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  • 1.
    Björk, Glenn R
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Jacobsson, Kerstin
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nilsson, Kristina
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Persson, Olof P
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A primordial tRNA modification required for the evolution of life?2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 1-2, p. 231-239Article in journal (Refereed)
    Abstract [en]

    The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

  • 2.
    Björklund, Stefan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Skogman, E
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit1992In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 11, no 13, p. 4953-4959Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.

  • 3. Bohmert, K.
    et al.
    Camus, I.
    Bellini, C.
    Bouchez, D.
    Caboche, M.
    Benning, C.
    AGO1 defines a novel locus of Arabidopsis controlling leaf development1998In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 17, no 1, p. 170-180Article in journal (Refereed)
    Abstract [en]

    An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated, The ago1 mutation pleotropically affects general plant architecture, The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop, Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation, Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced, Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA resulted in a partial mutant phenotype, Sense expression caused some transgenic lines to develop goblet-like leaves and petals, The cDNA encodes a putative 115 kDa protein with sequence similarity tea translation products of a novel gene family present in nematodes as,yell as humans, No specific function has been assigned to these genes, Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGOI belongs to a novel class of genes with a function specific to multicellular organisms.

  • 4.
    Boutté, Yohann
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Frescatada-Rosa, Márcia
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Men, Shuzhen
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Chow, Cheung-Ming
    Ebine, Kazuo
    Gustavsson, Anna
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Johansson, Lenore
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Ueda, Takashi
    Moore, Ian
    Jürgens, Gerd
    Grebe, Markus
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 3, p. 546-58Article in journal (Refereed)
    Abstract [en]

    Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.

  • 5.
    Byström, Anders S
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology. Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hjalmarsson, Karin J
    Umeå University, Faculty of Medicine, Molecular Biology.
    Wikström, P Mikael
    Umeå University, Faculty of Medicine, Molecular Biology.
    Björk, Glenn R
    Umeå University, Faculty of Medicine, Molecular Biology. Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide1983In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 2, no 6, p. 899-905Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.

  • 6. CAMPBELL, D
    et al.
    ZHOU, GQ
    Gustafsson, Petter
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Oquist, Gunnar
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    CLARKE, AK
    ELECTRON-TRANSPORT REGULATES EXCHANGE OF 2 FORMS OF PHOTOSYSTEM-II D1 PROTEIN IN THE CYANOBACTERIUM SYNECHOCOCCUS1995In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 14, no 22, p. 5457-5466Article in journal (Refereed)
    Abstract [en]

    Synechococcus sp, PCC 7942 modulates photosynthetic function by transiently replacing the constitutive D1 photosystem II protein, D1:1, with an alternate form, D1:2, to help counteract photoinhibition under excess light, We show that a temperature drop from 37 to 25 degrees C also drives D1:1/D1:2 exchange under constant, moderate light, Chilling or light-induced D1 exchange results from rapid loss of psbAI message coding for D1:1 and accumulation of psbAII and psbAIII messages coding for D1:2, During chilling, a large pool of a novel form, D1:2*, transiently accumulates, distinguishable from normal D1 by an increase in apparent molecular mass, D1:* is not phosphorylated and is probably a functionally inactive, incompletely processed precursor, After acclimation to 25 degrees C, D1:2* disappears and D1:1 again predominates, although substantial D1:2 remains, Partial inhibition of electron transport under constant, moderate light also triggers the D1 exchange process, These treatments all increase excitation pressure on photosystem II relative to electron transport, Therefore, information from photosynthetic electron transport regulates D1 exchange without any requirement for a change in light intensity or quality, possibly via a redox sensing mechanism proximal to photosystem II.

  • 7.
    Cava, Felipe
    et al.
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA ; CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    de Pedro, Miguel A
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Lam, Hubert
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA.
    Davis, Brigid M
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA.
    Waldor, Matthew K
    Department of Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School and HHMI, Boston, MA, USA.
    Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids2011In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, no 16, p. 3442-3453Article in journal (Refereed)
    Abstract [en]

    Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.

