cytosolic Fe/S proteins in a yeast strain in which the ATM1 gene had been deleted (Deltaatm1 cells) . Thus, ABC7 is a functional orthologue of Atm1p . In contrast, the expression of mutated ABC7 (E433K) or Atm1p (D398K) proteins in Deltaatm1 cells led to a low efficiency of cytosolic Fe/S protein maturation . These data demonstrate that both the molecular defect in XLSA/A and the impaired maturation of a cytosolic Fe/S protein result from an ABC7 mutation in the reported family. Protein Expr Purif, 2000 Nov, 20(2), 133 - 41 Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein; Guarna MM et al.; Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing . After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa) . However, the use of FXa is limited by its expense and availability from plasma . To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT . The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag . A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag . Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity . However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date . The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae . After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins . The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro . Chem Biol, 2000 Aug, 7(8), 601 - 10 Virtually unidirectional binding of TBP to the AdMLP TATA box within the quaternary complex with TFIIA and TFIIB; Kays AR et al.; BACKGROUND: The TATA box binding protein (TBP) is required by all three RNA polymerases for the promoter-specific initiation of transcription . All eukaryotic TBP-DNA complexes observed in crystal structures show the conserved C-terminal domain of TBP (TBPc) bound to the TATA box in a single orientation that is consistent with assembly of a preinitiation complex (PIC) possessing a unique polarity . The binding of TBP to the TATA box is believed to orient the PIC correctly on the promoter and can function as the rate-limiting step in PIC assembly . Previous work performed with TBP from Saccharomyces cerevisiae (yTBP) showed that, despite the oriented binding of eukaryotic TBP observed in crystal structures, yTBP in solution does not orient itself uniquely on the adenovirus major late promoter (AdMLP) TATA box . Instead, yTBP binds the AdMLP as a mixture of two orientational isomers that are related by a 180 degree rotation about the pseudo-dyad axis of the complex . In addition, these orientational isomers are not restricted to the 8 bp TATA box, but rather bind a distribution of sites that partially overlap the TATA box . Two members of the PIC, general transcription factor (TF) IIB and TFIIA individually enhance the orientational and axial specificity of yTBP binding to the TATA box, but fail to fix yTBP in a single orientation or a unique position on the promoter . RESULTS: We used an affinity cleavage assay to explore the combined effects of TFIIA and TFIIB on the axial and orientational specificity of yTBP . Our results show that the combination of TFIIA and TFIIB affixes yTBP in virtually a single orientation as well as a unique location on the AdMLP TATA box . Ninety-five percent of the quaternary TBP-TFIIA-TFIIB-TATA complex contained yTBP bound in the orientation expected on the basis of crystallographic and genetic experiments, and more than 70% is restricted axially to the 8 bp sequence TATAAAAG . CONCLUSIONS: Although yTBP itself binds to the TATA box without a high level of orientational or axial specificity, our data show that a small subset of general TFs are capable of uniquely orienting the PIC on the AdMLP . Our results, in combination with recent data concerning the pathway of PIC formation in yeast, suggest that transcription could be regulated during both early and late stages of PIC assembly by general factors (and the proteins to which they bind) that influence the position and orientation of TBP on the promoter. Mol Cell Biol, 2000 Nov, 20(22), 8623 - 33 Cell signaling switches HOX-PBX complexes from repressors to activators of transcription mediated by histone deacetylases and histone acetyltransferases; Saleh M et al.; The Hoxb1 autoregulatory element comprises three HOX-PBX binding sites . Despite the presence of HOXB1 and PBX1, this enhancer fails to activate reporter gene expression in retinoic acid-treated P19 cell monolayers . Activation requires cell aggregation in addition to RA . This suggests that HOX-PBX complexes may repress transcription under some conditions . Consistent with this, multimerized HOX-PBX binding sites repress reporter gene expression in HEK293 cells . We provide a mechanistic basis for repressor function by demonstrating that a corepressor complex, including histone deacetylases (HDACs) 1 and 3, mSIN3B, and N-CoR/SMRT, interacts with PBX1A . We map a site of interaction with HDAC1 to the PBX1 N terminus and show that the PBX partner is required for repression by the HOX-PBX complex . Treatment with the deacetylase inhibitor trichostatin A not only relieves repression but also converts the HOX-PBX complex to a net activator of transcription . We show that this activation function is mediated by the recruitment of the coactivator CREB-binding protein by the HOX partner . Interestingly, HOX-PBX complexes are switched from transcriptional repressors to activators in response to protein kinase A signaling or cell aggregation . Together, our results suggest a model whereby the HOX-PBX complex can act as a repressor or activator of transcription via association with corepressors and coactivators . The model implies that cell signaling is a direct determinant of HOX-PBX function in the patterning of the animal embryo. Mol Cell Biol, 2000 Nov, 20(22), 8602 - 12 Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis; Mendez J et al.; Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process . The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p . The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells . Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis . hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors . Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms . Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells. Mol Cell Biol, 2000 Nov, 20(22), 8489 - 98 Acetylation by PCAF enhances CIITA nuclear accumulation and transactivation of major histocompatibility complex class II genes; Spilianakis C et al.; The class II transactivator (CIITA), the master regulator of the tissue-specific and interferon gamma-inducible expression of major histocompatibility complex class II genes, synergizes with the histone acetylase coactivator CBP to activate gene transcription . Here we demonstrate that in addition to CBP, PCAF binds to CIITA both in vivo and in vitro and enhances CIITA-dependent transcriptional activation of class II promoters . Accordingly, E1A mutants defective for PCAF or CBP interaction show reduced ability in suppressing CIITA activity . Interestingly, CBP and PCAF acetylate CIITA at lysine residues within a nuclear localization signal . We show that CIITA is shuttling between the nucleus and cytoplasm . The shuttling behavior and activity of the protein are regulated by acetylation: overexpression of PCAF or inhibition of cellular deacetylases by trichostatin A increases the nuclear accumulation of CIITA in a manner determined by the presence of the acetylation target lysines . Furthermore, mutagenesis of the acetylated residues reduces the transactivation ability of CIITA . These results support a novel function for acetylation, i.e., to regulate gene expression by stimulating the nuclear accumulation of an activator. J Immunol, 2000 Nov 1, 165(9), 5127 - 32 Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor; Croze E et al.; The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor . We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase) . Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346 . RACK-1 binding to IFN-alphaRbetaL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2 . The interaction between RACK-1 and IFN-alphaRbetaL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-alphaRalpha), was also detected in human Daudi cells by coimmunoprecipitation . RACK-1 was shown to be constitutively associated with IFN-alphaRbetaL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-beta1b) . RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-beta1b . However, stimulation of cells with either IFN-beta1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1. Insect Biochem Mol Biol, 2000 Dec, 30(12), 1213 - 22 Cloning and characterization of a chitin synthase cDNA from the mosquito Aedes aegypti; Ibrahim GH et al.; Characterization of the enzymes involved in the chitin biosynthetic pathway in mosquitoes is critical due to the importance of chitin in the formation of the peritrophic matrix {PM} and its potential impact on vector competence . Chitin is the homopolymer of the amino sugar N-acetyl-D glucosamine {GlcNAc} . The final step of incorporation of GlcNAc into the chitin polymer is catalyzed by the enzyme chitin synthase {CS} . CS is a membrane bound enzyme, but the mechanism of its action in the biosynthesis of the PM is not understood . We have isolated and sequenced a CS-encoding cDNA clone from the mosquito Aedes aegypti, compared its sequence with CS from other organisms and studied its RNA expression . The cDNA is 3.5 kb in length with an open reading frame of 2.6 kb that encodes a protein of 865 amino acids with a predicted molecular mass of 99.5 kDa . The putative translation product shares 90% similarity to two CS proteins from Caenorhabditis elegans and 50% similarity to Saccharomyces cerevisiae in the catalytic domain of CS enzymes . Data suggest that CS is a single copy gene . RT-PCR analysis shows CS message in whole non-blood-fed females, whole blood-fed females, non-blood-fed midguts and in midguts dissected at different time points post-blood-feeding . In situ hybridization studies of midgut samples revealed that CS mRNA increases following a bloodmeal and is localized to the periphery of the epithelial cells facing the midgut lumen. J Biol Chem, 2001 Jan 12, 276(2), 1051 - 6 Biochemical analysis of the eIF2beta gamma complex reveals a structural function for eIF2alpha in catalyzed nucleotide exchange; Nika J et al.; Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner . Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2.GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B . The alpha-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction . We purified a form of eIF2 (eIF2betagamma) completely devoid of the alpha-subunit to further study the role of eIF2alpha in eIF2 function . These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2) . Removal of the alpha-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by eIF2 in vitro . Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2betagamma and the wild-type eIF2 heterotrimer . However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha-subunit increased K(m) for eIF2betagamma.GDP by an order of magnitude, with a smaller increase in V(max) . These data indicate that eIF2alpha is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange . We suggest that this function contributes to the ability of the alpha-subunit to control the rate of nucleotide exchange through reversible phosphorylation. Biochemistry, 2000 Oct 24, 39(42), 13034 - 43 Characterization of human copine III as a phosphoprotein with associated kinase activity; Caudell EG et al.; The copines, first described by Creutz et al . {(1998) J . Biol . Chem . 273, 1393-1402}, comprise a two C2 domain-containing protein family and are known to aggregate phosphatidylserine membranes in a calcium-dependent manner . No enzymatic function has been attributed to copines yet . Due to a cross-reacting activity of Mikbeta1, an antibody to the IL-2Rbeta chain, we were able to serendipitously purify, partially microsequence, and clone human copine III . The 5 kb copine III transcript is expressed ubiquitously as determined by a multitissue Northern blot analysis . Phosphoamino acid analysis revealed phosphorylation of copine III on serine and threonine residues . In vitro kinase assays were performed with immunoprecipitated endogenous copine III, chromatography-purified endogenous copine III, and recombinant copine III expressed in Saccharomyces cerevisiae . The exogenous substrate myelin basic protein was phosphorylated in all in vitro kinase assays containing copine III immunoprecipitate or purified copine III . A 60-kDa band was observed in corresponding in gel kinase assays with staurosporine-activated cells . Cell lines expressing high levels of copine III protein had correspondingly high kinase activity in copine III antiserum immunoprecipitate . However, the copine amino acid sequences lack the traditional kinase catalytic domain . Therefore, the data suggest copine III may possess an intrinsic kinase activity and represent a novel unconventional kinase family. Biochemistry, 2000 Oct 24, 39(42), 12862 - 74 Role of SRP19 in assembly of the Archaeoglobus fulgidus signal recognition particle; Diener JL et al.; Previous studies have shown that SRP19 promotes association of the highly conserved signal peptide-binding protein, SRP54, with the signal recognition particle (SRP) RNA in both archaeal and eukaryotic model systems . In vitro characterization of this process is now reported using recombinantly expressed components of SRP from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidis . A combination of native gel mobility shift, filter binding, and Ni-NTA agarose bead binding assays were used to determine the binding constants for binary and ternary complexes of SRP proteins and SRP RNA . Archaeal SRP54, unlike eukaryotic homologues, has significant intrinsic affinity for 7S RNA (K(D) approximately 15 nM), making it possible to directly compare particles formed in the presence and absence of SRP19 and thereby assess the precise role of SRP19 in the assembly process . Chemical modification studies using hydroxyl radicals and DEPC identify nonoverlapping primary binding sites for SRP19 and SRP54 corresponding to the tips of helix 6 and helix 8 (SRP19) and the distal loop and asymmetric bulge of helix 8 (SRP54) . SRP19 additionally induces conformational changes concentrated in the proximal asymmetric bulge of helix 8 . Selected nucleotides in this bulge become modified as a result of SRP19 binding but are subsequently protected from modification by formation of the complete complex with SRP54 . Together these results suggest a model for assembly in which bridging the ends of helix 6 and helix 8 by SRP19 induces a long-range structural change to present the proximal bulge in a conformation compatible with high-affinity SRP54 binding. Genes Dev, 2000 Oct 15, 14(20), 2650 - 63 Different human TFIIIB activities direct RNA polymerase III transcription from TATA-containing and TATA-less promoters; Schramm L et al.; Transcription initiation at RNA polymerase III promoters requires transcription factor IIIB (TFIIIB), an activity that binds to RNA polymerase III promoters, generally through protein-protein contacts with DNA binding factors, and directly recruits RNA polymerase III . Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide beta(") . Although human homologs for two of the TFIIIB subunits, the TATA box-binding protein TBP and the TFIIB-related factor BRF, have been characterized, a human homolog of yeast B(") has not been described . Moreover, human BRF, unlike yeast BRF, is not universally required for RNA polymerase III transcription . In particular, it is not involved in transcription from the small nuclear RNA (snRNA)-type, TATA-containing, RNA polymerase III promoters . Here, we characterize two novel activities, a human homolog of yeast B("), which is required for transcription of both TATA-less and snRNA-type RNA polymerase III promoters, and a factor equally related to human BRF and TFIIB, designated BRFU, which is specifically required for transcription of snRNA-type RNA polymerase III promoters . Together, these results contribute to the definition of the basal RNA polymerase III transcription machinery and show that two types of TFIIIB activities, with specificities for different classes of RNA polymerase III promoters, have evolved in human cells. Genes Dev, 2000 Oct 15, 14(20), 2635 - 49 High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation; Andrulis ED et al.; Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans . Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock . Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription . Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5' and 3' ends of heat shock genes . Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF . These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo. Genes Dev, 2000 Oct 15, 14(20), 2623 - 34 Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D . melanogaster; Kaplan CD et al.; The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription . They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells . In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo . To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization on Drosophila polytene chromosomes . We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock . Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II . These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo. Anal Biochem, 2000 Nov 1, 286(1), 173 - 8 Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose; Rabina J et al.; Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described . The methods are based on time-resolved fluorescence and microplate assay technology . The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase . In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate . The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM . In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell . The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster . We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc . The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner . J Cell Biol, 2000 Oct 16, 151(2), 453 - 66 Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion; Grote E et al.; Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex . High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking . The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes . Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase . Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S . Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane . In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells. J Cell Biol, 2000 Oct 16, 151(2), 367 - 80 Dnm1p GTPase-mediated mitochondrial fission is a multi-step process requiring the novel integral membrane component Fis1p; Mozdy AD et al.; Yeast Dnm1p is a soluble, dynamin-related GTPase that assembles on the outer mitochondrial membrane at sites where organelle division occurs . Although these Dnm1p-containing complexes are thought to trigger constriction and fission, little is known about their composition and assembly, and molecules required for their membrane recruitment have not been isolated . Using a genetic approach, we identified two new genes in the fission pathway, FIS1 and FIS2 . FIS1 encodes a novel, outer mitochondrial membrane protein with its amino terminus exposed to the cytoplasm . Fis1p is the first integral membrane protein shown to participate in a eukaryotic membrane fission event . In a related study (Tieu, Q., and J . Nunnari . 2000 . J . Cell Biol . 151:353-365), it was shown that the FIS2 gene product (called Mdv1p) colocalizes with Dnm1p on mitochondria . Genetic and morphological evidence indicate that Fis1p, but not Mdv1p, function is required for the proper assembly and distribution of Dnm1p-containing fission complexes on mitochondrial tubules . We propose that mitochondrial fission in yeast is a multi-step process, and that membrane-bound Fis1p is required for the proper assembly, membrane distribution, and function of Dnm1p-containing complexes during fission. J Cell Biol, 2000 Oct 16, 151(2), 353 - 66 Mdv1p is a WD repeat protein that interacts with the dynamin-related GTPase, Dnm1p, to trigger mitochondrial division; Tieu Q et al.; Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p, which assembles on the mitochondrial outer membrane into punctate structures associated with sites of membrane constriction and fission . We have identified additional nuclear genes required for mitochondrial fission, termed MDV (for mitochondrial division) . MDV1 encodes a predicted soluble protein, containing a coiled-coil motif and seven COOH-terminal WD repeats . Genetic and two-hybrid analyses indicate that Mdv1p interacts with Dnm1p to mediate mitochondrial fission . In addition, Mdv1p colocalizes with Dnm1p in fission-mediating punctate structures on the mitochondrial outer membrane . Whereas localization of Mdv1p to these structures requires Dnm1p, localization of Mdv1p to mitochondrial membranes does not . This indicates that Mdv1p possesses a Dnm1p-independent mitochondrial targeting signal . Dnm1p-independent targeting of Mdv1p to mitochondria requires MDV2 . Our data indicate that MDV2 also functions separately to regulate the assembly of Dnm1p into punctate structures . In contrast, Mdv1p is not required for the assembly of Dnm1p, but Dnm1p-containing punctate structures lacking Mdv1p are not able to complete division . Our studies suggest that mitochondrial fission is a multi-step process in which Mdv2p regulates the assembly of Dnm1p into punctate structures and together with Mdv1p functions later during fission to facilitate Dnm1p-dependent mitochondrial membrane constriction and/or division. J Cell Biol, 2000 Oct 16, 151(2), 341 - 52 The dynamin-related GTPase, Mgm1p, is an intermembrane space protein required for maintenance of fusion competent mitochondria; Wong ED et al.; Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear . To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles . Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments . This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion . Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells . However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating . This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion . Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space . Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events . In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion. J Cell Biol, 2000 Oct 16, 151(2), 333 - 40 Gag3p, an outer membrane protein required for fission of mitochondrial tubules; Fekkes P et al.; Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane . Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth) . Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures . Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide . The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission . GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay . Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane . This association requires neither the DNM1 nor GAG2 gene products . However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p . These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules. J Cell Biol, 2000 Oct 16, 151(2), 289 - 96 TRAPP stimulates guanine nucleotide exchange on Ypt1p; Wang W et al.; TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus . Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5 . Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP . Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p . Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p . Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p . Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP. J Cell Biol, 2000 Oct 16, 151(2), 263 - 76 The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway; Kirisako T et al.; Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles . Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature . We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself . First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG . Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes . This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme . Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4 . The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes . A reaction similar to ubiquitination is probably involved in the second modification . The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway. Plant Cell Physiol, 2000 Aug, 41(8), 940 - 7 Characterization and expression of monosaccharide transporters (osMSTs) in rice; Toyofuku K et al.; This study deals with the cloning and characterization of monosaccharide transporter cDNAs in rice . OsMST1-3 (Oryza sativa monosaccharide transporters 1-3) have two sets of putative six transmembrane domains separated by a central long hydrophilic region . Heterologous expression of OsMST3 in the yeast Saccharomyces cerevisiae indicated that OsMST3 has transport activity for some monosaccharides in an energy-dependent H+ co-transport manner . Northern blot and in situ hybridization analyses showed that OsMST3 mRNA is detectable in leaf blades, leaf sheaths, calli and roots, especially the xylem as well as in sclerenchyma cells in the root . These results suggested that OsMST3 is involved in the accumulation of monosaccharides required for cell wall synthesis at the stage of cell thickening. Curr Microbiol, 2000 Mar, 40(3), 169 - 75 Mcchs1, a member of a chitin synthase gene family in Mucor circinelloides, is differentially expressed during dimorphism; Lopez-Matas MA et al.; A complete chitin synthase gene and one chitin synthase gene fragment of the zygomycete Mucor circinelloides have been cloned and analyzed . Both genes encode zymogenic Class II chitin synthases . Hybridization analysis showed that there must exist at least another Class II chitin synthase gene in M . circinelloides highly homologous to the cloned Mcchs1 and Mcchs2 . The expression of these genes during the dimorphic growing stages was analyzed . Northern hybridizations showed that Mcchs1 transcript accumulates only during the exponentially growing hyphal stage, while no expression could be detected in the yeast form . Expression of Mcchs2 could not be detected at any stage . Accumulation of Mcchs1 transcript was not influenced by visible light . The existence of a multigene chitin synthase family and the observation that Mcchs1 transcription depends upon the dimorphic stage indicate that various chitin synthase activities may have different roles in the dimorphic growth of M . circinelloides. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12187 - 92 The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80; Tung KS et al.; In budding yeast, many mutants defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage of meiotic prophase . We recovered the NDT80 gene in a screen for genes whose overexpression bypasses the pachytene checkpoint . Ndt80 is a meiosis-specific transcription factor that promotes expression of genes required for exit from pachytene and entry into meiosis I . Herein, we show that the Ndt80 protein accumulates and is extensively phosphorylated during meiosis in wild type but not in cells arrested at the pachytene checkpoint . Our results indicate that inhibition of Ndt80 activity is one mechanism used to achieve pachytene arrest. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12068 - 73 A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation; Senkevich TG et al.; Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasmic DNA viruses for which substantial sequence information is available . Nevertheless, the roles of these proteins are imprecisely known . Multiple alignments of ERV1/ALR proteins indicated an invariant C-X-X-C motif, but no similarity to the thioredoxin fold was revealed by secondary structure predictions . We chose a virus model to investigate the role of these proteins as thiol oxidoreductases . When cells were infected with a mutant vaccinia virus in which the E10R gene encoding an ERV1/ALR family protein was repressed, the disulfide bonds of three other viral proteins-namely, the L1R and F9L proteins and the G4L glutaredoxin-were completely reduced . The same outcome occurred when Cys-43 or Cys-46, the putative redox cysteines of the E10R protein, was mutated to serine . These two cysteines were disulfide bonded during a normal virus infection but not if the synthesis of other viral late proteins was inhibited or the E10R protein was expressed by itself in uninfected cells, suggesting a requirement for an upstream viral thiol oxidoreductase . Remarkably, the cysteine-containing domains of the E10R and L1R viral membrane proteins and the glutaredoxin are in the cytoplasm, in which assembly of vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum . These data indicated a viral pathway of disulfide bond formation in which the E10R protein has a central role . By extension, the ERV1/ALR family may represent a ubiquitous class of cellular thiol oxidoreductases that interact with glutaredoxins or thioredoxins. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12356 - 60 Altered selectivity in an Arabidopsis metal transporter; Rogers EE et al.; Plants require metals for essential functions ranging from respiration to photosynthesis . These metals also contribute to the nutritional value of plants for both humans and livestock . Additionally, plants have the ability to accumulate nonessential metals such as cadmium and lead, and this ability could be harnessed to remove pollutant metals from the environment . Designing a transporter that specifically accumulates certain cations while excluding others has exciting applications in all of these areas . The Arabidopsis root membrane protein IRT1 is likely to be responsible for uptake of iron from the soil . Like other Fe(II) transporters identified to date, IRT1 transports a variety of other cations, including the essential metals zinc and manganese as well as the toxic metal cadmium . By heterologous expression in yeast, we show here that the replacement of a glutamic acid residue at position 103 in wild-type IRT1 with alanine increases the substrate specificity of the transporter by selectively eliminating its ability to transport zinc . Two other mutations, replacing the aspartic acid residues at either positions 100 or 136 with alanine, also increase IRT1 metal selectivity by eliminating transport of both iron and manganese . A number of other conserved residues in or near transmembrane domains appear to be essential for all transport function . Therefore, this study identifies at least some of the residues important for substrate selection and transport in a protein belonging to the ZIP gene family, a large transporter family found in a wide variety of organisms. J Biol Chem, 2001 Jan 12, 276(2), 1578 - 84 Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1; McLarren KW et al.; Hairy/Enhancer of split 1 (Hes1) is a mammalian transcriptional repressor that plays crucial roles in the regulation of several developmental processes, including neuronal differentiation . The aim of this study was to elucidate the molecular mechanisms that regulate the transcription repression activity of Hes1 . It is shown here that Hes1 associates with the nuclear matrix, the ribonucleoprotein network of the nucleus that plays important roles in transcriptional regulation . Nuclear matrix binding is mediated by the same Hes1 C-terminal domain that is also required for transcriptional repression . This domain contains the WRPW motif that acts as a binding site for the transcriptional corepressor Groucho, which also localizes to the nuclear matrix . Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of the WRPW motif, indicating that Groucho acts as a transcriptional corepressor for Hes1 . This corepressor role is not modulated by the Groucho-related gene product Grg5 . In contrast, the Runt-related protein RUNX2, which localizes to the nuclear matrix and interacts with Groucho and Hes1, can inhibit both the Groucho.Hes1 interaction and the transcription repression ability of Hes1 . Together, these observations suggest that transcriptional repression by Hes1 requires interactions with Groucho at the nuclear matrix and that RUNX proteins act as negative regulators of the repressive activity of Groucho.Hes1 complexes. J Biol Chem, 2001 Jan 12, 276(2), 875 - 83 A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes; Hirsch DS et al.; Cdc42, a Rho GTPase, regulates the organization of the actin cytoskeleton by its interaction with several distinct families of downstream effector proteins . Here, we report the identification of four new Cdc42-binding proteins that, along with MSE55, constitute a new family of effector proteins . These molecules, designated CEPs, contain three regions of homology, including a Cdc42 binding domain and two unique domains called CI and CII . Experimentally, we have verified that CEP2 and CEP5 bind Cdc42 . Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced pseudopodia formation . Fibroblasts coexpressing dominant negative Cdc42 with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation . In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at the adherens junctions with an increase in thin stress fibers that extended the length of the cell body . Keratinocytes expressing CEPs also showed an altered vinculin distribution and a loss of E-cadherin from adherens junctions . Similar effects were observed in keratinocytes expressing constitutively active Cdc42, but were not seen with a Cdc42/Rac interactive binding domain mutant of CEP2 . These results suggest that CEPs act downstream of Cdc42 to induce actin filament assembly leading to cell shape changes. J Cell Sci, 2000 Nov, 113 Pt 21, 3697 - 702 IRE1 and efferent signaling from the endoplasmic reticulum; Urano F et al.; Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response - UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease . In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one . Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them . Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP . The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified . Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6 . This indicates that the mammalian UPR is more complex than that found in yeast. J Exp Med, 2000 Oct 16, 192(8), 1165 - 74 FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation; Rochat-Steiner V et al.; Fas is a cell surface death receptor that signals apoptosis . Several proteins have been identified that bind to the cytoplasmic death domain of Fas . Fas-associated death domain (FADD), which couples Fas to procaspase-8, and Daxx, which couples Fas to the Jun NH(2)-terminal kinase pathway, bind independently to the Fas death domain . We have identified a 130-kD kinase designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fas-interacting protein . Binding to Fas is mediated by a conserved sequence in the COOH terminus of the protein . FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm . In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction . Although Fas ligand-induced activation of Jun NH(2)-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected . These results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas. Cancer Res, 2000 Oct 1, 60(19), 5529 - 35 A putative oncogenic role for MPP11 in head and neck squamous cell cancer; Resto VA et al.; Genetic alterations of chromosome 7 are common in human cancer . Furthermore, previous studies have supported the presence of a gene important in a broad range of cancers at 7q22-31.1 . There is evidence that supports an oncogenic function for this putative gene, as well as evidence that supports a tumor suppressive role . In this study, we used a cross-species candidate gene approach in combination with physical mapping to identify MPP11 as a candidate for the putative cancer-related activity at 7q22-31.1 . We then analyzed primary head and neck squamous cell tumors (HNSCCs) for loss of heterozygosity/allelic imbalance (LOH/AI) at the MPP11 genomic locus . Thirty-eight percent of tumors examined displayed LOH/AI involving the MPP11 genomic locus . Mutation analysis of MPP11 in the latter samples did not identify any inactivating mutations . However, immunohistochemical staining of primary tumor sections and Western blot analysis of HNSCC cell lines revealed a tumor-specific high level of expression of MPP11p . Fluorescence in situ hybridization analysis done on the cell lines identified increased chromosome 7 copy number with a concomitant increase in MPP11 copy number . These results suggest an oncogenic role for MPP11 in HNSCC. Cancer Res, 2000 Oct 1, 60(19), 5371 - 5 17q23 amplifications in breast cancer involve the PAT1, RAD51C, PS6K, and SIGma1B genes; Wu GJ et al.; Amplification of the 17q23 region occurs frequently in breast tumors . To characterize the structure of 17q23 amplicons and to identify oncogene targets associated with this alteration, we performed a copy number analysis of 87 17q23 localized expressed sequence tags in seven breast cancer cell lines . Three major regions of amplification were detected in the MCF7 and BT474 cell lines . Amplification of at least one of four known genes (PAT1, PS6K, RAD51C, and SIGMA1B) was detected in the cell lines and in 28% of 94 breast tumors . In most cases, these four genes were overexpressed when amplified, but there was a particularly good association between amplification of the SIGMA1B gene and elevated expression in tumors, which suggested a possible role for this gene in tumor progression . Our data show that this region contains at least four independent targets of amplification, which suggests that there is considerable variability in the structure of the 17q23 amplicon. Cancer Res, 2000 Oct 1, 60(19), 5349 - 53 Paclitaxel induces release of cytochrome c from mitochondria isolated from human neuroblastoma cells'; Andre N et al.; Paclitaxel is an antimicrotubule agent that induces mitotic block and apoptosis . We show for the first time that paclitaxel acts directly or mitochondria isolated from human cancer cells . In isolated yeast mito chondria, paclitaxel (15 microM) induced an 18% increase in the respiration rate, with no concomitant release of cytochrome c . In isolated neuroblas toma mitochondria, paclitaxel (10-100 microM) induced a 27-72% release o cytochrome c . Release was prevented by cyclosporin A, suggesting the involvement of the permeability transition pore . Doxorubicin did no induce cytochrome c release, whereas vinorelbine, another antimicrotu bule agent, did . Thus, antimicrotubule agents can directly affect mito chondria to induce apoptosis. Cancer Res, 2000 Oct 1, 60(19), 5340 - 4 Multiple genes at 17q23 undergo amplification and overexpression in breast cancer; Barlund M et al.; Studies by comparative genomic hybridization imply that amplification of the chromosomal region 17q22-q24 is common in breast cancer . Here, amplification and expression levels of six known genes located at 17q23 were examined in breast cancer cell lines . Four of them (RAD51C, S6K, PAT1, and TBX2) were found to be highly amplified and overexpressed . To investigate the involvement of these genes in vivo, fluorescence in situ hybridization analysis of a tissue microarray containing 372 primary breast cancers was used . S6K, PAT1, and TBX2 were coamplified in about 10% of tumors, whereas RADS1C amplification was seen in only 3% of tumors . Expression analysis in 12 primary tumors showed that RAD51C and S6K were consistently expressed in all cases in which they were amplified and also in some tumors without amplification . These data suggest that 17q23 amplification results in simultaneous up-regulation of several genes, whose increased biological activity may jointly contribute to the more aggressive clinical course observed in patients with 17q23-amplified tumors. FEBS Lett, 2000 Oct 13, 483(1), 43 - 6 In synergy with various cis-acting elements, plant insterstitial telomere motifs regulate gene expression in Arabidopsis root meristems; Manevski A et al.; The telo-box, an interstitial telomere motif, was shown to regulate gene expression in root meristems, in synergy with a cis-acting element involved in the activation of expression of plant eEF1A genes, encoding the translation elongation factor EF1A, and of several ribosomal protein genes . We demonstrate here that the telo-box is also required for transcription activation by two other cis elements present within the promoter of genes encoding the acidic ribosomal protein rp40 and the proliferating cell nuclear antigen respectively . The control of gene expression by telo-boxes during cell cycle progression in Arabidopsis root meristems is discussed . A parallel is drawn with the function of telomeric sequences in Saccharomyces cerevisiae. EMBO J, 2000 Oct 16, 19(20), 5552 - 61 Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination; Adair GM et al.; The XpF/Ercc1 structure-specific endonuclease performs the 5' incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae . In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination . To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1(+) and ERCC1(-) cell lines with insertion vectors having long or short terminal non-homologies flanking each side of a double-strand break . No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast . However, profound differences were observed in types of APRT(+) recombinants recovered from ERCC1(-) cells using targeting vectors with long terminal non-homologies-almost complete ablation of gap repair and single-reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1(+) cells . These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non-homologous tails from invading homologous strands. EMBO J, 2000 Oct 16, 19(20), 5514 - 24 She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p; Bohl F et al.; RNA localization is a widespread mechanism to achieve localized protein synthesis . In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching . ASH1 mRNA localization depends on four independently acting sequences ('zipcodes') within the mRNA . In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization . Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro . She2p can interact with She3 protein via She3p's C-terminus and becomes localized to the daughter cell tip upon ASH1 expression . The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p . She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA-motor protein adapters. EMBO J, 2000 Oct 16, 19(20), 5502 - 13 Importin-11, a nuclear import receptor for the ubiquitin-conjugating enzyme, UbcM2; Plafker SM et al.; Importins are members of a family of transport receptors (karyopherins) that mediate the nucleocytoplasmic transport of protein and RNA cargoes . We identified importin-11 as a potential new human member of this family, on the basis of limited similarity to the Saccharomyces cerevisiae protein, Lph2p, and cloned the complete open reading frame . Importin-11 interacts with the Ran GTPase, and constitutively shuttles between the nuclear and cytoplasmic compartments . A yeast dihybrid screen identified UbcM2, an E2-type ubiquitin-conjugating enzyme, as a binding partner and potential transport cargo for importin-11 . Importin-11 and UbcM2 interact directly, and the complex is disassembled by Ran:GTP but not by Ran:GDP . UbcM2 is constitutively nuclear and shuttles between the nuclear and cytoplasmic compartments . Nuclear import of UbcM2 requires Ran and importin-11, and is inhibited by wheatgerm agglutinin, energy depletion or dominant interfering mutants of Ran and importin-beta . These data establish importin-11 as a new member of the karyopherin family of transport receptors, and identify UbcM2 as a nuclear member of the E2 ubiquitin-conjugating enzyme family. EMBO J, 2000 Oct 16, 19(20), 5362 - 75 The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts; Wirbelauer C et al.; The ubiquitin protein ligase SCF(Skp2) is composed of Skp1, Cul1, Roc1/Rbx1 and the F-box protein Skp2, the substrate-recognition subunit . Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re-enter the cycle . Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen-independent S-phase entry . Hence, mechanisms must exist for limiting Skp2 protein expression during the G(0)/G(1) phases . Here we show that Skp2 is degraded by the proteasome in G(0)/G(1) and is stabilized when cells re-enter the cell cycle . Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum-deprived cells induces Skp2 expression . Furthermore, recombinant Cul1-Roc1/Rbx1-Skp1 complexes can catalyse Skp2 ubiquitylation in vitro . These results suggest that degradation of Skp2 in G(0)/G(1) is mediated, at least in part, by an autocatalytic mechanism involving a Skp2-bound Cul1-based core ubiquitin ligase and imply a role for this mechanism in the suppression of SCF(Skp2) ubiquitin protein ligase function during the G(0)/G(1) phases of the cell cycle. Cell, 2000 Sep 15, 102(6), 765 - 75 Identification of a regulated pathway for nuclear pre-mRNA turnover; Bousquet-Antonelli C et al.; We have identified a nuclear pathway that rapidly degrades unspliced pre-mRNAs in yeast . This involves 3'-->5' degradation by the exosome complex and 5'-->3' degradation by the exonuclease Rat1p . 3'-->5' degradation is normally the major pathway and is regulated in response to carbon source . Inhibition of pre-mRNA degradation resulted in increased levels of pre-mRNAs and spliced mRNAs . When splicing was inhibited by mutation of a splicing factor, inhibition of turnover resulted in 20- to 50-fold accumulation of pre-mRNAs, accompanied by increased mRNA production . Splicing of a reporter construct with a 3' splice site mutation was also increased on inhibition of turnover, showing competition between degradation and splicing . We propose that nuclear pre-mRNA turnover represents a novel step in the regulation of gene expression. Cell, 2000 Sep 15, 102(6), 745 - 51 swi1 and swi3 perform imprinting, pausing, and termination of DNA replication in S . pombe; Dalgaard JZ et al.; The developmental program of cell-type switching of S . pombe requires a strand-specific imprinting event at the mating-type locus (mat1) . Imprinting occurs only when mat1 is replicated in a specific direction and requires several trans-acting factors . This work shows (1) that the factors swi1p and swi3p act by pausing the replication fork at the imprinting site; and (2) that swi1p and swi3p are involved in termination at the mat1-proximal polar-terminator of replication (RTS1) . A genetic screen to identify termination factors identified an allele that separated pausing/imprinting and termination functions of swip . These results suggest that swi1p and swi3p promote imprinting in novel ways both by pausing replication at mat1 and by terminating replication at RTS1. Mol Cell, 2000 Sep, 6(3), 751 - 6 Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins; Zhou P et al.; The functional characterization of a specific gene, or its protein product, often relies on assessing the consequences of its elimination, usually accomplished by gene knockout, ribozyme, antisense, or RNA-mediated interference (RNAi) technologies . The selective degradation of cellular proteins is mediated primarily by the ubiquitin-proteasome pathway . Manipulation of the ubiquitin-dependent proteolytic machinery to eliminate specific gene products at the protein level has been previously attempted with some success in vitro; however, the in vivo efficacy of this approach has not yet been achieved . Here we report successful engineering of the substrate receptor of a major ubiquitin-proteolytic machinery to direct the degradation of otherwise stable cellular proteins both in yeast and in mammalian cells. Mol Cell, 2000 Sep, 6(3), 649 - 59 Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage; Costanzo V et al.; Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage . A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation . DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner . Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45 . The checkpoint is ATM dependent . Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15 . Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication . This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism. Eur J Biochem, 2000 Nov, 267(21), 6423 - 7 A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase; Nishida T et al.; A novel Smt3-specific isopeptidase, SMT3IP1, was cloned using a yeast two-hybrid screen with Smt3b as bait . The clone, named SMT3IP1 (Smt3-specific isopeptidase 1), which bound to Smt3b but not SUMO-1 in the two-hybrid system, was distantly related to budding yeast Saccharomyces cerevisiae Ulp1, human SENP1 or human SUSP1 . The catalytic domains in the C-terminal region were very similar, but the N-terminal region was quite different to other enzymes . The cysteine, histidine and asparatic acid residues in the catalytic domains were conserved . SMT3IP1 expressed by the baculovirus-expression system had the ability to cleave SUMO-1 or Smt3b from SUMO-1/RanGAP1 or Smt3b/RanGAP1 conjugates, respectively, and the activity was a little stronger towards the Smt3b conjugate than towards the SUMO-1 conjugate . Furthermore, the enzyme bound more strongly to Smt3a and Smt3b than to SUMO-1 in vitro . The enzyme did not cleave Nedd8 from Nedd8/cullin-1 . Nor did it cleave ubiquitin from ubiquitinated p53 . SMT3IP1 was localized almost exclusively at the nucleolus during interphase . The N-terminal sequence was responsible for the nucleolar localization of this enzyme . Whether SMT3IP1 functions in the nucleolus or just stays there before it functions in the nucleus, as shown in the case