bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Dissection and Hierarchy of Tubulin-folding Cofactor Homologues in Fission Yeast
Authors: Radcliffe Pippa A. , Hirata Dai , Vardy Leah , Toda Takashi
Published: July 2013

We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11Bcontains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21Edoes not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11Binteracts with both α-tubulin and Alp21E, but not with the cofactor D homologue Alp1, whereas Alp21Ealso interacts with Alp1D. The cellular amount of α-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11Bresults in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of α-tubulin. Both full-length Alp11Band the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to α-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21+or alp1+, whereas alp21deletion is rescued by overexpression ofalp1+but notalp11+. Finally, the alp1mutant is not complemented by either alp11+or alp21+. The results suggest that cofactors operate in a linear pathway (Alp11B-Alp21E-Alp1D), each with distinct roles.
A Role for the GSG Domain in Localizing Sam68 to Novel Nuclear Structures in Cancer Cell Lines
Authors: Chen Taiping , Boisvert François-Michel , Bazett-Jones David P. , Richard Stéphane
Published: July 2013

The GSG (GRP33, Sam68, GLD-1) domain is a protein module found in an expanding family of RNA-binding proteins. The numerous missense mutations identified genetically in the GSG domain support its physiological role. Although the exact function of the GSG domain is not known, it has been shown to be required for RNA binding and oligomerization. Here it is shown that the Sam68 GSG domain plays a role in protein localization. We show that Sam68 concentrates into novel nuclear structures that are predominantly found in transformed cells. These Sam68 nuclear bodies (SNBs) are distinct from coiled bodies, gems, and promyelocytic nuclear bodies. Electron microscopic studies show that SNBs are distinct structures that are enriched in phosphorus and nitrogen, indicating the presence of nucleic acids. A GFP-Sam68 fusion protein had a similar localization as endogenous Sam68 in HeLa cells, diffusely nuclear with two to five SNBs. Two other GSG proteins, the Sam68-like mammalian proteins SLM-1 and SLM-2, colocalized with endogenous Sam68 in SNBs. Different GSG domain missense mutations were investigated for Sam68 protein localization. Six separate classes of cellular patterns were obtained, including exclusive SNB localization and association with microtubules. These findings demonstrate that the GSG domain is involved in protein localization and define a new compartment for Sam68, SLM-1, and SLM-2 in cancer cell lines.
The Conserved Core of a HumanSIR2Homologue Functions in Yeast Silencing
Authors: Sherman Joyce M. , Stone Elisa M. , Freeman-Cook Lisa L. , Brachmann Carrie B. , Boeke Jef D. , Pillus Lorraine
Published: July 2013

Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure. Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres. Discovery of a gene family ofHomologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2’s silencing mechanism might be conserved. The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger. We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing. Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein’s core can substitute for that of Sir2p, implicating the core as a silencing domain. Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast–human chimeras’ ability to function at only a subset of Sir2p’s target loci. Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions.
Efficient Export of the Vesicular Stomatitis Virus G Protein from the Endoplasmic Reticulum Requires a Signal in the Cytoplasmic Tail That Includes Both Tyrosine-based and Di-acidic Motifs
Authors: Sevier Carolyn S. , Weisz Ora A. , Davis Mollie , Machamer Carolyn E.
Published: July 2013

The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxØ (where x is any amino acid and Ø is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.
Differential Induction of Two p24δ Putative Cargo Receptors upon Activation of a Prohormoneproducing Cell
Authors: Kuiper Roland P. , Waterham Hans R. , Rötter Jutta , Bouw Gerrit , Martens Gerard J. M.
Published: July 2013