  • 8.
    Chen, Peng
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Crain, Pamela F
    Näsvall, Joakim
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Pomerantz, Steven C
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    A "gain of function" mutation in a protein mediates production of novel modified nucleosides.2005In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 24, no 10, p. 1842-1851Article in journal (Refereed)
    Abstract [en]

    The mutation sufY204 mediates suppression of a +1 frameshift mutation in the histidine operon of Salmonella enterica serovar Typhimurium and synthesis of two novel modified nucleosides in tRNA. The sufY204 mutation, which results in an amino-acid substitution in a protein, is, surprisingly, dominant over its wild-type allele and thus it is a "gain of function" mutation. One of the new nucleosides is 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modified by addition of a C(10)H(17) side chain of unknown structure. Increased amounts of both nucleosides in tRNA are correlated to gene dosage of the sufY204 allele, to an increased efficiency of frameshift suppression, and to a decreased amount of the wobble nucleoside mnm(5)s(2)U34 in tRNA. Purified tRNA(Gln)(cmnm(5)s(2)UUG) in the mutant strain contains a modified nucleoside similar to the novel nucleosides and the level of aminoacylation of tRNA(Gln)(cmnm(5)s(2)UUG) was reduced to 26% compared to that found in the wild type (86%). The results are discussed in relation to the mechanism of reading frame maintenance and the evolution of modified nucleosides in tRNA.

  • 9. Davidson, Iain F.
    et al.
    Goetz, Daniela
    Zaczek, Maciej P.
    Molodtsov, Maxim I.
    in't Veld, Pim J. Huis
    Weissmann, Florian
    Litos, Gabriele
    Cisneros, David A.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Research Institute of Molecular Pathology (IMP), Vienna, Austria.
    Ocampo-Hafalla, Maria
    Ladurner, Rene
    Uhlmann, Frank
    Vaziri, Alipasha
    Peters, Jan-Michael
    Rapid movement and transcriptional re-localization of human cohesin on DNA2016In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 24, p. 2671-2685Article in journal (Refereed)
    Abstract [en]

    The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

  • 10. Davidson, Marta B.
    et al.
    Katou, Yuki
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sing, Tina L.
    Xia, Tian
    Ou, Jiongwen
    Vaisica, Jessica A.
    Thevakumaran, Neroshan
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Myers, Chad L.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Shirahige, Katsuhiko
    Brown, Grant W.
    Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 4, p. 895-907Article in journal (Refereed)
    Abstract [en]

    The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis. The EMBO Journal (2012) 31, 895-907. doi: 10.1038/emboj.2011.485; Published online 10 January 2012

  • 11. Delaney, Joseph R
    et al.
    Stöven, Svenja
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
    Uvell, Hanna
    Anderson, Kathryn V
    Engström, Ylva
    Mlodzik, Marek
    Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways.2006In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 25, no 13, p. 3068-77Article in journal (Refereed)
    Abstract [en]

    Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, we demonstrate that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals was rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. Our data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression.

  • 12. Gur, Gal
    et al.
    Rubin, Chanan
    Katz, Menachem
    Amit, Ido
    Citri, Ami
    Nilsson, Jonas
    Umeå University, Faculty of Medicine, Radiation Sciences, Oncology.
    Amariglio, Ninette
    Henriksson, Roger
    Umeå University, Faculty of Medicine, Radiation Sciences, Oncology.
    Rechavi, Gideon
    Hedman, Håkan
    Umeå University, Faculty of Medicine, Radiation Sciences, Oncology.
    Wides, Ron
    Yarden, Yosef
    LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation2004In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 23, no 16, p. 3270-3281Article in journal (Refereed)
  • 13. Hauge, Camilla
    et al.
    Antal, Torben L
    Hirschberg, Daniel
    Doehn, Ulrik
    Thorup, Katrine
    Idrissova, Leila
    Hansen, Klaus
    Jensen, Ole N
    Jørgensen, Thomas J
    Biondi, Ricardo M
    Frödin, Morten
    Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation.2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 9Article in journal (Refereed)
    Abstract [en]

