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Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting


Gristick, Harry Benjamin (2015) Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z95718ZD.


The proper targeting of membrane proteins is essential to the viability of all cells. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET pathway (Guided Entry of TA proteins). In the yeast pathway, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the central targeting factor, the Get3 ATPase. Although binding of Get4/5 to Get3 is critical for efficient TA targeting, the mechanisms by which Get4 regulates Get3 are unknown. To understand the molecular basis of Get4 function, we used a combination of structural biology, biochemistry, and cell biology. Get4/5 binds across the Get3 dimer interface, in an orientation only compatible with a closed Get3, providing insight into the role of nucleotide in complex formation. Additionally, this structure reveals two functionally distinct binding interfaces for anchoring and ATPase regulation, and loss of the regulatory interface leads to strong defects in vitro and in vivo. Additional crystal structures of the Get3-Get4/5 complex give rise to an alternate conformation, which represents an initial binding interaction mediated by electrostatics that facilitates the rate of subsequent inhibited complex formation. This interface is supported by an in-depth kinetic analysis of the Get3-Get4/5 interaction confirming the two-step complex formation. These results allow us to generate a refined model for Get4/5 function in TA targeting.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:X-ray crystallography; membrane proteins; Tail-anchored proteins; GET, protein targeting; Eukaryotes; biochemistry; biophysics
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Clemons, William M.
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Shan, Shu-ou
  • Chan, David C.
  • Clemons, William M.
Defense Date:29 May 2015
Record Number:CaltechTHESIS:06052015-162319516
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9003
Deposited By: Harry Gristick
Deposited On:08 Jun 2015 20:21
Last Modified:04 Oct 2019 00:08

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