Journal of Cell Biology

Modularity of the Hrd1 ERAD complex underlies its diverse client range.

Publication Date: 2010 Mar 8 PMID: 20212318
Authors: Kanehara, K. - Xie, W. - Ng, D. T.
Journal: J Cell Biol

Secretory protein folding is monitored by endoplasmic reticulum (ER) quality control mechanisms. Misfolded proteins are retained and targeted to ER-associated degradation (ERAD) pathways. At their core are E3 ubiquitin ligases, which organize factors that recognize, ubiquitinate, and translocate substrates. Of these, we report that the Hrd1 complex manages three distinct substrate classes. A core complex is required for all classes and is sufficient for some membrane proteins. The accessory factors Usa1p and Der1p adapt the complex to process luminal substrates. Their integration is sufficient to process molecules bearing glycan-independent degradation signals. The presence of Yos9p extends the substrate range by mediating the recognition of glycan-based degradation signals. This modular organization enables the Hrd1 complex to recognize topologically diverse substrates. The Hrd1 system does not directly evaluate the folding state of polypeptides. Instead, it does so indirectly, by recognizing specific embedded signals displayed upon misfolding.

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The SUMO protease SENP6 is essential for inner kinetochore assembly.

Publication Date: 2010 Mar 8 PMID: 20212317
Authors: Mukhopadhyay, D. - Arnaoutov, A. - Dasso, M.
Journal: J Cell Biol

We have analyzed the mitotic function of SENP6, a small ubiquitin-like modifier (SUMO) protease that disassembles conjugated SUMO-2/3 chains. Cells lacking SENP6 showed defects in spindle assembly and metaphase chromosome congression. Analysis of kinetochore composition in these cells revealed that a subset of proteins became undetectable on inner kinetochores after SENP6 depletion, particularly the CENP-H/I/K complex, whereas other changes in kinetochore composition mimicked defects previously reported to result from CENP-H/I/K depletion. We further found that CENP-I is degraded through the action of RNF4, a ubiquitin ligase which targets polysumoylated proteins for proteasomal degradation, and that SENP6 stabilizes CENP-I by antagonizing RNF4. Together, these findings reveal a novel mechanism whereby the finely balanced activities of SENP6 and RNF4 control vertebrate kinetochore assembly through SUMO-targeted destabilization of inner plate components.

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Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases.

Publication Date: 2010 Mar 8 PMID: 20212316
Authors: Jaqaman, K. - King, E. M. - Amaro, A. C. - Winter, J. R. - Dorn, J. F. - Elliott, H. L. - McHedlishvili, N. - McClelland, S. E. - Porter, I. M. - Posch, M. - Toso, A. - Danuser, G. - McAinsh, A. D. - Meraldi, P. - Swedlow, J. R.
Journal: J Cell Biol

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion.

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ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase.

Publication Date: 2010 Mar 8 PMID: 20212315
Authors: McNees, C. J. - Tejera, A. M. - Martinez, P. - Murga, M. - Mulero, F. - Fernandez-Capetillo, O. - Blasco, M. A.
Journal: J Cell Biol

Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.

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The Golgi and the centrosome: building a functional partnership.

Publication Date: 2010 Mar 8 PMID: 20212314
Authors: Sutterlin, C. - Colanzi, A.
Journal: J Cell Biol

The mammalian Golgi apparatus is characterized by a ribbon-like organization adjacent to the centrosome during interphase and extensive fragmentation and dispersal away from the centrosome during mitosis. It is not clear whether this dynamic association between the Golgi and centrosome is of functional significance. We discuss recent findings indicating that the Golgi-centrosome relationship may be important for directional protein transport and centrosome positioning, which are both required for cell polarization. We also summarize our current knowledge of the link between Golgi organization and cell cycle progression.

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New editorial board members 2010.

Publication Date: 2010 Mar 8 PMID: 20212313
Authors: Misteli, T.
Journal: J Cell Biol

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Isabelle Vernos: Motoring around the mitotic spindle.

Publication Date: 2010 Mar 8 PMID: 20212312
Authors: Sedwick, C.
Journal: J Cell Biol

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Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase.

Publication Date: 2010 Mar 8 PMID: 20194642
Authors: Beck, H. - Nahse, V. - Larsen, M. S. - Groth, P. - Clancy, T. - Lees, M. - Jorgensen, M. - Helleday, T. - Syljuasen, R. G. - Sorensen, C. S.
Journal: J Cell Biol

Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.

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The interphase microtubule aster is a determinant of asymmetric division orientation in Drosophila neuroblasts.

