bluuebunny/crossref_metadata_2025 huggingface dataset:
A Fission Yeast Homolog of Int-6, the Mammalian Oncoprotein and eIF3 Subunit, Induces Drug Resistance when Overexpressed
Authors: Crane Richard , Craig Randa , Murray Rachael , Dunand-Sauthier Isabelle , Humphrey Tim , Norbury Chris
Published: July 2013

Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.
Fission Yeast Int6 Is Not Essential for Global Translation Initiation, but Deletion ofint6+Causes Hypersensitivity to Caffeine and Affects Spore Formation
Authors: Bandyopadhyay Amitabha , Matsumoto Tomohiro , Maitra Umadas
Published: July 2013

Mammalian INT6 protein has been considered to be a subunit of the eukaryotic translation initiation factor, eIF3. The Int6 locus is also known as a common integration site of mousemammary tumor virus (MMTV). However, the function of Int6 in translation initiation and the mechanism of Int6-mediated tumor induction are yet to be explored. In this study, the fission yeast, Schizosaccharomyces pombe, int6+, which is 43% identical to the mammalian counterpart, was deleted. Despite the evidence that the majority of Int6 protein was associated with 40S particles in this organism, strains lackingint6+(Δ int6) were viable and showed only moderate inhibition in the rate of in vivo global protein synthesis. Polysome profile analysis showed no apparent defects in translation initiation. Δ int6 exhibited a hypersensitivity to caffeine, which could be suppressed by the addition of sorbitol to the growth medium. This and other phenotypes would imply that int6+is required for the integrity of cell membrane. In meiosis, Δ int6produced incomplete tetrads frequently. High dosage expression of a truncated mutant of int6+conferred a hypersensitivity to caffeine, but did not cause the defect in meiosis. A possible link between the function ofint6+and theΔ int6-phenotypes is discussed.
Requirements of Multiple Domains of SLI-1, aCaenorhabditis elegansHomologue of c-Cbl, and an Inhibitory Tyrosine in LET-23 in Regulating Vulval Differentiation
Authors: Yoon Charles H. , Chang Chieh , Hopper Neil A. , Lesa Giovanni M. , Sternberg Paul W.
Published: July 2013

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin–protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss ofsli-1 but not of another negative regulator,ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin–ligase domain.
Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells
Authors: Herbig Utz , Griffith Jason W. , Fanning Ellen
Published: July 2013

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.
Self-association of Coilin Reveals a Common Theme in Nuclear Body Localization
Authors: Hebert Michael D. , Matera A. Gregory
Published: July 2013

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e.g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.
Identification of Ribonucleotide Reductase Protein R1 as an Activator of Microtubule Nucleation inXenopusEgg Mitotic Extracts
Authors: Takada Saeko , Shibata Takehiko , Hiraoka Yasushi , Masuda Hirohisa
Published: July 2013

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared fromXenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopussperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to γ- and α/β-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.
Mammalian Meiotic Telomeres: Protein Composition and Redistribution in Relation to Nuclear Pores
Authors: Scherthan Harry , Jerratsch Martin , Li Bibo , Smith Susan , Hultén Maj , Lock Tycho , de Lange Titia
Published: July 2013

Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.
The Carboxy-terminal Cysteine of the Tetraspanin L6 Antigen Is Required for Its Interaction with SITAC, a Novel PDZ Protein
Authors: Borrell-Pagès Maria , Fernández-Larrea Juan , Borroto Aldo , Rojo Federico , Baselga José , Arribas Joaquı́n
Published: July 2013

PDZ domains are protein modules that mediate protein-protein interactions. Here, we present the identification and characterization of a protein similar to the recently identified PDZ-containing protein TACIP18, which we have named SITAC (similar to TACIP18). SITAC is preferentially expressed in cells of the digestive tract, associated with intracellular membranes. Despite the high degree of sequence identity between the PDZ domains of TACIP18 and those of SITAC, none of the known ligands of the former shows interaction with the latter, as judged by two-hybrid analysis. SITAC interacts with peptides containing bulky hydrophobic amino acids two positions upstream of the C-terminal residue. Surprisingly, SITAC also shows interaction with peptides ending in C, a previously unacknowledged ability of PDZ domains. The sequence -Y-X-C-COOH, bound in vitro by SITAC, is present in the member of the tetraspanin superfamily, the L6 antigen. Coimmunoprecipitation experiments show that SITAC interacts with L6A, but not with an L6A C-terminal mutant, confirming the capacity of SITAC to interact with proteins ending in C. Confocal analysis shows that the interaction between L6A and SITAC is necessary for the precise colocalization of both molecules in the same subcellular compartment. In summary, the characterization of the protein SITAC has unveiled novel sequences recognized by PDZ domains, and it suggests that L6A is a natural ligand of this PDZ protein.
Schizosaccharomyces pombeRho2p GTPase Regulates Cell Wall α-Glucan Biosynthesis through the Protein Kinase Pck2p
Authors: Calonge Teresa M. , Nakano Kentaro , Arellano Manuel , Arai Ritsuko , Katayama Satoshi , Toda Takashi , Mabuchi Issei , Perez Pilar
Published: July 2013

