Section: Research

Reid Gilmore, Ph.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Biochemistry and Molecular Pharmacology

Other Affiliation(s):
   Interdisciplinary Graduate Program

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 ellucidate 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 ribosomes synthesizing proteins with RER signal sequences to the Sec61 complex. The Sec61 complex is the central core of the protein translocation channel. We have developed assays to monitor the GTP-dependant insertion of the nascent chain into the Sec61 complex. The signal sequence binding subunit of SRP (SRP54) and both subunits of the SRP receptor are GTPases. The GTP hydrolysis cycles of SRP54, SRa, and SRb are being investigated to determine how GTP-binding to each of these GTPases regulates the affinity between SRP, the SRP receptor, the signal sequence and the translocation channel.

Asparagine-linked glycosylation of proteins occurs whithin the lumen of the RER. Oligosaccharyltransferase (OST), catalyzes th cotranslational transfer of a preassembled high-mannose oligosaccharide onto N-X-T or N-X-S acceptor sites as the nascent polypeptide is transported into the RER lumen. The oligosaccharide donor for the enzyme is the dolichol pyrophosphate linked oligosaccharide GlcNAC₂Man₉Glc₃. We have purified the canine and yeast oligosaccharyltransferases. In yeast, the OST is a heteroligomeric membrane protein composed of eight non identical subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Stt3p, Wbp1p and Swp1p). Of the eight yeast subunits, five are encoded by essential yeast genes, while three (Ost3p/Ost6p, Ost4p and Ost5p) are non-essential.

Current studies are directed towards determining which subunits of the enzyme are involved in the recognition of the nascent polypeptide and the dolichol oligosaccharide. For many proteins, N-linked glycosylation is an obligate event prior to protein folding, oligomeric assembly and subsequent vesicular transport via the exocytic pathway. In yeast, defects in N-linked glycosylation cause reduced incorporation of glycoproteins into the cell wall resulting in osmotic fragility. Hypoglycosylation of proteins induces the unfolded protein response (UPR) pathway, which is responsible for regulating the ER content of protein folding chaperones. Yeast provide an attractive model system to analyze the functional interaction between lumenal ER chaperones and hypoglycosylated proteins.

Office: Research 915, LAB N-P
Phone: 508-856-5894
Fax: 508-856-6231
E-mail: Reid.Gilmore@umassmed.edu
Keywords: Intracellular Trafficking, Clinical Research, Biochemistry

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