The goal of this research is to develop a detailed molecular understanding of a newly discovered post-translational targeting pathway that directs insertion of tail-anchored (TA) membrane proteins into the endoplasmic reticulum (ER) membrane. TA proteins, which account for nearly 5% of all eukaryotic membrane proteins, are anchored to the lipid bilayer by a single C-terminal transmembrane domain (TMD) and have a cytosolic-facing amino-terminal domain. These proteins are found in virtually all cell membranes where they mediate numerous essential cellular processes. Post-translational targeting and insertion of TA proteins is mediated by the newly discovered 'guided entry of tail-anchored protein'(GET) pathway. Over the past few years, a basic framework for the pathway has been defined. First, a 'pre-targeting'factor captures a newly synthesized TA protein in the cytosol, and transfers it to a soluble ATPase, called Get3. This targeting complex is directed to the ER membrane via an interaction with the Get1/2 receptor complex which is both necessary and sufficient to drive TA substrate release and insertion into the membrane. Our research goal is to define the biochemical and biophysical principles that underlie TA protein insertion. First, we will elucidate the molecular identity of the Get3-TA substrate targeting complex at defined steps along the targeting pathway (Aim 1). Second, we will determine whether TA substrates are integrated directly into the bilayer or if insertion requires a chaperoning role for the the Get1/2 receptor complex (Aim 2). Finally, we will define how the static and dynamic properties of the Get1/2 complex allow it to orchestrate the essential steps of TA substrate recruitment, release and insertion (Aim 3).
These Aims will be accomplished using a powerful interdisciplinary approach that combines structure-function analyses with state-of- the-art spectroscopic studies. By defining common themes between known insertion pathways, this work will deepen our understanding of the fundamental cell biological and biophysical process of TMD insertion. By developing new tools and experimental strategies, this work promises to enable analysis of other complex membrane-associated processes. Finally, because defects in TA protein biogenesis are linked to much human pathology, these studies promise insight that may lead to new therapeutic strategies for use in the fight against human disease.

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 newly 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 will enable the development of new therapies in the fight against human diseases including diabetes, neurodegenerative and heart disease, and cancer.

National Institute of Health (NIH)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Ainsztein, Alexandra M
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University of Chicago
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Sherrill, John; Mariappan, Malaiyalam; Dominik, Pawel et al. (2011) A conserved archaeal pathway for tail-anchored membrane protein insertion. Traffic 12:1119-23
Hegde, Ramanujan S; Keenan, Robert J (2011) Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat Rev Mol Cell Biol 12:787-98
Mariappan, Malaiyalam; Mateja, Agnieszka; Dobosz, Malgorzata et al. (2011) The mechanism of membrane-associated steps in tail-anchored protein insertion. Nature 477:61-6