    The growth factor/insulin-stimulated AGC kinases share an activation mechanism based on three phosphorylation sites. Of these, only the role of the activation loop phosphate in the kinase domain and the hydrophobic motif (HM) phosphate in a C-terminal tail region are well characterized. We investigated the role of the third, so-called turn motif phosphate, also located in the tail, in the AGC kinases PKB, S6K, RSK, MSK, PRK and PKC. We report cooperative action of the HM phosphate and the turn motif phosphate, because it binds a phosphoSer/Thr-binding site above the glycine-rich loop within the kinase domain, promoting zipper-like association of the tail with the kinase domain, serving to stabilize the HM in its kinase-activating binding site. We present a molecular model for allosteric activation of AGC kinases by the turn motif phosphate via HM-mediated stabilization of the alphaC helix. In S6K and MSK, the turn motif phosphate thereby also protects the HM from dephosphorylation. Our results suggest that the mechanism described is a key feature in activation of upto 26 human AGC kinases.

  • 14.
    Karlsson, Jan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Clarke, Adrian K
    Chen, Zhi-Yuan
    Hugghins, Stephanie Y
    Park, Youn-II
    Husic, H David
    Moroney, James V
    Samuelsson, Göran
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    A novel α-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO21998In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 17, no 5, p. 1208-1216Article in journal (Refereed)
    Abstract [en]

    A 29.5 kDa intracellular α-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.

  • 15.
    Laptenko, Oleg
    et al.
    Department of Cell Biology, School of Osteopathic Medicine at Stratford, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA.
    Kim, Seung-Sup
    Department of Biochemistry, New York University School of Medicine, New York, NY, USA.
    Lee, Jookyung
    Department of Cell Biology, School of Osteopathic Medicine at Stratford, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA.
    Starodubtseva, Marina
    Department of Cell Biology, School of Osteopathic Medicine at Stratford, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA.
    Cava, Felipe
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Berenguer, Jose
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Kong, Xiang-Peng
    Department of Biochemistry, New York University School of Medicine, New York, NY, USA.
    Borukhov, Sergei
    Department of Cell Biology, School of Osteopathic Medicine at Stratford, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA.
    pH-dependent conformational switch activates the inhibitor of transcription elongation2006In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 25, no 10, p. 2131-2141Article in journal (Refereed)
    Abstract [en]

    Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N-terminal coiled-coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C-terminal domain (CTD). We show that depending on pH, Gfh1-CTD exists in two alternative orientations. At pH above 7, it assumes an inactive 'flipped' orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1-CTD switches to an active 'Gre-like' orientation, which enables Gfh1 to bind to and inhibit RNAP.

  • 16. Leanderson, T
    et al.
    Sundström, S
    Mårtensson, I L
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundgren, E
    Interferon-specific effects on protein synthesis in P3HR-1 cells.1982In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 1, no 12, p. 1505-11Article in journal (Refereed)
    Abstract [en]

    The effect of interferon (IFN) on protein synthesis was studied in the Burkitt's lymphoma cell line P3HR-1 by [35S]methionine labelling of the cells, followed by two-dimensional gel electrophoresis of cell extracts. De novo synthesis of three proteins (mol. wts. 33 000, 62 000, and 98 000, respectively) and alterations in the rate of synthesis for a small number of additional proteins were observed during the first 12 h of treatment, while the rate of overall protein synthesis was unaffected. Treatment of P3HR-1 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or hydrocortisone (HC), which induce similar changes in cell cycle distribution as does IFN, did not induce comparable changes in the rates of protein synthesis. Thus, the effects were specific for IFN and not induced by the change in cell cycle distribution per se, i.e., accumulation in G0. Treatment of cells with 2'-5' pA core did not mimic the effect of IFN at the translational level. A substrain of P3HR-1 cells, selected for resistance to the anti-proliferative effect of IFN, lacked six proteins found in the wild-type. The 62 000 mol. wt. protein was induced in this substrain as well as in native P3HR-1 cells on addition of IFN. The resistant substrain still developed an anti-viral effect in response to IFN. Thus, it seems as if the anti-proliferative and anti-viral effects of IFN, at least in some cells are mediated by different intracellular molecular mechanisms.

