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    James Reid Gilmore PhD

    TitleProfessor
    InstitutionUniversity of Massachusetts Medical School
    DepartmentBiochemistry and Molecular Pharmacology
    AddressUniversity of Massachusetts Medical School
    364 Plantation Street, LRB
    Worcester MA 01605
    Phone508-856-5894
      Other Positions
      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentBiochemistry and Molecular Pharmacology

      InstitutionUMMS - Graduate School of Biomedical Sciences
      DepartmentInterdisciplinary Graduate Program

        Overview 
        Narrative

        PhD, University of Illinois, Urbana, 1980
        Postdoctoral Training
        The Rockefeller University, 1980-1983

        Molecular mechanism of secretory protein translocation

        Reid Gilmore

        The objective of research in our laboratory is to understand how proteins reach their final destinations within a cell. Specifically, we are investigating the biosynthesis, translocation, processing and folding of proteins in the rough endoplasmic reticulum.

        A major goal of our lab is to elucidate the mechanism of protein translocation across the rough endoplasmic reticulum (RER) membrane. The signal recognition particle (SRP), a ribonucleoprotein that binds to the polypeptide exit site on the ribosome, and the membrane bound SRP receptor (SR) function together to selectively attach a ribosome synthesizing a protein with an RER signal sequences to the Sec61 complex. The Sec61 heterotrimer forms an evolutionarily conserved channel for translocation of secreted proteins and integration of membrane proteins. We are using the Saccharomyces cerevisiae experimental to analyze the mechanism of translocation channel gating by ribosome-nascent chain complexes. Novel Sec61 mutants are designed based upon the structure of the Methanococcus janaschii SecYEb complex. The in vivo kinetics of protein translocation channel gating are being analyzed using ubiquitin translocation assay (UTA) reporters.

        Asparagine-linked glycosylation of proteins occurs within the lumen of the RER. Oligosaccharyltransferase (OST) catalyzes the transfer of a preassembled high-mannose oligosaccharide onto N-X-T or N-X-S acceptor sites (seqons) as the nascent polypeptide is transported into the RER lumen through the Sec61 complex. The oligosaccharide donor for the enzyme is the dolichol pyrophosphate linked oligosaccharide GlcNAc2Man9Glc3. The oligosaccharyltransferase is a hetero-octameric integral membrane protein in higher eukaryotes. Many unicellular protists have simpler OSTs with either a single subunit (e.g. Trypanosoma cruzi) or four subunits (e.g. Entamoeba histolytica). The active site subunit of the OST is the STT3 protein. Interestingly, the genomes of insects, plants and vertebrates encode two STT3 proteins. Biochemical experiments have revealed that the two SST3 proteins (STT3A and STT3B) assemble with a shared set of non-catalytic subunits into two separate OST complexes with different kinetic properties. We are analyzing the roles of these two distinct OST complexes in tissue culture cells by selectively knocking down STT3A or STT3B expression using siRNAs. Current evidence indicates that the STT3A isoform of the OST is primarily responsible for cotranslational glycosylation of nascent glycoproteins as the acceptor sequons exit the luminal face of the translocation channel. The STT3B isoform of the OST can modify skipped sequons on unfolded proteins in the lumen of the ER.



        Rotation Projects

        Rotation Project Background



        In yeast cells, protein translocation across the endoplasmic reticulum can occur by cotranslational or posttranslational pathways.  The objective of this project is to develop a rapid method to inactivate the posttranslational pathway in vivo in yeast cells by appending the CMV-ribosome stalling sequence to the C-terminus of a typical posttranslational translocation substrate (carboxypeptidase Y).  Complexes between the posttranslational translocation channel (SEC complex) and the stalled ribosomes will be purified for structural and functional analysis.  