Publication Date: 2010 Mar 8 PMID: 20194641
Authors: Januschke, J. - Gonzalez, C.
Journal: J Cell Biol

The mechanisms that maintain the orientation of cortical polarity and asymmetric division unchanged in consecutive mitoses in Drosophila melanogaster neuroblasts (NBs) are unknown. By studying the effect of transient microtubule depolymerization and centrosome mutant conditions, we have found that such orientation memory requires both the centrosome-organized interphase aster and centrosome-independent functions. We have also found that the span of such memory is limited to the last mitosis. Furthermore, the orientation of the NB axis of polarity can be reset to any angle with respect to the surrounding tissue and is, therefore, cell autonomous.

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The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation.

Publication Date: 2010 Mar 8 PMID: 20194640
Authors: Kim, S. - Wairkar, Y. P. - Daniels, R. W. - Diantonio, A.
Journal: J Cell Biol

Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.

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Integration of contractile forces during tissue invagination.

Publication Date: 2010 Mar 8 PMID: 20194639
Authors: Martin, A. C. - Gelbart, M. - Fernandez-Gonzalez, R. - Kaschube, M. - Wieschaus, E. F.
Journal: J Cell Biol

Contractile forces generated by the actomyosin cytoskeleton within individual cells collectively generate tissue-level force during epithelial morphogenesis. During Drosophila mesoderm invagination, pulsed actomyosin meshwork contractions and a ratchet-like stabilization of cell shape drive apical constriction. Here, we investigate how contractile forces are integrated across the tissue. Reducing adherens junction (AJ) levels or ablating actomyosin meshworks causes tissue-wide epithelial tears, which release tension that is predominantly oriented along the anterior-posterior (a-p) embryonic axis. Epithelial tears allow cells normally elongated along the a-p axis to constrict isotropically, which suggests that apical constriction generates anisotropic epithelial tension that feeds back to control cell shape. Epithelial tension requires the transcription factor Twist, which stabilizes apical myosin II, promoting the formation of a supracellular actomyosin meshwork in which radial actomyosin fibers are joined end-to-end at spot AJs. Thus, pulsed actomyosin contractions require a supracellular, tensile meshwork to transmit cellular forces to the tissue level during morphogenesis.

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Centrosomes remember which way is up.

Publication Date: 2010 Mar 8 PMID: 20194638
Authors: Leslie, M.
Journal: J Cell Biol

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Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements.

Publication Date: 2010 Mar 8 PMID: 20194637
Authors: Kawamura, R. - Pope, L. H. - Christensen, M. O. - Sun, M. - Terekhova, K. - Boege, F. - Mielke, C. - Andersen, A. H. - Marko, J. F.
Journal: J Cell Biol

We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II-specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.

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Anchorage of VEGF to the extracellular matrix conveys differential signaling responses to endothelial cells.

Publication Date: 2010 Feb 22 PMID: 20176926
Authors: Chen, T. T. - Luque, A. - Lee, S. - Anderson, S. M. - Segura, T. - Iruela-Arispe, M. L.
Journal: J Cell Biol

VEGF can be secreted in multiple isoforms with variable affinity for extracellular proteins and different abilities to induce vascular morphogenesis, but the molecular mechanisms behind these effects remain unclear. Here, we show molecular distinctions between signaling initiated from soluble versus matrix-bound VEGF, which mediates a sustained level of VEGFR2 internalization and clustering. Exposure of endothelial cells to matrix-bound VEGF elicits prolonged activation of VEGFR2 with differential phosphorylation of Y1214, and extended activation kinetics of p38. These events require association of VEGFR2 with beta1 integrins. Matrix-bound VEGF also promotes reciprocal responses on beta1 integrin by inducing its association with focal adhesions; a response that is absent upon exposure to soluble VEGF. Inactivation of beta1 integrin blocks the prolonged phosphorylation of Y1214 and consequent activation of p38. Combined, these results indicate that when in the context of extracellular matrix, activation of VEGFR2 is distinct from that of soluble VEGF in terms of recruitment of receptor partners, phosphorylation kinetics, and activation of downstream effectors.

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Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster.

Publication Date: 2010 Feb 22 PMID: 20176925
Authors: Hamel, S. - Fantini, J. - Schweisguth, F.
Journal: J Cell Biol

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the alpha1,4-N-acetylgalactosaminyltransferase-1 (alpha4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of alpha4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that alpha4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by alpha4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL-protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.

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A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila.

