Post-translational protein targeting by the GET pathway

How post-translational protein targeting pathways escort hydrophobic membrane proteins to their correct target site is a fundamental mechanistic challenge. In the recently discovered Guided Entry of Tail-anchor (GET) pathway, seemingly ‘simple’ TA proteins are delivered to the ER by a puzzlingly complex cascade of protein interactions that revolves around the dimeric ATPase Get3. The GET pathway provides an excellent opportunity to probe post-translational mechanisms of protein targeting and address fundamental questions: How are hydrophobic membrane proteins effectively protected during post-translational modes of targeting? What provides spatiotemporal control of the complex series of molecular events in this pathway? What molecular signals direct TA proteins to distinct organelles in eukaryotic cells? How do distinct post-translational protein targeting pathways intersect with one another and with quality control machineries?

Conformational Changes in the Get3 ATPase Dimer Drive the Post-Translational Targeting of Tail- Anchored (TA) Proteins.

T denotes ATP; D denotes ADP. (A) Nucleotide-, substrate-, and effector-driven conformational changes during the Get3 ATPase cycle, as described in the text. The perpendicular symbol (?) denotes the effect of Get4/5 in delaying ATPase activation. Top right panel: crystal structure of closed Get3 bound with ADP–AlF4– (2WOJ). The two Get3 subunits in the dimer are in blue and tan, respectively. The bound nucleotides are in spacefill. Right panel: crystal structure of Get4/5- and ATP-bound Get3 (4PWX). Bottom panel: crystal structure of Get3 bound with ADP–AlF4– and a TA peptide (4XTR). Top left panel: crystal structure of a wide-‘open’ Get3 bound to the Get1 cytosolic domain (3SJB). (B) ATPase rearrangements in Get3 drive distinct molecular steps during TA protein targeting. The steps are numbered to be consistent with (A). Get4/5 bridges between Sgt2 and Get3, and primes Get3 into the optimal conformation and nucleotide state for TA loading (Step 0). TA binding drives Get3 dissociation from Get4/5 (Step 3) and activates ATP hydrolysis (Step 4). The Get3–TA complex is probably captured by Get2 at the endoplasmic reticulum membrane (Step 4). ADP release initiates interaction of the Get3–TA complex with Get1 (Step 5), which drives disassembly of TA from Get3 (Step 6). Finally, ATP, together with Get4/5, displaces Get3 from Get1 and reinitiates the cycle (Steps 1 and 2). TMD, transmembrane domain.

Selected Publications:

  • Rome ME, Rao M, Clemons WM Jr., and Shan SO. (2013) Proc. Natl. Acad. Sci. “Precise timing of ATPase activation drives targeting of tail-anchored proteins.” PMID: 23610396.
  • Rome ME, Chio US, Rao M, Gristick, HB, and Shan SO. (2014) Proc. Natl. Acad. Sci. "Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway." PMID: 25368153.
  • Gristick HB, Rao M, Chartron JW, Rome ME, Shan SO, and Clemons WM Jr. (2014) Nat. Struct. Mol. Biol. "Crystal structure of ATP-bound Get3–Get4–Get5 complex reveals regulation of Get3 by Get4." PMID: 24211265.
  • Rao M, Okreglak V, Chio US, Cho H, Walter P, and Shan SO. (2016) eLife “Multiple selection filters ensure accurate tail-anchor membrane protein targeting.” PMID: 27925580.