In eukaryotic cells the early steps of polytopic membrane protein biogenesis take place at the endoplasmic reticulum (ER). Complex polytopic membrane proteins with multiple membrane-spanning segments (MS) co-translationally insert into the ER membrane and fold into native states prior to being recognized as cargo for incorporation, or packaging, into COPII-coated ER-derived secretory vesicles. Successful progression through these early steps is a requisite for the ultimate delivery and functional expression of membrane proteins at appropriate cellular membranes. The list of biogenesis factors that contribute to membrane protein biogenesis is expanding, and due to their diversity, it is imperative to investigate their individual functions. Highly specialized membrane-localized chaperones (MLC) have been identified that are required for the functional expression of discrete sets of related membrane protein substrates. This thesis focuses on Shr3 in Saccharomyces cerevisiae. Shr3 was the first MLC defined and was identified based on it being required for the biogenesis of all eighteen members of the Amino Acid Permease (AAP) protein family, each comprised of 12 membrane-spanning (MS) segments. Despite numerous independent findings corroborating the co-translational nature of the Shr3-AAP interactions during AAP translation, ER membrane insertion and folding, direct formal proof has been elusive. Specifically, a detailed mechanistic understanding of how Shr3 facilitates AAP folding has been lacking, and the underlying mechanisms governing its strict ER membrane localization have not been rigorously investigated. The studies documented in this thesis were aimed to obtain a deeper mechanistic understanding of how Shr3 facilitates AAP folding (Paper I) and its strict ER localization (Paper II) and to directly probe the co-translational interaction of Shr3 with its AAP folding substrates (Paper III).

Specifically, in Paper I, the chaperone folding function and temporal requirement of Shr3 was investigated. Strikingly, the experiments revealed that relatively few amino acids within the 4 MS segments of Shr3 contribute to substrate specific interactions, however, mutations within the 2 luminal loops of Shr3 did manifest substrate specific effects. Further, a split-ubiquitin approach was used to probe interactions between Shr3 and MS segments of AAP. The data indicate that Shr3 interacts early with the first few MS of AAP, presumably directly as they partition into the ER membrane. The experiments documented in Paper II were directed at identifying ER retention determinants present in the cytoplasmic carboxy-terminal tail of Shr3. Retention was found to depend on multiple motifs that function in an additive fashion. Surprisingly, one of the motifs was close to the membrane domain. This unexpected finding provided a novel tool that was exploited to obtain the first evidence of a mechanistic coupling between Shr3-dependent folding and packaging of AAP into COPII-coated vesicles. Paper III describes the use of a translational arrest peptide capable of stalling AAP translation on the ribosome. Strikingly, Shr3 was found associated with a nascent AAP chain with 10 membrane-spanning segments. This finding provides formal proof that Shr3 indeed functions in a co-translational manner. Collectively, these studies offer a significantly enhanced mechanistic understanding of MLC and their requirement during the biogenesis of complex polytopic membrane proteins

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.

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein 56 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

Polytopic membrane proteins with multiple transmembrane segments (TMS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Discrete sets of polytopic membrane proteins in Saccharomyces cerevisiae require ER membrane-localized chaperones (MLC) to prevent aggregation and fold properly. Shr3, the best characterized MLC, is specifically required for the functional expression of amino acid permeases (AAP), a family of transport proteins comprised of twelve TMS. We performed comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with split-ubiquitin approaches to probe chaperone-substrate (Shr3-AAP) interactions in vivo. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions, which initiate after the first 2 to 4 TMS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 TMS are inserted. Thus, Shr3 acts transiently in a co-translationally manner to prevent TMS of translation intermediates from engaging in non-productive interactions, thereby preventing AAP misfolding during biogenesis.