Publication Date: 2010 Feb 22 PMID: 20176924
Authors: Owald, D. - Fouquet, W. - Schmidt, M. - Wichmann, C. - Mertel, S. - Depner, H. - Christiansen, F. - Zube, C. - Quentin, C. - Korner, J. - Urlaub, H. - Mechtler, K. - Sigrist, S. J.
Journal: J Cell Biol

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, we identified Drosophila Syd-1 (DSyd-1) as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-alpha, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-alpha at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-alpha. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-alpha.

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Condensin complexes regulate mitotic progression and interphase chromatin structure in embryonic stem cells.

Publication Date: 2010 Feb 22 PMID: 20176923
Authors: Fazzio, T. G. - Panning, B.
Journal: J Cell Biol

In an RNA interference screen interrogating regulators of mouse embryonic stem (ES) cell chromatin structure, we previously identified 62 genes required for ES cell viability. Among these 62 genes were Smc2 and -4, which are core components of the two mammalian condensin complexes. In this study, we show that for Smc2 and -4, as well as an additional 49 of the 62 genes, knockdown (KD) in somatic cells had minimal effects on proliferation or viability. Upon KD, Smc2 and -4 exhibited two phenotypes that were unique to ES cells and unique among the ES cell-lethal targets: metaphase arrest and greatly enlarged interphase nuclei. Nuclear enlargement in condensin KD ES cells was caused by a defect in chromatin compaction rather than changes in DNA content. The altered compaction coincided with alterations in the abundance of several epigenetic modifications. These data reveal a unique role for condensin complexes in interphase chromatin compaction in ES cells.

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Directly probing the mechanical properties of the spindle and its matrix.

Publication Date: 2010 Feb 22 PMID: 20176922
Authors: Gatlin, J. C. - Matov, A. - Danuser, G. - Mitchison, T. J. - Salmon, E. D.
Journal: J Cell Biol

Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.

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Metabolic control of G1-S transition: cyclin E degradation by p53-induced activation of the ubiquitin-proteasome system.

Publication Date: 2010 Feb 22 PMID: 20176921
Authors: Mandal, S. - Freije, W. A. - Guptan, P. - Banerjee, U.
Journal: J Cell Biol

Cell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Previous genetic analysis in Drosophila melanogaster has shown that disruption of the mitochondrial electron transport chain activates a G1-S checkpoint as a result of a control of cyclin E by p53. This regulation does not involve activation of the p27 homologue dacapo in flies. We demonstrate that regulation of cyclin E is not at the level of transcription or translation. Rather, attenuated mitochondrial activity leads to transcriptional upregulation of the F-box protein archipelago, the Fbxw7 homologue in flies. We establish that archipelago and the proteasomal machinery contribute to degradation of cyclin E in response to mitochondrial dysfunction. Our work provides in vivo genetic evidence for p53-mediated integration of metabolic stress signals, which modulate the activity of the ubiquitin-proteasome system to degrade cyclin E protein and thereby impose cell cycle arrest.

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Automated microscopy for high-content RNAi screening.

Publication Date: 2010 Feb 22 PMID: 20176920
Authors: Conrad, C. - Gerlich, D. W.
Journal: J Cell Biol

Fluorescence microscopy is one of the most powerful tools to investigate complex cellular processes such as cell division, cell motility, or intracellular trafficking. The availability of RNA interference (RNAi) technology and automated microscopy has opened the possibility to perform cellular imaging in functional genomics and other large-scale applications. Although imaging often dramatically increases the content of a screening assay, it poses new challenges to achieve accurate quantitative annotation and therefore needs to be carefully adjusted to the specific needs of individual screening applications. In this review, we discuss principles of assay design, large-scale RNAi, microscope automation, and computational data analysis. We highlight strategies for imaging-based RNAi screening adapted to different library and assay designs.

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Graham Warren: Gaining ground on the Golgi. Interview by Caitlin Sedwick.

Publication Date: 2010 Feb 22 PMID: 20176919
Authors: Warren, G.
Journal: J Cell Biol

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Unconventional secretion by autophagosome exocytosis.

Publication Date: 2010 Feb 22 PMID: 20156968
Authors: Pfeffer, S. R.
Journal: J Cell Biol

In this issue, Duran et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911154) and Manjithaya et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200911149) use yeast genetics to reveal a role for autophagosome intermediates in the unconventional secretion of an acyl coenzyme A (CoA)-binding protein that lacks an endoplasmic reticulum signal sequence. Medium-chain acyl CoAs are also required and may be important for substrate routing to this pathway.

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Unconventional secretion of Acb1 is mediated by autophagosomes.