Schizosaccharomyces pombe rho1+andrho2+genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the twoS. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)β-d-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2Δ andrho2Δ strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of α-d-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2+in wild-type or pck1Δ cells is lethal and causes morphological alterations, actin depolarization, and an increase in α-d-glucan biosynthesis, all of these effects are suppressed in a pck2Δ strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1–3)α-d-glucan synthase. Thus, a rho2Δ mutation, like pck2Δ, is synthetically lethal with mok1–664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1+inrho2Δ cells causes a lethal phenotype that is completely different from that of mok1+overexpression in wild-type cells, and the increase in α-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating α-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.
The TRAPP Complex Is a Nucleotide Exchanger for Ypt1 and Ypt31/32
Authors: Jones Sara , Newman Christina , Liu Fengli , Segev Nava
Published: July 2013

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.
Sbe2p and Sbe22p, Two Homologous Golgi Proteins Involved in Yeast Cell Wall Formation
Authors: Santos Beatriz , Snyder Michael
Published: July 2013

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Althoughsbe2 and sbe22 null mutants are viable,sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First,sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37°C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p–GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 andSBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.
Detergent-insoluble GPI–anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting
Authors: Lipardi Concetta , Nitsch Lucio , Zurzolo Chiara
Published: July 2013

In contrast to Madin–Darby canine kidney cells, Fischer rat thyroid cells deliver the majority of endogenous glycosylphosphatidyl inositol (GPI)–anchored proteins to the basolateral surface. However, we report here that the GPI proteins Placental Alkaline Phosphatase (PLAP) and Neurotrophin Receptor–Placental Alkaline Phosphatase (NTR-PLAP) are apically localized in transfected Fischer rat thyroid cells. In agreement with the “raft hypothesis,” which postulates the incorporation of GPI proteins into glycosphingolipids and cholesterol-enriched rafts, we found that both of these proteins were insoluble in Triton X-100 and floated into the lighter fractions of sucrose density gradients. However, disruption of lipid rafts by removal of cholesterol did not cause surface missorting of PLAP and NTR-PLAP, and the altered surface sorting of these proteins after Fumonisin B1 treatment did not correlate with reduced levels in Triton X-100 –insoluble fractions. Furthermore, in contrast to the GPI-anchored forms of both of these proteins, the secretory and transmembrane forms (in the absence of a basolateral cytoplasmic signal) were sorted to the apical surface without association with lipid microdomains. Together, these data demonstrate that the GPI anchor is required to mediate raft association but is not sufficient to determine apical sorting. They also suggest that signals present in the ectodomain of the proteins play a major role and that lipid rafts may facilitate the recognition of these signals in thetrans-Golgi network, even though they are not required for apical sorting.
Conserved Composition of Mammalian Box H/ACA and Box C/D Small Nucleolar Ribonucleoprotein Particles and Their Interaction with the Common Factor Nopp140
Authors: Yang Yunfeng , Isaac Cynthia , Wang Chen , Dragon François , Pogac̆ić Vanda , Meier U. Thomas
Published: July 2013

