The mechanism of membrane-associated steps in tail-anchored protein insertion

• Published in: Nature (London) Vol. 477; no. 7362; pp. 61 - 66
• Main Authors: Mariappan, Malaiyalam, Mateja, Agnieszka, Dobosz, Malgorzata, Bove, Elia, Hegde, Ramanujan S., Keenan, Robert J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-09-2011; Nature Publishing Group
• Subjects: 631/45/535; 631/45/612/1237; 631/57/2271; Adenosine triphosphatase; Adenosine Triphosphate - metabolism; Biological and medical sciences; Cell physiology; E coli; Endoplasmic Reticulum - metabolism; Fundamental and applied biological sciences. Psychology; Humanities and Social Sciences; Membrane and intracellular transports; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membranes; Models, Molecular; Molecular and cellular biology; multidisciplinary; Physiological aspects; Protein binding; Protein Transport; Proteins; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae - physiology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Science; Science (multidisciplinary); Yeast; United States; United Kingdom; Intracellular transport; Membrane protein; Cell organelle; Insertion; Molecular interaction; Endoplasmic reticulum; Mechanism; Protein; Molecular chaperone
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature10362

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.
Tail-anchored protein targeting
Around 5% of eukaryotic membrane proteins — known as tail-anchored or TA proteins — are anchored to the lipid bilayer by a single C-terminal transmembrane domain. The Get3 ATPase targets TA proteins destined for the endoplasmic reticulum (ER), and substrate–Get3 complexes bind to two membrane proteins in the ER known as Get1 and Get2. In this study, Keenan and colleagues reconstitute the TA protein-insertion cycle with purified components and present crystal structures of key complexes. A TA substrate protein bound to the chaperone Get3 is initially recruited to the membrane through an interaction with Get2. Once recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent manner. This work provides a detailed structural and mechanistic framework for the minimal TA protein-insertion cycle.