The goal of this project is to establish a detailed molecular understanding for how tail-anchored (TA) membrane proteins are post-translationally inserted into the endoplasmic reticulum (ER) membrane. TA proteins, which account for nearly 5% of all eukaryotic membrane proteins, are found in virtually all cell membranes where they play essential roles in diverse cellular processes including intracellular traf?cking, protein translocation, enzyme catalysis and protein quality control. Defects in TA protein biogenesis are linked to many human pathologies, and thus a better understanding of function and dysfunction in these systems may lead to new therapeutic strategies for myriad disease states. Post-translational targeting and insertion of TA proteins into the ER membrane is a multi-step process mediated by the `Guided Entry of Tail-anchored proteins' (GET) pathway, ?rst discovered in early 2007. Since then, my lab has made fundamental contributions towards understanding the molecular basis of TA protein biogenesis in yeast and in mammals. Our rigorous studies performed during the previous granting period recapitulated the early, `pre-targeting' steps of the pathway using completely puri?ed components and established that the essential transmembrane `insertase' (called Get1/2) functions as a heterodimeric complex. In addition, we determined the ?rst high-resolution structures of a functional membrane protein targeting complex; this work resolved what was an ongoing controversy about the nature of the Get3-TA protein complex and de?ned a new paradigm for how transmembrane domains (TMDs) are shielded during transit through the aqueous cytosol. During the course of this project we have assembled a valuable suite of reagents, high-resolution structures, and functional assays that exploit yeast and cell-free systems. Indeed, we have now reconstituted every step in the pathway?from TA protein synthesis to TA protein insertion?using a set of puri?ed, recombinant soluble and membrane components. The power of this system lies in our ability to manipulate each component and step in the pathway, using recombinant and chemical tools. Thus, we are in a unique position to de?ne the structural, biochemical and biophysical principles that underlie every step in the pathway. Here we build on this technical and conceptual foundation to address two central questions that remain poorly understood in the ?eld.
In Aim 1, we will de?ne how the Get1/2 transmembrane complex coordinates TA protein insertion into the ER membrane.
In Aim 2 we will de?ne how the pre-targeting machinery captures TA proteins and transfers them onto the Get3 targeting factor. We will do this using a multi-disciplinary approach that combines functional analysis with a hybrid computational and experimental structural analysis of soluble and membrane protein complexes.

Public Health Relevance

Eukaryotic cells contain hundreds of different tail-anchored (TA) membrane proteins that are essential for growth and survival. We are studying a recently discovered pathway that directs the insertion of newly synthesized TA proteins into the endoplasmic reticulum membrane. Understanding how the cellular machinery coordinates this process is critical to understanding how healthy cells function, and may enable the development of new therapies in the ?ght against human diseases including neurodegenerative and heart disease, diabetes, and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086487-10
Application #
9686749
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Flicker, Paula F
Project Start
2010-04-05
Project End
2022-03-31
Budget Start
2019-04-01
Budget End
2020-03-31
Support Year
10
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Mateja, Agnieszka; Keenan, Robert J (2018) A structural perspective on tail-anchored protein biogenesis by the GET pathway. Curr Opin Struct Biol 51:195-202
Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T et al. (2017) Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins. Mol Cell 67:194-202.e6
Zalisko, Benjamin E; Chan, Charlene; Denic, Vladimir et al. (2017) Tail-Anchored Protein Insertion by a Single Get1/2 Heterodimer. Cell Rep 20:2287-2293
Srivastava, Renu; Zalisko, Benjamin E; Keenan, Robert J et al. (2017) The GET System Inserts the Tail-Anchored Protein, SYP72, into Endoplasmic Reticulum Membranes. Plant Physiol 173:1137-1145
Anghel, S Andrei; McGilvray, Philip T; Hegde, Ramanujan S et al. (2017) Identification of Oxa1 Homologs Operating in the Eukaryotic Endoplasmic Reticulum. Cell Rep 21:3708-3716
Meyer, Peter A; Socias, Stephanie; Key, Jason et al. (2016) Data publication with the structural biology data grid supports live analysis. Nat Commun 7:10882
Dominik, Pawel K; Borowska, Marta T; Dalmas, Olivier et al. (2016) Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs. Structure 24:300-9
Borowska, Marta T; Dominik, Pawel K; Anghel, S Andrei et al. (2015) A YidC-like Protein in the Archaeal Plasma Membrane. Structure 23:1715-1724
Mateja, Agnieszka; Paduch, Marcin; Chang, Hsin-Yang et al. (2015) Protein targeting. Structure of the Get3 targeting factor in complex with its membrane protein cargo. Science 347:1152-5
Hegde, Ramanujan S; Keenan, Robert J (2011) Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat Rev Mol Cell Biol 12:787-98

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