By: Andreas Ring, MBW

Title: "In vivo analysis of amino acid permease folding in yeast"

Abstract

In eukaryotic cells polytopic membrane proteins, comprised of multiple membrane segments (MS), are co-translationally inserted into the lipid bilayer of the endoplasmic reticulum (ER). Current information suggests that folding occurs concomitantly with membrane insertion. Little is known regarding the processes enabling effective partitioning of individual membrane segments into the ER membrane and how the MS of partially translated polytopic proteins are shielded from engaging in non-productive interactions or off-pathway folding events. We have shown that yeast possess specialized ER membrane-localized chaperones that facilitate the folding of specific sets of related polytopic membrane proteins. In strains individually lacking one of these chaperones, Shr3, Gsf2, Pho86, or Chs7, only their cognate substrates, i.e., amino acid permeases (AAP), hexose transporters, phosphate transporters and chitin synthase III, respectively, accumulate in the ER as large detergent-resistant aggregates. Shr3, the best characterized of these chaperones, is comprised of two distinct domains; a membrane domain with four MS and a hydrophilic cytoplasmically oriented C-terminal tail. The membrane domain of Shr3 is essential and sufficient to prevent AAP aggregation and to facilitate the functional assembly of co-expressed N- and C-terminal fragments of the general amino acid permease (Gap1). Specifically, Shr3 interacts with the first five MS of Gap1, preventing aggregation of the N-terminal fragment and enhancing its stability. When expressed alone the C-terminal fragment, with seven MS, aggregates and is unstable independent of Shr3. The data are consistent with Shr3 acting to maintain the N-terminal MS of Gap1 in a conformation that can post-translationally assemble with the C-terminal membrane segments. To more fully evaluate Shr3 function, we have used leucine- and alanine-scanning to identify critical amino acid residues in MS and extra-membrane loops, respectively. Using this approach we have identified residues critical for function but also residues affecting substrate specificity. We anticipate that ongoing studies analyzing the mutations  affecting  substrate  specificity  will  provide  novel  insight  into  the  molecular interactions underlying the function of membrane-localized chaperones.