bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid Composition of Outer Leaflet of Chloroplast Outer Envelope Determines Topology of OEP7
Authors: Schleiff Enrico , Tien Roselynn , Salomon Michael , Soll Jürgen
Published: July 2013

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native Nin-Coutorientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like Nin-Coutorientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.
Integrin-mediated Adhesion Regulates Cell Polarity and Membrane Protrusion through the Rho Family of GTPases
Authors: Cox Elisabeth A. , Sastry Sarita K. , Huttenlocher Anna
Published: July 2013

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through α5β1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.
Na,K-ATPase β-Subunit Is Required for Epithelial Polarization, Suppression of Invasion, and Cell Motility
Authors: Rajasekaran Sigrid A. , Palmer Lawrence G. , Quan Karina , Harper Jeffrey F. , Ball William J. , Bander Neil H. , Soler Alejandro Peralta , Rajasekaran Ayyappan K.
Published: July 2013

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an α- and β-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin–mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and β1-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase β1-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin–mediated cell-cell adhesion requires the Na,K-ATPase β-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the β1-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.
Genome-wide Responses to Mitochondrial Dysfunction
Authors: Epstein Charles B. , Waddle James A. , Hale Walker , Davé Varshal , Thornton Janet , Macatee Timothy L. , Garner Harold R. , Butow Ronald A.
Published: July 2013

Mitochondrial dysfunction can lead to diverse cellular and organismal responses. We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations inSaccharomyces cerevisiae. We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin. We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle. The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis. Transcript profiling of cells harboring null alleles ofRTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.
Division of Mitochondria Requires a NovelDNM1-interacting Protein, Net2p
Authors: Cerveny Kara L. , McCaffery J. Michael , Jensen Robert E.
Published: July 2013

Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood. Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission. Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p. Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1Δ cells. NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats. Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein. Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules. Our results suggest that Net2p is a new component of the mitochondrial division machinery.
Characterization of Syntenin, a Syndecan-binding PDZ Protein, as a Component of Cell Adhesion Sites and Microfilaments
Authors: Zimmermann Pascale , Tomatis Daniela , Rosas Marcela , Grootjans Johan , Leenaerts Iris , Degeest Gisèle , Reekmans Gunter , Coomans Christien , David Guido
Published: July 2013

Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, β-catenin, and α-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes.
Phosphorylation of a Conserved Integrin α3 QPSXXE Motif Regulates Signaling, Motility, and Cytoskeletal Engagement
Authors: Zhang Xin A. , Bontrager Alexa L. , Stipp Christopher S. , Kraeft Stine-Kathrein , Bazzoni Gianfranco , Chen Lan Bo , Hemler Martin E.
Published: July 2013

Integrin α3A cytoplasmic tail phosphorylation was mapped to amino acid S1042, as determined by mass spectrometry, and confirmed by mutagenesis. This residue occurs within a “QPSXXE” motif conserved in multiple α chains (α3A, α6A, α7A), from multiple species. Phosphorylation of α3A and α6A did not appear to bedirectly mediated by protein kinase C (PKC) α, β, γ, δ, ε, ζ, or μ, or by any of several other known serine kinases, although PKC has an indirect role in promoting phosphorylation. A S1042A mutation did not affect α3-Chinese hamster ovary (CHO) cell adhesion to laminin-5, but did alter 1) α3-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin (in the presence or absence of phorbol 12-myristate 13 acetate stimulation), and p130CAS(in the absence of phorbol 12-myristate 13 acetate stimulation), 2) the shape of cells spread on laminin-5, and 3) α3-dependent random CHO cell migration on laminin-5. In addition, S1042A mutation altered the PKC-dependent, ligand-dependent subcellular distribution of α3 and F-actin in CHO cells. Together, the results demonstrate clearly that α3A phosphorylation is functionally relevant. In addition, the results strongly suggest that α3 phosphorylation may regulate α3 integrin interaction with the cytoskeleton.
Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals
Authors: Anand Monika , Rush Jeffrey S. , Ray Sutapa , Doucey Marie-Agnes , Weik Jennifer , Ware Felecia E. , Hofsteenge Jan , Waechter Charles J. , Lehrman Mark A.
Published: July 2013