Small nucleolar ribonucleoprotein particles (snoRNPs) mainly catalyze the modification of rRNA. The two major classes of snoRNPs, box H/ACA and box C/D, function in the pseudouridylation and 2′-O-methylation, respectively, of specific nucleotides. The emerging view based on studies in yeast is that each class of snoRNPs is composed of a unique set of proteins. Here we present a characterization of mammalian snoRNPs. We show that the previously characterized NAP57 is specific for box H/ACA snoRNPs, whereas the newly identified NAP65, the rat homologue of yeast Nop5/58p, is a component of the box C/D class. Using coimmunoprecipitation experiments, we show that the nucleolar and coiled-body protein Nopp140 interacts with both classes of snoRNPs. This interaction is corroborated in vivo by the exclusive depletion of snoRNP proteins from nucleoli in cells transfected with a dominant negative Nopp140 construct. Interestingly, RNA polymerase I transcription is arrested in nucleoli depleted of snoRNPs, raising the possibility of a feedback mechanism between rRNA modification and transcription. Moreover, the Nopp140-snoRNP interaction appears to be conserved in yeast, because depletion of Srp40p, the yeast Nopp140 homologue, in a conditional lethal strain induces the loss of box H/ACA small nucleolar RNAs. We propose that Nopp140 functions as a chaperone of snoRNPs in yeast and vertebrate cells.
VPS21Controls Entry of Endocytosed and Biosynthetic Proteins into the Yeast Prevacuolar Compartment
Authors: Gerrard Sonja R. , Bryant Nia J. , Stevens Tom H.
Published: July 2013

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class DVPS genes include VPS21,PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.
Essential Functions of Protein Tyrosine Phosphatases Ptp2 and Ptp3 and Rim11 Tyrosine Phosphorylation inSaccharomyces cerevisiaeMeiosis and Sporulation
Authors: Zhan Xiao-Li , Hong Yulong , Zhu Tianqing , Mitchell Aaron P. , Deschenes Robert J. , Guan Kun-Liang
Published: July 2013

Tyrosine phosphorylation plays a central role in eukaryotic signal transduction. In yeast, MAP kinase pathways are regulated by tyrosine phosphorylation, and it has been speculated that other biochemical processes may also be regulated by tyrosine phosphorylation. Previous genetic and biochemical studies demonstrate that protein tyrosine phosphatases (PTPases) negatively regulate yeast MAP kinases. Here we report that deletion of PTP2 and PTP3results in a sporulation defect, suggesting that tyrosine phosphorylation is involved in regulation of meiosis and sporulation. Deletion of PTP2 and PTP3 blocks cells at an early stage of sporulation before premeiotic DNA synthesis and induction of meiotic-specific genes. We observed that tyrosine phosphorylation of several proteins, including 52-, 43-, and 42-kDa proteins, was changed in ptp2Δptp3Δ homozygous deletion cells under sporulation conditions. The 42-kDa tyrosine-phosphorylated protein was identified as Mck1, which is a member of the GSK3 family of protein kinases and previously known to be phosphorylated on tyrosine. Mutation of MCK1 decreases sporulation efficiency, whereas mutation of RIM11, another GSK3 member, specifically abolishes sporulation; therefore, we investigated regulation of Rim11 by Tyr phosphorylation during sporulation. We demonstrated that Rim11 is phosphorylated on Tyr-199, and the Tyr phosphorylation is essential for its in vivo function, although Rim11 appears not to be directly regulated by Ptp2 and Ptp3. Biochemical characterizations indicate that tyrosine phosphorylation of Rim11 is essential for the activity of Rim11 to phosphorylate substrates. Our data demonstrate important roles of protein tyrosine phosphorylation in meiosis and sporulation
Physical State of the Extracellular Matrix Regulates the Structure and Molecular Composition of Cell-Matrix Adhesions
Authors: Katz Ben-Zion , Zamir Eli , Bershadsky Alexander , Kam Zvi , Yamada Kenneth M. , Geiger Benjamin
Published: July 2013

This study establishes that the physical state of the extracellular matrix can regulate integrin-mediated cytoskeletal assembly and tyrosine phosphorylation to generate two distinct types of cell-matrix adhesions. In primary fibroblasts, α5β1integrin associates mainly with fibronectin fibrils and forms adhesions structurally distinct from focal contacts, independent of actomyosin-mediated cell contractility. These “fibrillar adhesions” are enriched in tensin, but contain low levels of the typical focal contact components paxillin, vinculin, and tyrosine-phosphorylated proteins. However, when the fibronectin is covalently linked to the substrate, α5β1integrin forms highly tyrosine-phosphorylated, “classical” focal contacts containing high levels of paxillin and vinculin. These experiments indicate that the physical state of the matrix, not just its molecular composition, is a critical factor in defining cytoskeletal organization and phosphorylation at adhesion sites. We propose that molecular organization of adhesion sites is controlled by at least two mechanisms: 1) specific integrins associate with their ligands in transmembrane complexes with appropriate cytoplasmic anchor proteins (e.g., fibronectin–α5β1integrin–tensin complexes), and 2) physical properties (e.g., rigidity) of the extracellular matrix regulate local tension at adhesion sites and activate local tyrosine phosphorylation, recruiting a variety of plaque molecules to these sites. These mechanisms generate structurally and functionally distinct types of matrix adhesions in fibroblasts.
Polarized Sphingolipid Transport from the Subapical Compartment Changes during Cell Polarity Development
Authors: van IJzendoorn Sven C.D. , Hoekstra Dick
Published: July 2013

