The ER membrane chaperone Shr3 co-translationally assists biogenesis of related plasma membrane transport proteins
AbstractProteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. In Saccharomyces cerevisiae, Shr3 is an ER membrane-localized chaperone (MLC) that is specifically required for the functional expression of amino acid permeases (AAP), a family of eighteen transporters comprised of 12 MS. Here, comprehensive scanning mutagenesis and deletion analysis of Shr3, combined with a modified split-ubiquitin approach, were used to probe chaperone-substrate (Shr3-AAP) interactions in vivo. A surprisingly low level of sequence specificity in Shr3 underlies Shr3-AAP interactions, which initiate early as the first 2 MS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 MS partition into the membrane. Thus, Shr3 acts transiently in a co-translational manner to prevent MS of AAP translation intermediates from engaging in non-productive interactions, effectively preventing AAP misfolding during biogenesis.