Suloway, Christian J. M. (2012) Structural insights into tail-anchored protein targeting by Get3. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:02282012-151846967
Translocation of membrane proteins from the point of synthesis to their integration in the membrane is critical to the function of the cell. Tail-anchored (TA) proteins are an important class of membrane proteins with a single transmembrane domain (TMD) close to the carboxyl-terminus. They are defined topologically by having their amino-terminus in the cytosol and their carboxyl-terminus on the exterior side of the membrane. Since the TMD is sequestered by the ribosome during translation, co-translational translocation of TA proteins by the SRP-dependent pathway is not possible. The Guided Entry of Tail-anchored proteins (GET) pathway post-translationally targets TA proteins to the endoplasmic reticulum (ER) membrane. The conserved nucleotide hydrolase Get3 is the central protein in the pathway that specifically binds the TMD of TA proteins to chaperone them from a sorting complex of Get4, Get5, Sgt2 and other chaperones to an ER membrane receptor formed by Get1 and Get2. We have created a model for the mechanism of Get3 TA protein binding coupled to nucleotide state using X-ray crystallography, structural modeling and mutagenesis experiments. We then demonstrate expression, purification and crystallization of complexes of Get3 with TA proteins for structural studies. Finally, we present a crystal structure of a tetrameric archaeal Get3 homologue that forms a central hydrophobic chamber and is capable of binding TA proteins. Using small-angle X-ray scattering, the structure is comparable to a tetrameric fungal Get3 complex with TA protein, which is capable of TA protein membrane integration in vitro. This suggests a model in which a tetramer of Get3 binds TA proteins for delivery to the membrane.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Protein translocation; Tail-anchored proteins; GET; TRC; Get3; TRC40; ArsA; nucleotide hydrolase; structural biology; X-ray crystallography; SAXS|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||21 February 2012|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Christian Suloway|
|Deposited On:||06 Mar 2012 23:52|
|Last Modified:||14 Feb 2013 18:46|
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