Molecular Cell
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Aug 27, 2010

Volume 39Issue 4p485-656
Open Archive
On the cover: In this issue, Facchinetti et al. (595–605) report their findings from a global analysis of replication termination sites in the budding yeast Sacchormyces cerevisiase. Termination occurs at pausing sites and is assisted by the Rrm3 helicase and the Top2 topoisomerase. Their study unmasks the mechanisms controlling DNA integrity at termination zones. The cover illustrates converging replication forks (crossroads). One polymerase stalls while the other progresses thanks to Rrm3. Top2 is important in resolving the topological constraints. Artwork by Daniele Fachinetti....
On the cover: In this issue, Facchinetti et al. (595–605) report their findings from a global analysis of replication termination sites in the budding yeast Sacchormyces cerevisiase. Termination occurs at pausing sites and is assisted by the Rrm3 helicase and the Top2 topoisomerase. Their study unmasks the mechanisms controlling DNA integrity at termination zones. The cover illustrates converging replication forks (crossroads). One polymerase stalls while the other progresses thanks to Rrm3. Top2 is important in resolving the topological constraints. Artwork by Daniele Fachinetti.


  • GTP: Gatekeeper for Autophagy

    • Isabelle Beau,
    • Patrice Codogno
    Chaperone-mediated autophagy (CMA) is involved in cellular quality control, and in the response to stress, deterioration in CMA contributes to the aging phenotype and to various disorders. The paper by Bandyopadhyay et al. in this issue of Molecular Cell demonstrates the role of GTP binding by elongation factor 1 alpha (EF1α) in regulating the lysosomal uptake of CMA substrates.
  • Termination at sTop2

    • Robert C. Alver,
    • Anja-Katrin Bielinsky
    In this issue of Molecular Cell, Fachinetti et al. provide the first comprehensive map of replication termination sites (TERs) in Saccharomyces cerevisiae (Fachinetti et al., 2010). Strikingly, the majority of TERs are occupied by topoisomerase 2, which shields these regions against genomic instability.
  • Apollo—Taking the Lead in Telomere Protection

    • Jay F. Sarthy,
    • Peter Baumann
    The single-stranded overhangs at the ends of telomeres are thought to be critical for telomere maintenance, but how they are generated has been largely unclear. Two studies (one in this issue of Molecular Cell, Wu et al., 2010) have now implicated the Apollo nuclease in maintaining the overhang specifically at those telomeres generated by leading-strand DNA synthesis.


  • STAT3 Activation of miR-21 and miR-181b-1 via PTEN and CYLD Are Part of the Epigenetic Switch Linking Inflammation to Cancer

    • Dimitrios Iliopoulos,
    • Savina A. Jaeger,
    • Heather A. Hirsch,
    • Martha L. Bulyk,
    • Kevin Struhl
    A transient inflammatory signal can initiate an epigenetic switch from nontransformed to cancer cells via a positive feedback loop involving NF-κB, Lin28, let-7, and IL-6. We identify differentially regulated microRNAs important for this switch and putative transcription factor binding sites in their promoters. STAT3, a transcription factor activated by IL-6, directly activates miR-21 and miR-181b-1. Remarkably, transient expression of either microRNA induces the epigenetic switch. MiR-21 and miR-181b-1, respectively, inhibit PTEN and CYLD tumor suppressors, leading to increased NF-κB activity required to maintain the transformed state.
  • Hsp12 Is an Intrinsically Unstructured Stress Protein that Folds upon Membrane Association and Modulates Membrane Function

    • Sylvia Welker,
    • Birgit Rudolph,
    • Elke Frenzel,
    • Franz Hagn,
    • Gerhard Liebisch,
    • Gerd Schmitz,
    • Johannes Scheuring,
    • Andreas Kerth,
    • Alfred Blume,
    • Sevil Weinkauf,
    • Martin Haslbeck,
    • Horst Kessler,
    • Johannes Buchner
    Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure.
  • Combined Functional Genomic and Proteomic Approaches Identify a PP2A Complex as a Negative Regulator of Hippo Signaling

    • Paulo S. Ribeiro,
    • Filipe Josué,
    • Alexander Wepf,
    • Michael C. Wehr,
    • Oliver Rinner,
    • Gavin Kelly,
    • Nicolas Tapon,
    • Matthias Gstaiger
    The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the progrowth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth.
  • Identification of Regulators of Chaperone-Mediated Autophagy

