Mechanistic Studies of Tail-Anchored Membrane Protein Targeting to the ER

Citation

Fry, Michelle Yen (2022) Mechanistic Studies of Tail-Anchored Membrane Protein Targeting to the ER. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/b3yj-c572. https://resolver.caltech.edu/CaltechTHESIS:08232021-203647268

Abstract

The successful biogenesis – synthesis, delivery, and insertion into designated membranes – of membrane proteins is a crucial cellular process. One particular class of membrane proteins, tail-anchored (TA) proteins have a single transmembrane domain (TMD) that this located at their C-termini and are targeted to membranes post-translationally. Multiple pathways have been identified to target TA proteins to the ER membranes, but designated pathways for targeting TA proteins to the mitochondria remain elusive. The most well understood ER TA protein pathway is the Guided Entry of Tail-anchored proteins (GET) pathway, consisting of six (fungal) or seven (metazoans) proteins, SGTA, Get1-5, and Bag6 (metazoans only), has nearly been studied exclusively in Opisthokants (fungi and metazoans). Here we employed a combination of x-ray crystallography, cryo-electron microscopy, computational modeling, cellular biology, fluorescent imaging, and bioinformatics in order to understand the underlying factors that regulate the targeting of these TA proteins to their correct membranes. Our work reveals that ER-bound TA proteins tend to have a hydrophobic face whereas mitochondria-bound TA proteins contain a charge following their TMD. This finding corroborates our observation that the first component of the GET pathway to interact with TA proteins, SGTA, falls in a category of other hydrophobic segment binding domains, dubbed STI1-domains. Structures presented here demonstrate that the overall structure of Get3 is conserved in organisms as distant as Excavats and Opistokonts, and slight conformational changes in the ATPase allows the described chaperone cascade of the GET pathway to progress. Together these results refine the model for TA protein targeting to the ER membrane.

Thesis Files