The ER membrane chaperone Shr3 acts in a progressive manner to assist the folding of related plasma membrane transport proteins

Proteins 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 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 interactions with seven different AAP in vivo. Our findings indicate that Shr3 specifically recognizes AAP substrates, largely independent of sequence-specific interactions involving membrane and luminally oriented domains. Shr3 selectively and robustly interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate polytopic sugar transporters. Strikingly, Shr3-AAP interactions initiate with the first 4 MS of AAP and successively strengthen, but abruptly weaken when all 12 MS are present. The data are consistent with Shr3 acting in a temporal manner as a scaffold preventing AAP translation intermediates from engaging in non-productive interactions.