The p24 family consists of type I transmembrane proteins that are present abundantly in transport vesicles, may play a role in endoplasmic reticulum-to-Golgi cargo transport, and have been classified into subfamilies named p24α, -β, -γ, and -δ. We previously identified a member of the p24δ subfamily that is coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of the intermediate pituitary during black background adaptation of the amphibian Xenopus laevis(∼30-fold increase in POMC mRNA). In this study, we report on the characterization of this p24δ member (Xp24δ2) and on the identification and characterization of a second member (Xp24δ1) that is also expressed in the melanotrope cells and that has 66% amino acid sequence identity to Xp24δ2. The two p24δ members are ubiquitously expressed, but Xp24δ2is neuroendocrine enriched. During black background adaptation, the amount of the Xp24δ2protein in the intermediate pituitary was increased ∼25 times, whereas Xp24δ1protein expression was increased only 2.5 times. Furthermore, the level of Xp24δ2mRNA was ∼5-fold higher in the melanotrope cells of black-adapted animals than in those of white-adapted animals, whereas Xp24δ1mRNA expression was not induced. Therefore, the expression of Xp24δ2specifically correlates with the expression of POMC. Together, our findings suggest that p24δ proteins have a role in selective protein transport in the secretory pathway.
Evidence for a Novel Affinity Mechanism of Motor-assisted Transport Along Microtubules
Authors: Wada Yuuko , Hamasaki Toshikazu , Satir Peter
Published: July 2013

In microtubule (MT) translocation assays, using colloidal gold particles coupled to monoclonal tubulin antibodies to mark positions along MTs, we found that relative motion is possible between the gold particle and an MT, gliding on dynein or kinesin. Such motion evidently occurred by an affinity release and rebinding mechanism that did not require motor activity on the particle. As the MTs moved, particles drifted to the trailing edge of the MT and then were released. Sometimes the particles transferred from one MT to another, moving orthogonally. Although motion of the particles was uniformly rearward, movement was toward the (−) or (+) end of the MT, depending on whether dynein or kinesin, respectively, was used in the assay. These results open possibilities for physiological mechanisms of organelle and other movement that, although dependent on motor-driven microtubule transport, do not require direct motor attachment between the organelle and the microtubule. Our observations on the direction of particle drift and time of release may also provide confirmation in a dynamic system for the conclusion that β tubulin is exposed at the (+) end of the MT.
Organization of the Yeast Golgi Complex into at Least Four Funtionally Distinct Compartments
Authors: Brigance William T. , Barlowe Charles , Graham Todd R.
Published: July 2013

Pro-α-factor (pro-αf) is posttranslationally modified in the yeast Golgi complex by the addition of α1,6-, α1,2-, and α1,3-linked mannose to N-linked oligosaccharides and by a Kex2p-initiated proteolytic processing event. Previous work has indicated that the α1,6- and α1,3-mannosylation and Kex2p-dependent processing of pro-αf are initiated in three distinct compartments of the Golgi complex. Here, we present evidence that α1,2-mannosylation of pro-αf is also initiated in a distinct Golgi compartment. Linkage-specific antisera and an endo-α1,6-d-mannanase (endoM) were used to quantitate the amount of each pro-αf intermediate during transport through the Golgi complex. We found that α1,6-, α1,2-, and α1,3-mannose were sequentially added to pro-αf in a temporally ordered manner, and that the intercompartmental transport factor Sec18p/N-ethylmaleimide-sensitive factor was required for each step. The Sec18p dependence implies that a transport event was required between each modification event. In addition, most of the Golgi-modified pro-αf that accumulated in brefeldin A-treated cells received only α1,6-mannosylation as did ∼50% of pro-αf transported to the Golgi in vitro. This further supports the presence of an early Golgi compartment that houses an α1,6-mannosyltransferase but lacks α1,2-mannosyltransferase activity in vivo. We propose that the α1,6-, α1,2-, and α1,3-mannosylation and Kex2p-dependent processing events mark the cis, medial,trans, and trans-Golgi network of the yeast Golgi complex, respectively.
Kinetic Analysis of a Molecular Model of the Budding Yeast Cell Cycle
Authors: Chen Katherine C. , Csikasz-Nagy Attila , Gyorffy Bela , Val John , Novak Bela , Tyson John J.
Published: July 2013

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast.
Association of Mouse Actin-binding Protein 1 (mAbp1/SH3P7), an Src Kinase Target, with Dynamic Regions of the Cortical Actin Cytoskeleton in Response to Rac1 Activation
Authors: Kessels Michael M. , Engqvist-Goldstein Åsa E. Y. , Drubin David G.
Published: July 2013

