Membrane proteins comprise ~30% of a cell's proteome, and their efficient and accurate localization is essential for the structure and proper functioning of all cells. Compared to the well-studied co-translational protein targeting pathway, post-translational membrane protein targeting poses additional challenges due to the presence of highly hydrophobic transmembrane domains on the substrate protein. Deciphering the molecular strategies to escort such aggregation-prone substrates to the correct target site is a fundamental mechanistic challenge. In the Guided Entry of Tail-anchor (GET) pathway, a complex cascade of protein interactions mediates the post-translational delivery of TA proteins to the endoplasmic reticulum membrane, providing an excellent opportunity to address these questions. Our general goal is to decipher, at the biochemical and biophysical level, the molecular mechanisms underlying the targeting of TA proteins by this novel pathway. Our specific goal is to understand how Get3, the central ATPase in this pathway, uses its ATPase cycle to drive and coordinate the complex cascade of protein interactions during the GET pathway. To this end, we will establish a precise framework for the Get3 ATPase cycle and identify conformational changes that occur during this cycle. We will define when, where and how the upstream and downstream interaction partners of Get3 regulate its ATPase cycle and reciprocally, how this ATPase cycle drives an ordered cascade of interactions of Get3 with its effector proteins. We will develop novel assays to dissect individual steps of the targeting reaction in real time and use this to decipher how highly specific substrate selection is achieved by the pathway. These studies will significantly advance our understanding of the molecular mechanisms that underlie the post- translational targeting of membrane proteins. Further, Get3 represents the first eukaryotic ATPase that belongs to a novel class of 'dimerization-activated'nucleotide hydrolases;studies of this ATPase dimer will be instrumental to test, expand, and generalize the regulatory principles for this growing class of novel cellular regulators.

Public Health Relevance

Membrane proteins impart essential functionality to the cellular membrane and their proper localization is essential for all cells. Mislocalization of tail anchored proteins cause impaired cellular function, especially under stress conditions. The proposed studies will significantly advance our understanding of the mechanism of membrane protein localization and biogenesis within the cell, and contribute profoundly to our general understanding of physiology and pathology of all living cells at a molecular level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM107368-01A1
Application #
8762368
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2014-08-01
Project End
2018-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Gristick, Harry B; Rome, Michael E; Chartron, Justin W et al. (2015) Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting. J Biol Chem 290:30006-17
Rome, Michael E; Chio, Un Seng; Rao, Meera et al. (2014) Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway. Proc Natl Acad Sci U S A 111:E4929-35
Gristick, Harry B; Rao, Meera; Chartron, Justin W et al. (2014) Crystal structure of ATP-bound Get3-Get4-Get5 complex reveals regulation of Get3 by Get4. Nat Struct Mol Biol 21:437-42
Rome, Michael E; Rao, Meera; Clemons, William M et al. (2013) Precise timing of ATPase activation drives targeting of tail-anchored proteins. Proc Natl Acad Sci U S A 110:7666-71