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Updated: Jul 22, 2025

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Published on: December 12, 2017
ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis
Ioanna Myronidi1, Andreas Ring1, Fei Wu2
1Department of Molecular Biosciences, The Wenner-Gren Institute, SciLifeLab, Stockholm University, Stockholm, Sweden.
Abstract:
Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane-localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone-substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3-AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3-AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.
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