Yeast Abp1p is a cortical actin cytoskeleton protein implicated in cytoskeletal regulation, endocytosis, and cAMP-signaling. We have identified a gene encoding a mouse homologue of Abp1p, and it is identical to SH3P7, a protein shown recently to be a target of Src tyrosine kinases. Yeast and mouse Abp1p display the same domain structure including an N-terminal actin-depolymerizing factor homology domain and a C-terminal Src homology 3 domain. Using two independent actin-binding domains, mAbp1 binds to actin filaments with a 1:5 saturation stoichiometry. In stationary cells, mAbp1 colocalizes with cortical F-actin in fibroblast protrusions that represent sites of cellular growth. mAbp1 appears at the actin-rich leading edge of migrating cells. Growth factors cause mAbp1 to rapidly accumulate in lamellipodia. This response can be mimicked by expression of dominant-positive Rac1. mAbp1 recruitment appears to be dependent on de novo actin polymerization and occurs specifically at sites enriched for the Arp2/3 complex. mAbp1 is a newly identified cytoskeletal protein in mice and may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics.
Protection of Telomeres by the Ku Protein in Fission Yeast
Authors: Baumann Peter , Cech Thomas R.
Published: July 2013

Schizosaccharomyces pombe cells survive loss of telomeres by a unique pathway of chromosome circularization. Factors potentially involved in this survival mechanism include the heterodimeric Ku protein and ligase IV, both of which are involved in the repair of DNA double-strand breaks in mammalian cells. Furthermore, Ku plays a role in telomere maintenance as well as in DNA double-strand break repair in Saccharomyces cerevisiae. We have identified Ku and ligase IV homologues in S. pombe and analyzed their functions during normal growth and in cells undergoing senescence. In the absence of either a Ku subunit (pku70+) or ligase IV (lig4+), nonhomologous DNA end-joining was severely reduced. Lack of functional Ku led to shorter but stable telomeres and caused striking rearrangements of telomere-associated sequences, indicating a function for Ku in inhibiting recombinational activities near chromosome ends. In contrast to S. cerevisiae, concurrent deletion ofpku70+and the gene for the catalytic subunit of telomerase (trt1+) was not lethal, allowing for the first time the dissection of the roles of Ku during senescence. Our results support a model in which Ku protects chromosome termini from nucleolytic and recombinational activities but is not involved in the formation of chromosome end fusions during senescence. The conclusion that nonhomologous end-joining is not required for chromosome circularization was further supported by analysis of survivors in strains lacking the genes for bothtrt1+and lig4+.
The Diabetes Autoantigen ICA69 and ItsCaenorhabditis elegansHomologue,ric-19, Are Conserved Regulators of Neuroendocrine Secretion
Authors: Pilon Marc , Peng Xiao-Rong , Spence Andrew M. , Plasterk Ronald H.A. , Dosch Hans-Michael
Published: July 2013

ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting β-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into theric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.
Ajuba, a Cytosolic LIM Protein, Shuttles into the Nucleus and Affects Embryonal Cell Proliferation and Fate Decisions
Authors: Kanungo Jyotshnabala , Pratt Stephen J. , Marie Helene , Longmore Gregory D.
Published: July 2013

Cellular adhesive events affect cell proliferation and differentiation decisions. How cell surface events mediating adhesion transduce signals to the nucleus is not well understood. After cell–cell or cell–substratum contact, cytosolic proteins are recruited to clustered adhesion receptor complexes. One such family of cytosolic proteins found at sites of cell adhesion is the Zyxin family of LIM proteins. Here we demonstrate that the family member Ajuba was recruited to the cell surface of embryonal cells, upon aggregate formation, at sites of cell–cell contact. Ajuba contained a functional nuclear export signal and shuttled into the nucleus. Importantly, accumulation of the LIM domains of Ajuba in the nucleus of P19 embryonal cells resulted in growth inhibition and spontaneous endodermal differentiation. The differentiating effect of Ajuba mapped to the third LIM domain, whereas regulation of proliferation mapped to the first and second LIM domains. Ajuba-induced endodermal differentiation of these cells correlated with the capacity to activate c-Jun kinase and required c-Jun kinase activation. These results suggest that the cytosolic LIM protein Ajuba may provide a new mechanism to transduce signals from sites of cell adhesion to the nucleus, regulating cell growth and differentiation decisions during early development.
Forced Expression of Keratin 16 Alters the Adhesion, Differentiation, and Migration of Mouse Skin Keratinocytes
Authors: Wawersik Matthew , Coulombe Pierre A.
Published: July 2013

