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.