  • 17. Lee, J Y
    et al.
    Edlund, T
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Faye, I
    Boman, H G
    Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia.1983In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 2, no 4, p. 577-81Article in journal (Refereed)
    Abstract [en]

    Diapausing pupae of the Cecropia moth (Hyalophora cecropia) respond to an injection of live bacteria by the selective synthesis of certain types of RNA and immune proteins (designated P1-P9). The in vitro translation products of RNA from both injured and infected pupae showed specific patterns with a defined number of extra bands. Some proteins characteristic of the normal RNA were reduced in the immune RNA translation products. Antibody reaction was used to show the selective synthesis of immune proteins P4 and P5 with mRNA from pupae subjected to injury or infection. The protein synthesized in vitro, which cross-reacted with P5 antibodies, is most likely a precursor of the attacins described in the preceding paper. A cDNA clone bank was prepared and two clones were isolated and shown to contain 750 bp corresponding to P4 and 250 bp of attacin information. These clones were used to estimate the sizes of the mRNAs by Northern blotting and to estimate, by RNA/DNA hybridization, the levels of P4 and P5 mRNA. In vivo incorporation of [35S]methionine into attacins and P4 during different conditions was compared with the levels of the corresponding mRNA.

  • 18. Magyar, Zoltan
    et al.
    Horvath, Beatrix
    Khan, Safina
    Mohammed, Binish
    Henriques, Rossana
    De Veylder, Lieven
    Bako, Laszlo
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Scheres, Ben
    Boegre, Laszlo
    Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 6, p. 1480-1493Article in journal (Refereed)
    Abstract [en]

    Post-embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB-RBR1 (retinoblastoma-related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FA(Delta RB)) lacking the RBR1-binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FADRB and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways. The EMBO Journal (2012) 31, 1480-1493. doi:10.1038/emboj.2012.13; Published online 3 February 2012

  • 19. Martin, Nadine
    et al.
    Popov, Nikolay
    Aguilo, Francesca
    O'Loghlen, Ana
    Raguz, Selina
    Snijders, Ambrosius P
    Dharmalingam, Gopuraja
    Li, Side
    Thymiakou, Efstathia
    Carroll, Thomas
    Zeisig, Bernd B
    So, Chi Wai Eric
    Peters, Gordon
    Episkopou, Vasso
    Walsh, Martin J
    Gil, Jesús
    Interplay between Homeobox proteins and Polycomb repressive complexes in p16INK⁴a regulation.2013In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 32, no 7Article in journal (Refereed)
    Abstract [en]

    The INK4/ARF locus regulates senescence and is frequently altered in cancer. In normal cells, the INK4/ARF locus is found silenced by Polycomb repressive complexes (PRCs). Which are the mechanisms responsible for the recruitment of PRCs to INK4/ARF and their other target genes remains unclear. In a genetic screen for transcription factors regulating senescence, we identified the homeodomain-containing protein HLX1 (H2.0-like homeobox 1). Expression of HLX1 extends cellular lifespan and blunts oncogene-induced senescence. Using quantitative proteomics, we identified p16(INK4a) as the key target mediating the effects of HLX1 in senescence. HLX1 represses p16(INK4a) transcription by recruiting PRCs and HDAC1. This mechanism has broader implications, as HLX1 also regulates a subset of PRC targets besides p16(INK4a). Finally, sampling members of the Homeobox family, we identified multiple genes with ability to repress p16(INK4a). Among them, we found HOXA9 (Homeobox A9), a putative oncogene in leukaemia, which also recruits PRCs and HDAC1 to regulate p16(INK4a). Our results reveal an unexpected and conserved interplay between homeodomain-containing proteins and PRCs with implications in senescence, development and cancer.

  • 20.
    Nissan, Tracy A
    et al.
    Biochemie-Zentrum Heidelberg, Universität Heidelberg, Germany.
    Bassler, Jochen
    Petfalski, Elisabeth
    Tollervey, David
    Hurt, Ed
    60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm2002In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 21, no 20, p. 5539-5547Article in journal (Refereed)
    Abstract [en]

    60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.