    • Urmi Bandyopadhyay,
    • Sunandini Sridhar,
    • Susmita Kaushik,
    • Roberta Kiffin,
    • Ana Maria Cuervo
    Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of cytosolic proteins in lysosomes that contributes to cellular quality control and becomes an additional source of amino acids when nutrients are scarce. A chaperone complex delivers CMA substrates to a receptor protein at the lysosomal membrane that assembles into multimeric translocation complexes. However, the mechanisms regulating this process remain, for the most part, unknown. In this work, we have identified two regulatory proteins, GFAP and EF1α, that mediate a previously unknown inhibitory effect of GTP on CMA.
  • Catalysis of Lysine 48-Specific Ubiquitin Chain Assembly by Residues in E2 and Ubiquitin

    • Monica C. Rodrigo-Brenni,
    • Scott A. Foster,
    • David O. Morgan
    Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains.
  • The Three-Dimensional Organization of Polyribosomes in Intact Human Cells

    • Florian Brandt,
    • Lars-Anders Carlson,
    • F. Ulrich Hartl,
    • Wolfgang Baumeister,
    • Kay Grünewald
    Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel.
  • Nuclear IKKβ Is an Adaptor Protein for IκBα Ubiquitination and Degradation in UV-Induced NF-κB Activation

    • Yoshihiro Tsuchiya,
    • Tomoichiro Asano,
    • Keiko Nakayama,
    • Tomohisa Kato Jr.,
    • Michael Karin,
    • Hideaki Kamata
    Proinflammatory cytokines activate NF-κB using the IκB kinase (IKK) complex that phosphorylates inhibitory proteins (IκBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-κB by an unknown mechanism through IκBα degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKβ in UV-induced IκBα degradation. UV irradiation induces the nuclear translocation of IκBα and association with IKKβ, which constitutively interacts with β-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IκBα ubiquitination and degradation.
  • RNA Polymerase I Contains a TFIIF-Related DNA-Binding Subcomplex

    • Sebastian R. Geiger,
    • Kristina Lorenzen,
    • Amelie Schreieck,
    • Patrizia Hanecker,
    • Dirk Kostrewa,
    • Albert J.R. Heck,
    • Patrick Cramer
    The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core.
  • Replication Termination at Eukaryotic Chromosomes Is Mediated by Top2 and Occurs at Genomic Loci Containing Pausing Elements

    • Daniele Fachinetti,
    • Rodrigo Bermejo,
    • Andrea Cocito,
    • Simone Minardi,
    • Yuki Katou,
    • Yutaka Kanoh,
    • Katsuhiko Shirahige,
    • Anna Azvolinsky,
    • Virginia A. Zakian,
    • Marco Foiani
    Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs).
  • Apollo Contributes to G Overhang Maintenance and Protects Leading-End Telomeres

    • Peng Wu,
    • Megan van Overbeek,
    • Sean Rooney,
    • Titia de Lange
    Mammalian telomeres contain a single-stranded 3′ overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions.
  • A Proteomic Screen for Nucleolar SUMO Targets Shows SUMOylation Modulates the Function of Nop5/Nop58

    • Belinda J. Westman,
    • Céline Verheggen,
    • Saskia Hutten,
    • Yun Wah Lam,
    • Edouard Bertrand,
    • Angus I. Lamond
    Posttranslational SUMO modification is an important mechanism of regulating protein function, especially in the cell nucleus. The nucleolus is the subnuclear organelle responsible for rRNA synthesis, processing, and assembly of the large and small ribosome subunits. Here, we have used SILAC-based quantitative proteomics to identify nucleolar SUMOylated proteins. This reveals a role for SUMOylation in the biogenesis and/or function of small nucleolar ribonucleoprotein complexes (snoRNPs) via the targeting of Nhp2 and Nop58.

Short Articles

  • MMXD, a TFIIH-Independent XPD-MMS19 Protein Complex Involved in Chromosome Segregation

    • Shinsuke Ito,
    • Li Jing Tan,
    • Daisuke Andoh,
    • Takashi Narita,
    • Mineaki Seki,
    • Yasuhiro Hirano,
    • Keiko Narita,
    • Isao Kuraoka,
    • Yasushi Hiraoka,
    • Kiyoji Tanaka
    Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes.


  • Site-Specific Identification of SUMO-2 Targets in Cells Reveals an Inverted SUMOylation Motif and a Hydrophobic Cluster SUMOylation Motif

    • Ivan Matic,
    • Joost Schimmel,
    • Ivo A. Hendriks,
    • Maria A. van Santen,
    • Frans van de Rijke,
    • Hans van Dam,
    • Florian Gnad,
    • Matthias Mann,
    • Alfred C.O. Vertegaal
    Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry is still very challenging. We have developed a generic method for the identification of SUMO acceptor lysines in target proteins. We have identified 103 SUMO-2 acceptor lysines in endogenous target proteins.