        Bibliographic 
        selected publications
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        1. Cherepanova NA, Shrimal S, Gilmore R. Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins. J Cell Biol. 2014 Aug 18; 206(4):525-39.
          View in: PubMed
        2. Shrimal S, Gilmore R. Glycosylation of closely spaced acceptor sites in human glycoproteins. J Cell Sci. 2013 Dec 1; 126(Pt 23):5513-23.
          View in: PubMed
        3. Shrimal S, Ng BG, Losfeld ME, Gilmore R, Freeze HH. Mutations in STT3A and STT3B cause two congenital disorders of glycosylation. Hum Mol Genet. 2013 Nov 15; 22(22):4638-45.
          View in: PubMed
        4. Shrimal S, Trueman SF, Gilmore R. Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST. J Cell Biol. 2013 Apr 1; 201(1):81-95.
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        5. Mandon EC, Trueman SF, Gilmore R. Protein translocation across the rough endoplasmic reticulum. Cold Spring Harb Perspect Biol. 2013 Feb; 5(2).
          View in: PubMed
        6. Trueman SF, Mandon EC, Gilmore R. A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function. J Cell Biol. 2012 Dec 10; 199(6):907-18.
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        7. Gilmore R, Mandon EC. Understanding integration of a-helical membrane proteins: the next steps. Trends Biochem Sci. 2012 Aug; 37(8):303-8.
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        8. Zhang X, Garbett K, Veeraraghavalu K, Wilburn B, Gilmore R, Mirnics K, Sisodia SS. A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2. J Neurosci. 2012 Jun 20; 32(25):8633-48.
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        9. Trueman SF, Mandon EC, Gilmore R. Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity. Mol Biol Cell. 2011 Sep; 22(17):2983-93.
          View in: PubMed
        10. Gilmore R. Structural biology: Porthole to catalysis. Nature. 2011 Jun 16; 474(7351):292-3.
          View in: PubMed
        11. Bas T, Gao GY, Lvov A, Chandrasekhar KD, Gilmore R, Kobertz WR. Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease. J Biol Chem. 2011 Aug 12; 286(32):28150-9.
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        12. Becker T, Bhushan S, Jarasch A, Armache JP, Funes S, Jossinet F, Gumbart J, Mielke T, Berninghausen O, Schulten K, Westhof E, Gilmore R, Mandon EC, Beckmann R. Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science. 2009 Dec 4; 326(5958):1369-73.
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        13. Mandon EC, Trueman SF, Gilmore R. Translocation of proteins through the Sec61 and SecYEG channels. Curr Opin Cell Biol. 2009 Aug; 21(4):501-7.
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        14. Ruiz-Canada C, Kelleher DJ, Gilmore R. Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms. Cell. 2009 Jan 23; 136(2):272-83.
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        15. Magnelli P, Cipollo JF, Ratner DM, Cui J, Kelleher D, Gilmore R, Costello CE, Robbins PW, Samuelson J. Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica. J Biol Chem. 2008 Jun 27; 283(26):18355-64.
          View in: PubMed
        16. Jiang Y, Cheng Z, Mandon EC, Gilmore R. An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation. J Cell Biol. 2008 Mar 24; 180(6):1149-61.
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        17. Banerjee S, Vishwanath P, Cui J, Kelleher DJ, Gilmore R, Robbins PW, Samuelson J. The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation. Proc Natl Acad Sci U S A. 2007 Jul 10; 104(28):11676-81.
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        18. Kelleher DJ, Banerjee S, Cura AJ, Samuelson J, Gilmore R. Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms. J Cell Biol. 2007 Apr 9; 177(1):29-37.
          View in: PubMed
        19. Mandon EC, Gilmore R. The tail end of membrane insertion. Cell. 2007 Mar 23; 128(6):1031-2.
          View in: PubMed
        20. Cheng Z, Gilmore R. Slow translocon gating causes cytosolic exposure of transmembrane and lumenal domains during membrane protein integration. Nat Struct Mol Biol. 2006 Oct; 13(10):930-6.
          View in: PubMed
        21. Kelleher DJ, Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology. 2006 Apr; 16(4):47R-62R.
          View in: PubMed
        22. Karamyshev AL, Kelleher DJ, Gilmore R, Johnson AE, von Heijne G, Nilsson I. Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains. J Biol Chem. 2005 Dec 9; 280(49):40489-93.
          View in: PubMed
        23. Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, Robbins PW. The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci U S A. 2005 Feb 1; 102(5):1548-53.
          View in: PubMed
        24. Cheng Z, Jiang Y, Mandon EC, Gilmore R. Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation. J Cell Biol. 2005 Jan 3; 168(1):67-77.
          View in: PubMed
        25. Mandon EC, Gilmore R. GTPase twins in the SRP family. Nat Struct Mol Biol. 2004 Feb; 11(2):115-6.
          View in: PubMed
        26. Mandon EC, Jiang Y, Gilmore R. Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum. J Cell Biol. 2003 Aug 18; 162(4):575-85.
          View in: PubMed
        27. Wu X, Rush JS, Karaoglu D, Krasnewich D, Lubinsky MS, Waechter CJ, Gilmore R, Freeze HH. Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij. Hum Mutat. 2003 Aug; 22(2):144-50.
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        28. Kelleher DJ, Karaoglu D, Mandon EC, Gilmore R. Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties. Mol Cell. 2003 Jul; 12(1):101-11.
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        29. Nilsson I, Kelleher DJ, Miao Y, Shao Y, Kreibich G, Gilmore R, von Heijne G, Johnson AE. Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J Cell Biol. 2003 May 26; 161(4):715-25.
          View in: PubMed
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