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.
Positive Regulation of Wee1 by Chk1 and 14-3-3 Proteins
Authors: Lee Joon , Kumagai Akiko , Dunphy William G.
Published: July 2013

Wee1 inactivates the Cdc2–cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted fromXenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.
Replication-dependent Histone Gene Expression Is Related to Cajal Body (CB) Association but Does Not Require Sustained CB Contact
Authors: Shopland Lindsay S. , Byron Meg , Stein Janet L. , Lian Jane B. , Stein Gary S. , Lawrence Jeanne B.
Published: July 2013

Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3′ end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3′ end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci.
Multifaceted Physiological Response Allows Yeast to Adapt to the Loss of the Signal Recognition Particle-dependent Protein-targeting Pathway
Authors: Mutka Sarah C. , Walter Peter
Published: July 2013

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.
GIPC and GAIP Form a Complex with TrkA: A Putative Link between G Protein and Receptor Tyrosine Kinase Pathways
Authors: Lou Xiaojing , Yano Hiroko , Lee Francis , Chao Moses V. , Farquhar Marilyn Gist
Published: July 2013

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-γ1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.
A Family of Small Coiled-Coil–forming Proteins Functioning at the Late Endosome in Yeast
Authors: Kranz Andreas , Kinner Andrea , Kölling Ralf
Published: July 2013

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32,VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10(more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil–forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a “class E vps phenotype.” Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the “Snf7-family” members are involved in a cargo-specific event.
Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations
Authors: Rescan Claude , Coutant Alexandre , Talarmin Hélène , Theret Nathalie , Glaise Denise , Guguen-Guillouzo Christiane , Baffet Georges
Published: July 2013

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occured at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin β1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin β1 and fibronectin in a MEK-ERK–dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.
Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis
Authors: Wu Jian-Qiu , Bähler Jürg , Pringle John R.
Published: July 2013

Eukaryotic cells contain many actin-interacting proteins, including the α-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an α-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin–dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin–dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.
Transforming Growth Factor-β Induces Nuclear Import of Smad3 in an Importin-β1 and Ran-dependent Manner
Authors: Kurisaki Akira , Kose Shingo , Yoneda Yoshihiro , Heldin Carl-Henrik , Moustakas Aristidis
Published: July 2013

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-β (TGF-β) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-β type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-β1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-β1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-β1. 4) Importin-β1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-β receptor leads to enhanced interaction with importin-β1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.
O-Mannosylation Protects Mutant Alpha-Factor Precursor from Endoplasmic Reticulum-associated Degradation
Authors: Harty Carol , Strahl Sabine , Römisch Karin
Published: July 2013

Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by proteinO-mannosyl transferase 2 (Pmt2p).O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion ofPMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.
Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98
Authors: Takahashi Yuji , Bigler Dora , Ito Yasuhiko , White Judith M.
Published: July 2013

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions.
Deletion of Yeast p24 Genes Activates the Unfolded Protein Response
Authors: Belden William J. , Barlowe Charles
Published: July 2013

Yeast cells lacking a functional p24 complex accumulate a subset of secretory proteins in the endoplasmic reticulum (ER) and increase the extracellular secretion of HDEL-containing ER residents such as Kar2p/BiP. We report that a loss of p24 function causes activation of the unfolded protein response (UPR) and leads to increasedKAR2 expression. The HDEL receptor (Erd2p) is functional and traffics in p24 deletion strains as in wild-type strains, however the capacity of the retrieval pathway is exceeded. Other conditions that activate the UPR and elevate KAR2 expression also lead to extracellular secretion of Kar2p. Using an in vitro assay that reconstitutes budding from the ER, we detect elevated levels of Kar2p in ER-derived vesicles from p24 deletion strains and from wild-type strains with an activated UPR. Silencing the UPR byIRE1 deletion diminished Kar2p secretion under these conditions. We suggest that activation of the UPR plays a major role in extracellular secretion of Kar2p.
Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin
Authors: Rusan Nasser M. , Fagerstrom Carey J. , Yvon Anne-Marie C. , Wadsworth Patricia
Published: July 2013

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.
Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria
Authors: Rapaport Doron , Taylor Rebecca D. , Käser Michael , Langer Thomas , Neupert Walter , Nargang Frank E.
Published: July 2013