Injury to the skin results in an induction of keratins K6, K16, and K17 concomitant with activation of keratinocytes for reepithelialization. Forced expression of human K16 in skin epithelia of transgenic mice causes a phenotype that mimics several aspects of keratinocyte activation. Two types of transgenic keratinocytes, with forced expression of either human K16 or a K16-C14 chimeric cDNA, were analyzed in primary culture to assess the impact of K16 expression at a cellular level. High K16-C14-expressing and low K16-expressing transgenic keratinocytes behave similar to wild type in all aspects tested. In contrast, high K16-expressing transgenic keratinocytes show alterations in plating efficiency and calcium-induced differentiation, but proliferate normally. Migration of keratinocytes is reduced in K16 transgenic skin explants compared with controls. Finally, a subset of high K16-expressing transgenic keratinocytes develops major changes in the organization of keratin filaments in a time- and calcium concentration-dependent manner. These changes coincide with alterations in keratin content while the steady-state levels of K16 protein remain stable. We conclude that forced expression of K16 in progenitor skin keratinocytes directly impacts properties such as adhesion, differentiation, and migration, and that these effects depend upon determinants contained within its carboxy terminus.
Regulation of Macropinocytosis by p21-activated Kinase-1
Authors: Dharmawardhane Suranganie , Schürmann Annette , Sells Mary Ann , Chernoff Jonathan , Schmid Sandra L. , Bokoch Gary M.
Published: July 2013

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5′-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.
Matricellular Proteins as Modulators of Cell–Matrix Interactions: Adhesive Defect in Thrombospondin 2-null Fibroblasts is a Consequence of Increased Levels of Matrix Metalloproteinase-2
Authors: Yang Zhantao , Kyriakides Themis R. , Bornstein Paul
Published: July 2013

Thrombospondin 2 (TSP2)-null mice, generated by disruption of theThbs2 gene, display a variety of connective tissue abnormalities, including fragile skin and the presence of abnormally large collagen fibrils with irregular contours in skin and tendon. In this study we demonstrate that TSP2-null skin fibroblasts show a defect in attachment to a number of matrix proteins, and a reduction in cell spreading. To investigate the molecular mechanisms responsible for these abnormal cell–matrix interactions, we compared the levels of matrix metalloproteinases (MMPs) in wild-type and mutant fibroblasts. Isolation and analysis of gelatinases from conditioned media by gelatin-agarose affinity chromatography and gelatinolytic assays demonstrated that TSP2-null fibroblasts produce a 2-fold increase in gelatinase A (MMP2) compared with wild-type cells. The adhesive defect was corrected by treatment of TSP2-null fibroblasts with soluble TSP2, with the MMP inhibitors BB94 and tissue inhibitor of metalloproteinase-2, and with a neutralizing antibody to MMP2. Moreover, stable transfection of TSP2-null fibroblasts with mouse TSP2 cDNA corrected both the adhesive defect and the altered expression of MMP2. Finally, MMP2 was shown to interact with TSP2 in a direct-binding plate assay. We conclude that TSP2 plays an important role in cell–matrix interactions, and that a deficiency in the protein results in increased levels of MMP2 that contribute to the adhesive defect in TSP2-null fibroblasts and could play a role in the complex phenotype of TSP2-null mice.
The Doa4 Deubiquitinating Enzyme Is Functionally Linked to the Vacuolar Protein-sorting and Endocytic Pathways
Authors: Amerik Alexander Y. , Nowak Jonathan , Swaminathan Sowmya , Hochstrasser Mark
Published: July 2013

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 clonedDID (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.
Identification of a New Vertebrate Nucleoporin, Nup188, with the Use of a Novel Organelle Trap Assay
Authors: Miller Brian R. , Powers Maureen , Park Minkyu , Fischer Wolfgang , Forbes Douglass J.
Published: July 2013