Publication Date: 2010 Feb 22 PMID: 20156967
Authors: Duran, J. M. - Anjard, C. - Stefan, C. - Loomis, W. F. - Malhotra, V.
Journal: J Cell Biol

Starving Dictyostelium discoideum cells secrete AcbA, an acyl coenzyme A-binding protein (ACBP) that lacks a conventional signal sequence for entering the endoplasmic reticulum (ER). Secretion of AcbA in D. discoideum requires the Golgi-associated protein GRASP. In this study, we report that starvation-induced secretion of Acb1, the Saccharomyces cerevisiae ACBP orthologue, also requires GRASP (Grh1). This highlights the conserved function of GRASP in unconventional secretion. Although genes required for ER to Golgi or Golgi to cell surface transport are not required for Acb1 secretion in yeast, this process involves autophagy genes and the plasma membrane t-SNARE, Sso1. Inhibiting transport to vacuoles does not affect Acb1 secretion. In sum, our experiments reveal a unique secretory pathway where autophagosomes containing Acb1 evade fusion with the vacuole to prevent cargo degradation. We propose that these autophagosome intermediates fuse with recycling endosomes instead to form multivesicular body carriers that then fuse with the plasma membrane to release cargo.

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BAG1 restores formation of functional DJ-1 L166P dimers and DJ-1 chaperone activity.

Publication Date: 2010 Feb 22 PMID: 20156966
Authors: Deeg, S. - Gralle, M. - Sroka, K. - Bahr, M. - Wouters, F. S. - Kermer, P.
Journal: J Cell Biol

Mutations in the gene coding for DJ-1 protein lead to early-onset recessive forms of Parkinson's disease. It is believed that loss of DJ-1 function is causative for disease, although the function of DJ-1 still remains a matter of controversy. We show that DJ-1 is localized in the cytosol and is associated with membranes and organelles in the form of homodimers. The disease-related mutation L166P shifts its subcellular distribution to the nucleus and decreases its ability to dimerize, impairing cell survival. Using an intracellular foldase biosensor, we found that wild-type DJ-1 possesses chaperone activity, which is abolished by the L166P mutation. We observed that this aberrant phenotype can be reversed by the expression of the cochaperone BAG1 (Bcl-2-associated athanogene 1), restoring DJ-1 subcellular distribution, dimer formation, and chaperone activity and ameliorating cell survival.

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Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein.

Publication Date: 2010 Feb 22 PMID: 20156965
Authors: Rane, N. S. - Chakrabarti, O. - Feigenbaum, L. - Hegde, R. S.
Journal: J Cell Biol

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.

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Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling.

Publication Date: 2010 Feb 22 PMID: 20156964
Authors: Koivusalo, M. - Welch, C. - Hayashi, H. - Scott, C. C. - Kim, M. - Alexander, T. - Touret, N. - Hahn, K. M. - Grinstein, S.
Journal: J Cell Biol

Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.

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Plakophilin 1 stimulates translation by promoting eIF4A1 activity.

Publication Date: 2010 Feb 22 PMID: 20156963
Authors: Wolf, A. - Krause-Gruszczynska, M. - Birkenmeier, O. - Ostareck-Lederer, A. - Huttelmaier, S. - Hatzfeld, M.
Journal: J Cell Biol

Plakophilins 1-3 (PKP1-3) are desmosomal proteins of the p120(ctn) family of armadillo-related proteins that are essential for organizing the desmosomal plaque. Recent findings identified PKPs in stress granules, suggesting an association with the translational machinery. However, a role of PKPs in controlling translation remained elusive so far. In this study, we show a direct association of PKP1 with the eukaryotic translation initiation factor 4A1 (eIF4A1). PKP1 stimulated eIF4A1-dependent translation via messenger RNA cap and encephalomyocarditis virus internal ribosomal entry site (IRES) structures, whereas eIF4A1-independent translation via hepatitis C virus IRES was not affected. PKP1 copurified with eIF4A1 in the cap complex, and its overexpression stimulated eIF4A1 recruitment into cap-binding complexes. At the molecular level, PKP1 directly promoted eIF4A1 adenosine triphosphatase activity. The stimulation of translation upon PKP1 overexpression correlated with the up-regulation of proliferation and cell size. In conclusion, these findings identify PKP1 as a regulator of translation and proliferation via modulation of eIF4A1 activity and suggest that PKP1 controls cell growth in physiological and pathological conditions.

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Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation.

Publication Date: 2010 Feb 22 PMID: 20156962
Authors: Manjithaya, R. - Anjard, C. - Loomis, W. F. - Subramani, S.
Journal: J Cell Biol

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)-binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2-like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.

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