The molecular mechanisms that accomplish co-translational protein translocation across and integration into the membrane of the endoplasmic reticulum (ER) at sites termed translocons are complex, in part because of the intrinsic difficulty of each process and in part because each process must be completed without compromising the permeability barrier established by the ER membrane. We have previously investigated protein trafficking from the point of view of the nascent protein chain by incorporating fluorescent dyes or photoreactive groups into the nascent chain as it is being made by the ribosome. By examining functional, fully-assembled, and intact translocation or integration intermediates with probes in the nascent chain, we have elucidated several important structural and mechanistic aspects of these processes. We now propose to extend our unique fluorescence and fluorescence resonance energy transfer (FRET) investigations of trafficking by addressing questions that include: Which domains and functions of BiP are required for the BiP-dependent closing of the pore and for the opening of the pore when BiP is released? Does the ribosome induce all nascent chain transmembrane (TM) sequences to fold into an alpha-helix (or nearly so) far inside the exit tunnel? Do TM sequences oriented in the bilayer in opposite directions regulate ribosome-translocon-BiP interactions differently, with N(lum)-C(cyto) TM sequences initiating BiP-mediated closure of the lumenal end of the pore via a long transmembrane signal transduction pathway and N(cyto)-C(lum) TM sequences alternately triggering the closure of the cytosolic end of the pore by the ribosome? When do the TM sequences of a multi-spanning membrane protein (MSMP) begin to assemble into their native structure? We will also use a novel variation of the photocrosslinking approach to determine whether the TM segments in a MSMP move through the translocon and into the bilayer singly, in pairs, or all at once. In addition, we will create a reconstituted system with fluorescent-labeled substrates of the dislocation or ER-associated degradation (ERAD) pathway to characterize and quantify various aspects of the retrotranslocation of misfolded and unassembled proteins from the ER lumen to the cytosol, and thereby assess the role of various cytosolic, lumenal, and membrane proteins on ERAD targeting and retrotranslocation. This basic research will elucidate aspects of protein sorting at the molecular level in normal cells and provide a context for identifying irregularities in abnormal cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM026494-25
Application #
6823159
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
1979-04-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
25
Fiscal Year
2004
Total Cost
$356,475
Indirect Cost
Name
Texas A&M University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
141582986
City
College Station
State
TX
Country
United States
Zip Code
77845
Mayerhofer, Peter U; Bañó-Polo, Manuel; Mingarro, Ismael et al. (2016) Human Peroxin PEX3 Is Co-translationally Integrated into the ER and Exits the ER in Budding Vesicles. Traffic 17:117-30
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Cain, Peter; Holdermann, Iris; Sinning, Irmgard et al. (2011) Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain. Biochem J 437:149-55

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