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 Xenopusegg 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 containN-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 XenopusNup93 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 theN-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.
A Role for the START Gene–specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction
Authors: Tournier Sylvie , Millar Jonathan B.A.
Published: July 2013

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature. Eleven of 17 mutants that suppress this phenotype define a single complementation group, mcs1. The mcs1-77mutant also suppresses lethal inactivation of the Wee1 and Mik1 tyrosine kinases and thus delays mitosis independently of Cdc2 tyrosine phosphorylation. We have cloned mcs1 by isolating suppressors of the cell cycle arrest phenotype of mcs1-77 cdc25-22 cells and found that it encodes Res2, a component of the START gene–specific transcription factor complex MBF (also known as DSC-1). The mcs1-77 mutant bears a single point mutation in the DNA-binding domain of Res2 that causes glycine 68 to be replaced by a serine residue. Importantly, two substrates of the anaphase-promoting complex (APC), the major B-type cyclin, Cdc13, and the anaphase inhibitor, Cut2, are unstable in G2-phasemcs1-77 cells. Consistent with this, we observe abnormal sister chromatid separation in mcs1-77 cdc25-22 cells at the restrictive temperature. Mutation of either Cdc10 or Res1 also deregulates MBF-dependent transcription and causes a G2 delay. We find that this cell cycle delay is abolished in the absence of the APC regulator Ste9/Srw1 and that the periodic expression of Ste9/Srw1 is controlled by the MBF complex. These data suggest that in fission yeast the MBF complex plays a key role in the inactivation of cyclin B and Cut2 destruction by controlling the periodic production of APC regulators.
Proteasomal Proteomics: Identification of Nucleotide-sensitive Proteasome-interacting Proteins by Mass Spectrometric Analysis of Affinity-purified Proteasomes
Authors: Verma Rati , Chen Stephen , Feldman Renny , Schieltz David , Yates John , Dohmen Juergen , Deshaies Raymond J.
Published: July 2013

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-γ-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.
Constitutive Macropinocytosis in Oncogene-transformed Fibroblasts Depends on Sequential Permanent Activation of Phosphoinositide 3-Kinase and Phospholipase C
Authors: Amyere Mustapha , Payrastre Bernard , Krause Ulrike , Smissen Patrick Van Der , Veithen Alex , Courtoy Pierre J.
Published: July 2013

Macropinocytosis results from the closure of lamellipodia generated by membrane ruffling, thereby reflecting cortical actin dynamics. Both transformation of Rat-1 fibroblasts by v-Src or K-Ras and stable transfection for expression of dominant-positive, wild-type phosphoinositide 3-kinase (PI3K) regulatory subunit p85α constitutively led to stress fiber disruption, cortical actin recruitment, extensive ruffling, and macropinosome formation, as measured by a selective acceleration of fluid-phase endocytosis. These alterations closely correlated with activation of PI3K and phosphatidylinositol-specific phospholipase C (PI-PLC), as assayed by 3-phosphoinositide synthesis in situ and in vitro and inositol 1,4,5 trisphosphate steady-state levels, respectively; they were abolished by stable transfection of v-Src–transformed cells for dominant-negative truncated p85α expression and by pharmacological inhibitors of PI3K and PI-PLC, indicating a requirement for both enzymes. Whereas PI3K activation resisted PI-PLC inhibition, PI-PLC activation was abolished by a PI3K inhibitor and dominant-negative transfection, thus placing PI-PLC downstream of PI3K. Together, these data suggest that permanent sequential activation of both PI3K and PI-PLC is necessary for the dramatic reorganization of the actin cytoskeleton in oncogene-transformed fibroblasts, resulting in constitutive ruffling and macropinocytosis.
Overexpression of β-Catenin Induces Apoptosis Independent of Its Transactivation Function with LEF-1 or the Involvement of Major G1 Cell Cycle Regulators
Authors: Kim Kwonseop , Pang Ka Ming , Evans Michael , Hay Elizabeth D.
Published: July 2013