The subapical compartment (SAC) plays an important role in the polarized transport of proteins and lipids. In hepatoma-derived HepG2 cells, fluorescent analogues of sphingomyelin and glucosylceramide are sorted in the SAC. Here, evidence is provided that shows that polarity development is regulated by a transient activation of endogenous protein kinase A and involves a transient activation of a specific membrane transport pathway, marked by the trafficking of the labeled sphingomyelin, from the SAC to the apical membrane. This protein kinase A–regulated pathway differs from the apical recycling pathway, which also traverses SAC. After reaching optimal polarity, the direction of the apically activated pathway switches to one in the basolateral direction, without affecting the apical recycling pathway.
The p42/p44 MAP Kinase Pathway Prevents Apoptosis Induced by Anchorage and Serum Removal
Authors: Le Gall Maude , Chambard Jean-Claude , Breittmayer Jean-Philippe , Grall Dominique , Pouysségur Jacques , Van Obberghen-Schilling Ellen
Published: July 2013

Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor– and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin–Darby canine kidney cells stably expressing an estrogen-inducible activated–Raf-1 construct (ΔRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal–regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the ΔRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.
Phosphatidylinositol 4,5-Bisphosphate Regulates Two Steps of Homotypic Vacuole Fusion
Authors: Mayer Andreas , Scheglmann Dietrich , Dove Stephen , Glatz Alexandra , Wickner William , Haas Albert
Published: July 2013

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are important for fusion. Monoclonal antibodies to PI(4,5)P2, neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4,5)P2restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P2synthesis are not required. PI(4,5)P2is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (α-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P2is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P2controls two steps of vacuole fusion.
Clathrin-mediated Endocytosis of MUC1 Is Modulated by Its Glycosylation State
Authors: Altschuler Yoram , Kinlough Carol L. , Poland Paul A. , Bruns James B. , Apodaca Gerard , Weisz Ora A. , Hughey Rebecca P.
Published: July 2013

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylatedO-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.
Restoration of Tight Junction Structure and Barrier Function by Down-Regulation of the Mitogen-activated Protein Kinase Pathway in Ras-transformed Madin-Darby Canine Kidney Cells
Authors: Chen Yan-hua , Lu Qun , Schneeberger Eveline E. , Goodenough Daniel A.
Published: July 2013

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell–cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin–mediated cell–cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell–cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell–cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.
Mechanisms ofsod2Gene Amplification inSchizosaccharomyces pombe
Authors: Albrecht Elizabeth B. , Hunyady Aaron B. , Stark George R. , Patterson Thomas E.
Published: July 2013

Gene amplification in eukaryotes plays an important role in drug resistance, tumorigenesis, and evolution. TheSchizosaccharomyces pombe sod2 gene provides a useful model system to analyze this process. sod2 is near the telomere of chromosome I and encodes a plasma membrane Na+(Li+)/H+antiporter. Whensod2 is amplified, S. pombe survives otherwise lethal concentrations of LiCl, and >90% of the amplifiedsod2 genes are found in 180- and 225-kilobase (kb) linear amplicons. The sequence of the novel joint of the 180-kb amplicon indicates that it is formed by recombination between homologous regions near the telomeres of the long arm of chromosome I and the short arm of chromosome II. The 225-kb amplicon, isolated three times more frequently than the 180-kb amplicon, is a palindrome derived from a region near the telomere of chromosome I. The center of symmetry of this palindrome contains an inverted repeat consisting of two identical 134-base pair sequences separated by a 290-base pair spacer. LiCl-resistant mutants arise 200–600 times more frequently in strains deficient for topoisomerases or DNA ligase activity than in wild-type strains, but the mutant cells contain the same amplicons. These data suggest that amplicon formation may begin with DNA lesions such as breaks. In the case of the 225-kb amplicon, the breaks may lead to a hairpin structure, which is then replicated to form a double-stranded linear amplicon, or to a cruciform structure, which is then resolved to yield the same amplicon.
Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development
Authors: Terasaki Mark
Published: July 2013

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.
Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells
Authors: Kook Seunghyi , Shim Sang Ryeol , Choi Soo Jeon , Ahnn Joohong , Kim Jae Il , Eom Soo Hyun , Jung Yong Keun , Paik Sang Gi , Song Woo Keun
Published: July 2013

