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Gilmore, James Reid

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Property	Value
overview	PhD, University of Illinois, Urbana, 1980 Postdoctoral Training The Rockefeller University, 1980-1983 Molecular mechanism of secretory protein translocation 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	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.

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overview

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

Molecular mechanism of secretory protein translocation

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

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.

One or more keywords matched the following items that are connected to Gilmore, James Reid

Item Type	Name
Academic Article	Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties.
Academic Article	GTPase twins in the SRP family.
Academic Article	Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains.
Academic Article	An evolving view of the eukaryotic oligosaccharyltransferase.
Academic Article	Slow translocon gating causes cytosolic exposure of transmembrane and lumenal domains during membrane protein integration.
Academic Article	The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation.
Academic Article	Understanding integration of a-helical membrane proteins: the next steps.
Academic Article	A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function.
Academic Article	Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST.
Academic Article	Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex.
Academic Article	Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum.
Academic Article	Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation.
Academic Article	The tail end of membrane insertion.
Academic Article	An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation.
Academic Article	Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica.
Academic Article	Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms.
Academic Article	Translocation of proteins through the Sec61 and SecYEG channels.
Academic Article	Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.
Academic Article	Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease.
Academic Article	Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity.
Academic Article	Protein translocation across the rough endoplasmic reticulum.
Academic Article	Glycosylation of closely spaced acceptor sites in human glycoproteins.
Concept	Protein Binding
Concept	Protein Subunits
Concept	Protein Transport
Concept	Protein Folding
Concept	Protein Processing, Post-Translational
Concept	Protein Biosynthesis
Concept	Protein Precursors
Concept	Protein Interaction Domains and Motifs
Concept	Protein Structure, Secondary
Concept	Protein Structure, Tertiary
Concept	Protein Modification, Translational
Concept	Protein Multimerization
Concept	Receptor Protein-Tyrosine Kinases
Concept	Protein Sorting Signals
Concept	Protein Isoforms
Concept	Protein Conformation
Concept	Databases, Protein
Academic Article	Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins.
Academic Article	Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum.
Academic Article	Mammalian cells lacking either the cotranslational or posttranslocational oligosaccharyltransferase complex display substrate-dependent defects in asparagine linked glycosylation.
Academic Article	Reduced expression of the oligosaccharyltransferase exacerbates protein hypoglycosylation in cells lacking the fully assembled oligosaccharide donor.
Academic Article	Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells.
Academic Article	Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.
Academic Article	DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon.
Academic Article	Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum.
Academic Article	Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII.
Academic Article	One flexible loop in OST lassos both substrates.
Academic Article	Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations.
Academic Article	Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.
Academic Article	Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction.
Academic Article	Selective inhibition of N-linked glycosylation impairs receptor tyrosine kinase processing.
Academic Article	Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis.
Concept	Protein Domains
Concept	Protein Conformation, alpha-Helical
Concept	Protein Conformation, beta-Strand

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