  • 21.
    O'Neill, E
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikström, P
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An active role for a structured B-linker in effector control of the sigma54-dependent regulator DmpR.2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4Article in journal (Refereed)
    Abstract [en]

    The activities of many prokaryotic sigma54-dependent transcriptional activators are controlled by the N-terminal A-domain of the protein, which is linked to the central transcriptional activation domain via a short B-linker. It used to be thought that these B-linkers simply serve as flexible tethers. Here we show that the B-linker of the aromatic-responsive regulator DmpR and many other regulators of the family contain signature heptad repeats with regularly spaced hydrophobic amino acids. Mutant analysis of this region of DmpR demonstrates that B-linker function is dependent on the heptad repeats and is critical for activation of the protein by aromatic effectors. The phenotypes of DmpR mutants refute the existing model that the level of ATPase activity directly controls the level of transcription it promotes. The mutant analysis also shows that the B-linker is involved in repression of ATPase activity and that allosteric changes upon effector binding are transduced to alleviate both B-linker repression of ATP hydrolysis and A-domain repression of transcriptional activation. The mechanistic implications of these findings for DmpR and other family members are discussed.

  • 22. Ottmann, Christian
    et al.
    Yasmin, Lubna
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Weyand, Michael
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Palmer, Ruth H
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hauser, Alan R
    Wittinghofer, Alfred
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.2007In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 3, p. 902-913Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 A crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14-3-3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.

  • 23.
    Persson, Cathrine
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Carballeira, Nivia
    Umeå University, Faculty of Medicine, Molecular Biology.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The PTPase YopH inhibits uptake of Yersinia, tyrosine phosphorylation of p130Cas and FAK, and the associated accumulation of these proteins in peripheral focal adhesions1997In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 16, no 9, p. 2307-2318Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia resist uptake by eukaryotic cells by a mechanism involving the virulence protein YopH, a protein tyrosine phosphatase. We show that p130Cas and FAK are phosphorylated and recruited to peripheral focal complexes during bacterial uptake in HeLa cells. The inactive form of YopH interacts with the tyrosine phosphorylated forms of FAK and p130Cas and co-localizes with these proteins in focal adhesions. On the other hand, the presence of active YopH results in inhibition of uptake, dephosphorylation of p130Cas and FAK, and disruption of peripheral focal complexes. We suggest that p130Cas and FAK are substrates for YopH and that the dephosphorylation of these proteins impairs the uptake of Yersinia pseudotuberculosis into HeLa cells.

  • 24.
    Pinto do Ó, Perpétua
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology.
    Kolterud, Åsa
    Umeå University, Faculty of Medicine, Molecular Biology.
    Carlsson, Leif
    Umeå University, Faculty of Medicine, Molecular Biology.
    Expression of the LIM-homeobox gene LH2 generates immortalized Steel factor-dependent multipotent hematopoietic precursors1998In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 17, no 19, p. 5744-5756Article in journal (Refereed)
    Abstract [en]

    The genes controlling self-renewal and differentiation in the hematopoietic system are largely unknown. The LIM-homeobox genes are known to be important for asymmetric cell divisions and differentiation of specific cell types and organs. One member of this family, LH2, is expressed in fetal liver at the time of active hematopoiesis. Therefore, we have assessed the function of LH2 during the formation and initial expansion of the hematopoietic system by differentiating LH2-transduced embryonic stem (ES) cells in vitro. This procedure generated multipotent hematopoietic precursor cell (HPC) lines that required Steel factor for growth. HPC lines have been maintained in an undifferentiated state in culture for >7 months. Other growth factors tested efficiently induce terminal differentiation of HPCs into various mature myeloid lineages. Steel factor is also required and acts synergistically with the other growth factors to generate multilineage colonies from the HPCs. These HPC lines express transcription factors that are consistent with an immature progenitor, and the pattern of cell surface marker expression is similar to that of early fetal multipotent hematopoietic progenitors. Collectively, these data suggest that the HPC lines represent an early fetal multipotent hematopoietic progenitor, and suggest a role for LH2 in the control of cell fate decision and/or proliferation in the hematopoietic system.