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell–cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD416G and DSPD748G) in vitro, and point mutations substituting the Asp of DVPD416G and DSPD748G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD416G generated a 74-kDa fragment, which was in turn cleaved at DSPD748G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas–paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.
Apg5p Functions in the Sequestration Step in the Cytoplasm-to-Vacuole Targeting and Macroautophagy Pathways
Authors: George Michael D. , Baba Misuzu , Scott Sidney V. , Mizushima Noboru , Garrison Brian S. , Ohsumi Yoshinori , Klionsky Daniel J.
Published: July 2013

The cytoplasm-to-vacuole targeting (Cvt) pathway and macroautophagy are dynamic events involving the rearrangement of membrane to form a sequestering vesicle in the cytosol, which subsequently delivers its cargo to the vacuole. This process requires the concerted action of various proteins, including Apg5p. Recently, it was shown that another protein required for the import of aminopeptidase I (API) and autophagy, Apg12p, is covalently attached to Apg5p through the action of an E1-like enzyme, Apg7p. We have undertaken an analysis of Apg5p function to gain a better understanding of the role of this novel nonubiquitin conjugation reaction in these import pathways. We have generated the first temperature-sensitive mutant in the Cvt pathway, designated apg5ts. Biochemical analysis of API import in theapg5tsstrain confirmed that Apg5p is directly required for the import of API via the Cvt pathway. By analyzing the stage of API import that is blocked in theapg5tsmutant, we have determined that Apg5p is involved in the sequestration step and is required for vesicle formation and/or completion.
Sec24p and Iss1p Function Interchangeably in Transport Vesicle Formation from the Endoplasmic Reticulum inSaccharomyces cerevisiae
Authors: Kurihara Tatsuo , Hamamoto Susan , Gimeno Ruth E. , Kaiser Chris A. , Schekman Randy , Yoshihisa Tohru
Published: July 2013

The Sec23p/Sec24p complex functions as a component of the COPII coat in vesicle transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protein of 926 amino acids (YIL109C), and a close homologue, ISS1 (YNL049C), which is 55% identical to SEC24. SEC24 is essential for vesicular transport in vivo because depletion of Sec24p is lethal, causing exaggeration of the endoplasmic reticulum and a block in the maturation of carboxypeptidase Y. Overproduction of Sec24p suppressed the temperature sensitivity of sec23-2, and overproduction of both Sec24p and Sec23p suppressed the temperature sensitivity of sec16-2. SEC24 gene disruption could be complemented by overexpression ofISS1, indicating functional redundancy between the two homologous proteins. Deletion of ISS1 had no significant effect on growth or secretion; however, iss1Δ mutants were found to be synthetically lethal with mutations in the v-SNARE genes SEC22 and BET1. Moreover, overexpression of ISS1 could suppress mutations inSEC22. These genetic interactions suggest that Iss1p may be specialized for the packaging or the function of COPII v-SNAREs. Iss1p tagged with His6at its C terminus copurified with Sec23p. Pure Sec23p/Iss1p could replace Sec23p/Sec24p in the packaging of a soluble cargo molecule (α-factor) and v-SNAREs (Sec22p and Bet1p) into COPII vesicles. Abundant proteins in the purified vesicles produced with Sec23p/Iss1p were indistinguishable from those in the regular COPII vesicles produced with Sec23p/Sec24p.
Activation of B-Raf and Regulation of the Mitogen-activated Protein Kinase Pathway by the Goα chain
Authors: Antonelli Valeria , Bernasconi Francesca , Wong Yung H. , Vallar Lucia
Published: July 2013

Many receptors coupled to the pertussis toxin-sensitive Gi/oproteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the α chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Gαoto regulate MAPK activity by transient expression of the activated mutant Gαo-Q205L in Chinese hamster ovary cells. Gαo-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Gαo-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Gαostimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Gαo. Moreover, Gαo-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Gαocan regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Gαocaused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Gαoinduces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.
Time-Lapse Video Microscopy Analysis Reveals Astral Microtubule Detachment in the Yeast Spindle Pole Mutantcnm67
Authors: Hoepfner Dominic , Brachat Arndt , Philippsen Peter
Published: July 2013

Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.
A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1p
Authors: Paluh Janet L. , Nogales Eva , Oakley Berl R. , McDonald Kent , Pidoux Alison L. , Cande W. Z.
Published: July 2013

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp)Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component.