β-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed β-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous β-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5–8 d after transfection) cells overexpressing β-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous β-catenin does not induce apoptosis, even though some endogenous β-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length β-catenin is able to induce LEF-1–dependent transactivation, whereas Arm β-catenin totally abolishes the transactivating function. However, Arm β-catenin, containing deletions of known LEF-1–transactivating domains, has the same apoptotic effects as full-length β-catenin. Overexpressed β-catenin also induces apoptosis in cells transfected with nuclear localization signal–deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous β-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed δ-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to β-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed β-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing β-catenin might be a physiological mechanism to eliminate them from the population.
Mps1p Regulates Meiotic Spindle Pole Body Duplication in Addition to Having Novel Roles during Sporulation
Authors: Straight Paul D. , Giddings Thomas H. , Winey Mark
Published: July 2013

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 MPS1disrupt 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.
Coiled-Coil Trigger Motifs in the 1B and 2B Rod Domain Segments Are Required for the Stability of Keratin Intermediate Filaments
Authors: Wu Kenneth C. , Bryan Janine T. , Morasso Maria I. , Jang Shyh-Ing , Lee Jeung-Hoon , Yang Jun-Mo , Marekov Lyuben N. , Parry David A.D. , Steinert Peter M.
Published: July 2013

Many α-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A22mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.
Yeast Exocytic v-SNAREs Confer Endocytosis
Authors: Gurunathan Sangiliyandi , Chapman-Shimshoni Daphne , Trajkovic Selena , Gerst Jeffrey E.
Published: July 2013

In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1(SNC1ala43), are deficient in the endocytic uptake of components from the cell surface. We found that bothSNC and temperature-shiftedSNC1ala43yeast are deficient in their ability to deliver the soluble dye FM4–64 to the vacuole. Under conditions in which vesicles accumulate, FM4–64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, α-factor–stimulated endocytosis of the α-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1ala43was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane.
Mutant RBL Mast Cells Defective in FcεRI Signaling and Lipid Raft Biosynthesis Are Reconstituted by Activated Rho-family GTPases
Authors: Field Kenneth A. , Apgar John R. , Hong-Geller Elizabeth , Siraganian Reuben P. , Baird Barbara , Holowka David
Published: July 2013

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (FcεRI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both FcεRI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca2+influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-FcεRI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca2+response. FcεRI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca2+suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A2and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for FcεRI-mediated signaling that leads to Ca2+mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.
Bud6 Directs Sequential Microtubule Interactions with the Bud Tip and Bud Neck during Spindle Morphogenesis inSaccharomyces cerevisiae
Authors: Segal Marisa , Bloom Kerry , Reed Steven I.
Published: July 2013

In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule–cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule–neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Δmutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule–cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.
Activation of the Small GTPase Rac Is Sufficient to Disrupt Cadherin-dependent Cell-Cell Adhesion in Normal Human Keratinocytes
Authors: Braga Vania M.M. , Betson Martha , Li Xiaodong , Lamarche-Vane Nathalie
Published: July 2013

To achieve strong adhesion to their neighbors and sustain stress and tension, epithelial cells develop many different specialized adhesive structures. Breakdown of these structures occurs during tumor progression, with the development of a fibroblastic morphology characteristic of metastatic cells. During Ras transformation, Rac-signaling pathways participate in the disruption of cadherin-dependent adhesion. We show that sustained Rac activation per se is sufficient to disassemble cadherin-mediated contacts in keratinocytes, in a concentration- and time-dependent manner. Cadherin receptors are removed from junctions before integrin receptors, suggesting that pathways activated by Rac can specifically interfere with cadherin function. We mapped an important region for disruption of junctions to the putative second effector domain of the Rac protein. Interestingly, although this region overlaps the domain necessary to induce lamellipodia, we demonstrate that the disassembly of cadherin complexes is a new Rac activity, distinct from Rac-dependent lamellipodia formation. Because Rac activity is also necessary for migration, Rac is a good candidate to coordinately regulate cell-cell and cell-substratum adhesion during tumorigenesis.
Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo
Authors: Stage-Zimmermann Tracy , Schmidt Ute , Silver Pamela A.
Published: July 2013

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit inS. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120.