The research project is directed towards elucidating the mechanism responsible for translocation of proteins across the rough endoplasmic reticulum (RER). The first specific aim will be to determine how the signal recognition particle (SRP) and the SRP receptor (SR) mediate the efficient high-fidelity delivery of the ribosome-nascent chain (RNC) complex to the translocation channel when the ribosome, a high affinity ligand for the Sec61 core of the translocation channel, is presented in vast molar excess. Competition experiments will indicate whether synergism between the SR and the Sec61 complex accounts for the specificity and affinity for SRP-RNC targeting to the RER. Quantification of membrane bound ribosomes will reveal whether inactive ribosomes that occlude the Sec61 complex are displaces when SRP delivers a RNC complex to the Sec61 complex. Saturation binding of RNC complexes to Sec61 oligomers will indicate whether the SR interferes with the non-specific binding of ribosomes to adjacent vacant Sec61 complexes. The objective of the second specific aim of this proposal is to test the hypothesis that the SRbeta GTPase performs a crucial role in RNC targeting to the translocation channel. To define a role for SR beta, translocation assays will be conducted using SRP54 and SRalpha mutants that bind xanthosine triphosphate in preference to GTP. Functional assays that monitor individual reaction steps during protein translocation will be used to define the events that are dependent upon the GTP binding and hydrolysis activities of Srbeta. The nucleotide binding status of Srbeta will be evaluated in the context of protein translocation reaction. The GTPase cycle of SRbeta will be analyzed in vitro by identifying the protein factors that regulate ribonucleotide exchange and activate GTP hydrolysis. The objective of the third specific aim of this proposal is to test the hypothesis that cooperative binding of GTP to the SRP-SR complex initiates the transfer of the signal sequences from SRP54 to Sec61alpha. Cooperative binding of GTP to the SRP-SR complex will be evaluated using purified SR and a reconstituted SRP containing a SRP54 mutant that binds xanthosine triphosphate in preference to GTP. Transfer of the signal sequence from SRP54 to the Sec61 complex is blocked in N-ethyl maleimide modified microsomes. Dissociation of SRP from RNC complexes will be investigated using NEW-inactivated microsomes to determine whether dissociation of SRP54 from the signal sequence is contingent upon the presence of an active acceptor for the nascent polypeptide.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Cytology Study Section (CTY)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Massachusetts Medical School Worcester
Schools of Medicine
United States
Zip Code
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:
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 (2012) A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function. J Cell Biol 199:907-18
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

Showing the most recent 10 out of 32 publications