  • 25. Poli, Jerome
    et al.
    Tsaponina, Olga
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Crabbe, Laure
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pantesco, Veronique
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lengronne, Armelle
    Pasero, Philippe
    dNTP pools determine fork progression and origin usage under replication stress2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 4, p. 883-894Article in journal (Refereed)
    Abstract [en]

    Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

  • 26. Shimokawa, Noriaki
    et al.
    Haglund, Kaisa
    Hölter, Sabine M
    Grabbe, Caroline
    Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany.
    Kirkin, Vladimir
    Koibuchi, Noriyuki
    Schultz, Christian
    Rozman, Jan
    Hoeller, Daniela
    Qiu, Chun-Hong
    Londoño, Marina B
    Ikezawa, Jun
    Jedlicka, Peter
    Stein, Birgit
    Schwarzacher, Stephan W
    Wolfer, David P
    Ehrhardt, Nicole
    Heuchel, Rainer
    Nezis, Ioannis
    Brech, Andreas
    Schmidt, Mirko H H
    Fuchs, Helmut
    Gailus-Durner, Valerie
    Klingenspor, Martin
    Bogler, Oliver
    Wurst, Wolfgang
    Deller, Thomas
    de Angelis, Martin Hrabé
    Dikic, Ivan
    CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 14, p. 2421-32Article in journal (Refereed)
    Abstract [en]

    Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.

  • 27. van Weering, Jan R. T.
    et al.
    Sessions, Richard B.
    Traer, Colin J.
    Kloer, Daniel P.
    Bhatia, Vikram K.
    Stamou, Dimitrios
    Carlsson, Sven R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hurley, James H.
    Cullen, Peter J.
    Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules2012In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 23, p. 4466-4480Article in journal (Refereed)
    Abstract [en]

    Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX-BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX-BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 angstrom structure of a SNX1-BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip-loop' interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX-BAR-decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

  • 28.
    Wilczynska, Malgorzata
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lobov, Sergei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ohlsson, Per-Ingvar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization.2003In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 8, p. 1753-1761Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.

  • 29.
    Wilson, Sara I
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Target practice: Zic2 hits the bullseye!2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 18, p. 3037-3038Article in journal (Refereed)
  • 30. Wrzaczek, Michael
    et al.
    Vainonen, Julia P.
    Stael, Simon
    Tsiatsiani, Liana
    Help-Rinta-Rahko, Hanna
    Gauthier, Adrien
    Kaufholdt, David
    Bollhöner, Benjamin
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Lamminmaki, Airi
    Staes, An
    Gevaert, Kris
    Tuominen, Hannele
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Van Breusegem, Frank
    Helariutta, Yka
    Kangasjarvi, Jaakko
    GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis2015In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 31, no 1, p. 55-66Article in journal (Refereed)
    Abstract [en]

    Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor.

  • 31.
    Yamazaki, Yasuo
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schönherr, Christina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Varshney, Gaurav K.
    Dogru, Murat
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Palmer, Ruth H.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Goliath family E3 ligases regulate the recycling endosome pathway via VAMP3 ubiquitylation2013In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 32, no 4, p. 524-537Article in journal (Refereed)
    Abstract [en]

    Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, processes regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. In addition to sorting to the lysosome, cargo is recycled to the plasma membrane via recycling endosomes. Here, we describe a role of the goliath gene family of protease-associated (PA) domain E3 ligases in regulating recycling endosome trafficking. The two Drosophila members of this family-Goliath and Godzilla(CG10277) - are located on endosomes, and both ectopic expression and loss-of-function lead to the accumulation of Rab5-positive giant endosomes. Furthermore, the human homologue RNF167 exhibits similar behaviour. We show that the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein VAMP3 is a target of these ubiquitin ligases, and that recycling endosome trafficking is abrogated in response to their activity. Furthermore, mutation of the Godzilla ubiquitylation target lysines on VAMP3 abrogates the formation of enlarged endosomes induced by either Godzilla or RNF167. Thus, Goliath ubiquitin ligases play a novel role in regulating recycling endosome trafficking via ubiquitylation of the VAMP3 SNARE protein.

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