of CDC14 phosphatase, remains to be elucidated. Int J Oncol, 2000 Nov, 17(5), 947 - 54 Expression of cell cycle regulator genes in KB, a human squamous cell carcinoma cell line, after irradiation; Uno M et al.; DNA damage induced by irradiation causes overexpression of the p53 gene, and subsequently the upregulation of p53 downstream genes involved in cell cycle modification . Irradiated malignant cells which possess wild-type p53 have been known to undergo G1 arrest due to p21/Cip1/Waf1 upregulation . Other p53 downstream genes related to the modification of the cell cycle such as gadd45 may cause G2 arrest . Many of the genes which regulate the cell cycle progression have been identified, including the G1 phase specific ink4 family of cyclin-dependent kinase inhibitors (CDK-I), another group of CDK-Is, which affect the cyclin-CDK complexes ubiquitously, and S/G2 accelerator genes . The sequential changes in these cell cycle regulator genes after irradiation has not been clarified . We analyzed the appearance of the apoptotic fraction and cell cycle perturbation after irradiation using KB, a human squamous cell carcinoma line derived from oral floor, and examined the alteration of gene expression for cell cycle regulator genes . The KB cells proceeded to undergo apoptosis in a time and dose dependent manner after irradiation and showed G2 arrest accompanied by upregulation of p53, ubiquitous CDK-Is, and S and G2 accelerator genes. J Comb Chem, 2000 Sep-Oct, 2(5), 522 - 36 Solid-phase synthesis of a farnesylated CaaX peptide library: inhibitors of the Ras CaaX endoprotease; Dolence EK et al.; A solid-phase method, based on Kaiser's p-benzophenone oxime resin, was developed for the synthesis of a series of N-acetyl-S-(E, E-farnesylated) Ca(1)a(2)X tetrapeptides as potential inhibitors of recombinant Ras and a-factor converting enzyme (RCE) . N-Acetyl-S-(E, E-farnesyl)-L-cysteine was coupled to resin-bound a(1)a(2) dipeptide using HOBt/DCC activation in conjunction with N-BOC chemistry . The protected farnesylated tripeptide was cleaved from the resin with simultaneous addition of the X residue by treating the resin-bound farnesylated Ca(1)a(2) tripeptide with L-amino acid benzyl ester tosylates under mildly acidic conditions . The benzyl ester was saponified, and the resulting carboxylate precipitated by ether to afford a library of tetrapeptides as a mixture of diastereomers at the cysteine center . The peptides were evaluated as inhibitors of recombinant yeast RCE endoprotease (yRCE) to obtain information about the affinity of the enzyme for the a(1)a(2)X portion of the Ca(1)a(2)X moiety. Mol Biol Cell, 2000 Oct, 11(10), 3601 - 15 Role for the silencing protein Dot1 in meiotic checkpoint control; San-Segundo PA et al.; During the meiotic cell cycle, a surveillance mechanism called the "pachytene checkpoint" ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective . The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae . In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products . Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2 . In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus . In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids . In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1. Mol Biol Cell, 2000 Oct, 11(10), 3525 - 37 Mps1p regulates meiotic spindle pole body duplication in addition to having novel roles during sporulation; Straight PD et al.; Sporulation in yeast requires that a modified form of chromosome segregation be coupled to the development of a specialized cell type, a process akin to gametogenesis . Mps1p is a dual-specificity protein kinase essential for spindle pole body (SPB) duplication and required for the spindle assembly checkpoint in mitotically dividing cells . Four conditional mutant alleles of MPS1 disrupt sporulation, producing two distinct phenotypic classes . Class I alleles of mps1 prevent SPB duplication at the restrictive temperature without affecting premeiotic DNA synthesis and recombination . Class II MPS1 alleles progress through both meiotic divisions in 30-50% of the population, but the asci are incapable of forming mature spores . Although mutations in many other genes block spore wall formation, the cells produce viable haploid progeny, whereas mps1 class II spores are unable to germinate . We have used fluorescently marked chromosomes to demonstrate that mps1 mutant cells have a dramatically increased frequency of chromosome missegregation, suggesting that loss of viability is due to a defect in spindle function . Overall, our cytological data suggest that MPS1 is required for meiotic SPB duplication, chromosome segregation, and spore wall formation. Mol Biol Cell, 2000 Oct, 11(10), 3469 - 84 Identification of a novel saturable endoplasmic reticulum localization mechanism mediated by the C-terminus of a Dictyostelium protein disulfide isomerase; Monnat J et al.; Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms . Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood . We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum . Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope . Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes . The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms . Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization . Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D . discoideum and Saccharomyces cerevisiae. Mol Biol Cell, 2000 Oct, 11(10), 3425 - 39 Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes; Verma R et al.; Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex . Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells . Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1 . Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-gamma-S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap . To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry . Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex) . Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction . Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies. Mol Biol Cell, 2000 Oct, 11(10), 3381 - 96 Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay; Miller BR et al.; The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleoporins . We have developed a novel two-step "organelle trap" assay involving affinity selection and in vitro pore assembly . In the first step, soluble proteins derived from Xenopus egg extracts are applied to a column containing a ligand of interest . The bound proteins are then tagged by biotinylation and eluted . In the second step, potential nucleoporins are selected for by virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores . The incorporated proteins are then recognized by their biotin tag . Here we use the lectin wheat germ agglutinin (WGA) as ligand; WGA inhibits nuclear transport and has been shown to directly bind three known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues . Under reduced-stringency conditions, three additional proteins bind to WGA-Sepharose and are revealed by the organelle trap assay . We identified all three as partner nucleoporins . Two were discovered to be Xenopus Nup93 and Nup205 . The third is a novel vertebrate nucleoporin, Nup188 . This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205 . The Nup93-Nup188-Nup205 complex does not bind directly to WGA but binds indirectly via the N-acetylglucosamine-modified nucleoporins . A gene encoding human Nup188 was also identified . The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein-protein interactions illustrates the power of the two-step organelle trap assay and identifies new building blocks for constructing the nuclear pore. Mol Biol Cell, 2000 Oct, 11(10), 3365 - 80 The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways; Amerik AY et al.; The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates . Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome . Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned . Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway . In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole . Four of the six Did proteins are structurally related, suggesting an overlap in function . In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution . However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole . These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole. Cell Mol Life Sci, 2000 Aug, 57(8-9), 1184 - 92 Regulation of gene expression by transcription factor acetylation; Bannister AJ et al.; In the nucleus, DNA is tightly packaged into higher-order structures, generating an environment that is highly repressive towards DNA processes such as gene transcription . Acetylation of lysine residues within proteins has recently emerged as a major mechanism used by the cell to overcome this repression . Acetylation of non-histone proteins, including transcription factors, as well as histones, appears to be involved in this process . Like phosphorylation, acetylation is a dynamic process that can regulate protein-DNA and protein-protein interactions . Moreover, a conserved domain, the bromodomain, has been implicated in the binding of acetylated peptides, suggesting a role for acetylation in intracellular signalling. Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 1105 - 11 Ku70 can translocate to the nucleus independent of Ku80 translocation and DNA-PK autophosphorylation; Koike M et al.; Ku plays an important role in multiple nuclear processes, e.g., DNA repair, chromosome maintenance, and transcriptional regulation . Although some evidence suggests that the nuclear translocation of Ku plays a key role in regulating the function of Ku, the mechanism is poorly understood . Using the site-directed mutagenesis technique, we demonstrate here that Ku70 can translocate to the nucleus without heterodimerization with Ku80 . The nuclear accumulation of Ku70 mutants of the nuclear localization signal, which retained their binding ability with Ku80, was diminished . On the other hand, Ku70 mutants which lacked the ability to bind with Ku80 could translocate to the nuclei . Human Ku70, when transfected, accumulated within the nuclei of hamster xrs-6 cells which had undetectable DNA-PK activity and Ku80 . Ku70 and Ku80 mutants of DNA-PK phosphorylation sites showed normal heterodimerization and nuclear translocation . These findings also support the idea that Ku70 can translocate to the nucleus independent of DNA-PK autophosphorylation . Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 999 - 1004 A pivotal role of Zn-binding residues in the function of the copper chaperone for SOD1; Endo T et al.; A Cu chaperone for SOD1 (CCS) is required for the incorporation of copper ion into the protein . To investigate the roles of the conserved metal-binding residues in CCS, we introduced amino acid substitutions into human CCS and examined the function of the mutant CCS by transforming a mutant yeast strain, SY2950, which lacks the lys7 gene, a yeast orthologue of the mammalian CCS . Mutant CCS in which amino acid residues His147 and Asp167 were substituted by Ala exhibited a decreased ability to complement the growth of SY2950 under Lys-deficient conditions . This is because the mutations made the human CCS function in a less efficient manner, especially under metal-restricted conditions, leaving Cu,Zn-SOD in an apo-form . Since the His and Asp residues are both responsible for binding Zn which would serve to maintain the folded structure, the structural integrity supported by the coordinated Zn ion would be essential for CCS function . Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11377 - 82 A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse; Conforti L et al.; Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S) . Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively . They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S) . The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction . Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain . This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld . The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland . The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice . However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene . These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism. Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11303 - 6 Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis; Yamauchi T et al.; PCAF plays a role in transcriptional activation, cell-cycle arrest, and cell differentiation in cultured cells . PCAF contributes to transcriptional activation by acetylating chromatin and transcription factors through its intrinsic histone acetylase activity . In this report, we present evidence for the in vivo function of PCAF and the closely related PCAF-B/GCN5 . Mice lacking PCAF are developmentally normal without a distinct phenotype . In PCAF null-zygous mice, protein levels of PCAF-B/GCN5 are drastically elevated in lung and liver, where PCAF is abundantly expressed in wild-type mice, suggesting that PCAF-B/GCN5 functionally compensates for PCAF . In contrast, animals lacking PCAF-B/GCN5 die between days 9.5 and 11.5 of gestation . Normally, PCAF-B/GCN5 mRNA is expressed at high levels already by day 8, whereas PCAF mRNA is first detected on day 12.5, which may explain, in part, the distinct knockout phenotypes . These results provide evidence that PCAF and PCAF-B/GCN5 play distinct but functionally overlapping roles in embryogenesis. Mol Cell Biol, 2000 Nov, 20(21), 8254 - 63 Inhibition of p300 histone acetyltransferase by viral interferon regulatory factor; Li M et al.; Kaposi's sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi's sarcomas, body cavity-based lymphomas, and some forms of Castleman's disease . The K9 open reading frame of KSHV encodes a viral interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon-mediated signal transduction and as an oncogene to induce cell growth transformation . We demonstrate that KSHV vIRF directly interacts with cellular transcriptional coactivator p300 and displaces p300/CBP-associated factor from p300 complexes . This interaction inhibits the histone acetyltransferase activity of p300, resulting in drastic reduction of nucleosomal histone acetylation and alteration of chromatin structure . As a consequence, vIRF expression markedly alters cellular cytokine expression, which is regulated by acetylation of nucleosomal histones . These results demonstrate that KSHV vIRF interacts with and inhibits the p300 transcriptional coactivator to circumvent the host antiviral immune response and to induce a global alteration of cellular gene expression . These studies also illustrate how a cellular gene captured by a herpesvirus has evolved several functions that suit the needs of the virus. Mol Cell Biol, 2000 Nov, 20(21), 8230 - 43 Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA; van Hoof A et al.; One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body . Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype . In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA . Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome . In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation . We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome . Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein . This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability . In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation . We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene . These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation. Mol Cell Biol, 2000 Nov, 20(21), 8143 - 56 Loss of a protein phosphatase 2A regulatory subunit (Cdc55p) elicits improper regulation of Swe1p degradation; Yang H et al.; CDC55 encodes a Saccharomyces cerevisiae protein phosphatase 2A (PP2A) regulatory subunit . cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, but cdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology . Furthermore, cdc55-null cells exhibit a hyperphosphorylation of Y19 . For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation . We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences . By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrested cdc55-null cells . This excess Swe1p in cdc55-null cells is the result of ectopic stabilization of this protein during G(2) and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells . We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype. Mol Cell Biol, 2000 Nov, 20(21), 7845 - 52 The abundance of Met30p limits SCF(Met30p) complex activity and is regulated by methionine availability