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.
UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein
Authors: Oakley Gregory G. , Loberg Lisa I. , Yao Jiaqin , Risinger Mary A. , Yunker Remy L. , Zernik-Kobak Maria , Khanna Kum Kum , Lavin Martin F. , Carty Michael P. , Dixon Kathleen
Published: July 2013

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.
Isolation and Characterization of Effector-Loop Mutants ofCDC42in Yeast
Authors: Gladfelter Amy S. , Moskow John J. , Zyla Trevin R. , Lew Daniel J.
Published: July 2013

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of “effector-loop” mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants ofSaccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.
Fission Yeast Aip3p (spAip3p) Is Required for an Alternative Actin-directed Polarity Program
Authors: Jin Hui , Amberg David C.
Published: July 2013

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of anaip3Δ strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in bothSchizosaccharomyces pombe and Saccharomyces cerevisiae.
Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering
Authors: Rudolf Rüdiger , Salm Thorsten , Rustom Amin , Gerdes Hans-Hermann
Published: July 2013

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.
Dribble, theDrosophilaKRR1p Homologue, Is Involved in rRNA Processing
Authors: Chan H.Y. Edwin , Brogna Saverio , O'Kane Cahir J.
Published: July 2013

The Drosophila dribble (dbe) gene encodes a KH domain protein, homologous to yeast KRR1p. Expression ofdbe transcripts is ubiquitous during embryogenesis. Overexpressed Dribble protein is localized in the nucleus and in some cell types in a subregion of the nucleolus. Homozygousdbe mutants die at first instar larval stage. Clonal analyses suggest that dbe+is required for survival of dividing cells. In dbe mutants, a novel rRNA-processing defect is found and accumulation of an abnormal rRNA precursor is detected.
Ligand-dependent Degradation of Smad3 by a Ubiquitin Ligase Complex of ROC1 and Associated Proteins
Authors: Fukuchi Minoru , Imamura Takeshi , Chiba Tomoki , Ebisawa Takanori , Kawabata Masahiro , Tanaka Keiji , Miyazono Kohei
Published: July 2013

Smads are signal mediators for the members of the transforming growth factor-β (TGF-β) superfamily. Upon phosphorylation by the TGF-β receptors, Smad3 translocates into the nucleus, recruits transcriptional coactivators and corepressors, and regulates transcription of target genes. Here, we show that Smad3 activated by TGF-β is degraded by the ubiquitin–proteasome pathway. Smad3 interacts with a RING finger protein, ROC1, through its C-terminal MH2 domain in a ligand-dependent manner. An E3 ubiquitin ligase complex ROC1-SCFFbw1aconsisting of ROC1, Skp1, Cullin1, and Fbw1a (also termed βTrCP1) induces ubiquitination of Smad3. Recruitment of a transcriptional coactivator, p300, to nuclear Smad3 facilitates the interaction with the E3 ligase complex and triggers the degradation process of Smad3. Smad3 bound to ROC1-SCFFbw1ais then exported from the nucleus to the cytoplasm for proteasomal degradation. TGF-β/Smad3 signaling is thus irreversibly terminated by the ubiquitin–proteasome pathway.
TGF-β3-induced Palatogenesis Requires Matrix Metalloproteinases
Authors: Blavier Laurence , Lazaryev Alisa , Groffen John , Heisterkamp Nora , DeClerck Yves A. , Kaartinen Vesa
Published: July 2013

Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-β3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-β3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-β3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.
Regulation of Myosin Heavy Chain Expression during Rat Skeletal Muscle Development In Vitro
Authors: Torgan Carol E. , Daniels Mathew P.
Published: July 2013

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ∼10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN–nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.
Characterization of a Novel Human SMC Heterodimer Homologous to theSchizosaccharomyces pombeRad18/Spr18 Complex
Authors: Taylor Elaine M. , Moghraby Jeelan S. , Lees Jennifer H. , Smit Bep , Moens Peter B. , Lehmann Alan R.
Published: July 2013

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 andspr18 and designated them hSMC6 andhSMC5. Both proteins are about 1100 amino acids in length and are 27–28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.