The research described in this proposal is directed towards elucidating the molecular mechanism by which nascent polypeptides are translocated across on integrated into the rough endoplasmic reticulum (PER) membrane. Particular emphasis will be placed upon (a) analyzing the signal recognition particle (SRP) and SRP receptor-dependent steps in the translocation reaction that culminate with the selective attachment of a ribosome-nascent chain complex (RNC) to the translocation channel and (b) analyzing the function of the translocation channel during nascent polypeptide transport and membrane protein integration. One objective of the first specific aim is to characterize the interaction between the SRP receptor (SR) and the ribosome by indentifying the minimal SR domain that interacts with the ribosome. The binding site for the SR on the large ribosomal subunit will be determined using a combination of biochemical methods and cryoelectron microscopy. A second objective is to understand the role of cytoplasmic loops of Sec61p in the SRP- dependent cotranslational translocation pathway. The objective of the second specific aim is to investigate the in vivo kinetics of membrane protein integration in the yeast Saccharomyces cerevisiae using a series of ubiquitin-translocation assay (UTA) reporters. We will test the hypothesis that the lateral movement of a nascent transmembrane span within the protein translocation channel is a slow event relative to the protein synthesis elongation rate. These experiments should provide insight into the in vivo dynamics of the ribosome-channel junction. Several crucial questions about the mechanism of protein translocation were raised by the recently solved X-ray crystal structure of the M. jannaschii SecYEb complex. Yeast molecular genetics methods will be used to test current models of protein translocation by conducting a structure- function analysis of the auxiliary Sshlp translocation channel. The three research objectives outlined above address poorly understood aspects of the protein translocation reaction. The accurate and efficient biosynthesis of integral membrane proteins, secreted proteins and lysosomal proteins is an essential function in human cells, as well as in simple model organisms like budding yeast. Defects in the modification or folding of proteins in the rough endoplasmic reticulum are responsible for a growing list of human diseases that are termed """"""""ER-quality control"""""""" diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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University of Massachusetts Medical School Worcester
Schools of Medicine
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Braunger, Katharina; Pfeffer, Stefan; Shrimal, Shiteshu et al. (2018) Structural basis for coupling protein transport and N-glycosylation at the mammalian endoplasmic reticulum. Science 360:215-219
Mandon, Elisabet C; Butova, Cameron; Lachapelle, Amber et al. (2018) Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations. J Biol Chem 293:13662-13672
Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid et al. (2017) Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins. J Biol Chem 292:8007-8018
Mandon, Elisabet C; Trueman, Steven F; Gilmore, Reid (2013) Protein translocation across the rough endoplasmic reticulum. Cold Spring Harb Perspect Biol 5:
Trueman, Steven F; Mandon, Elisabet C; Gilmore, Reid (2012) A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function. J Cell Biol 199:907-18
Gilmore, Reid; Mandon, Elisabet C (2012) Understanding integration of ?-helical membrane proteins: the next steps. Trends Biochem Sci 37:303-8
Trueman, Steven F; Mandon, Elisabet C; Gilmore, Reid (2011) Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity. Mol Biol Cell 22:2983-93
Becker, Thomas; Bhushan, Shashi; Jarasch, Alexander et al. (2009) Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 326:1369-73
Mandon, Elisabet C; Trueman, Steven F; Gilmore, Reid (2009) Translocation of proteins through the Sec61 and SecYEG channels. Curr Opin Cell Biol 21:501-7
Jiang, Ying; Cheng, Zhiliang; Mandon, Elisabet C et al. (2008) An interaction between the SRP receptor and the translocon is critical during cotranslational protein translocation. J Cell Biol 180:1149-61

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