Membrane proteins play important roles in various life processes, for example, those in the inner mitochondrial membrane (IMM), endoplasmic reticulum (ER) membrane, and plasma membrane (PM). Oxidative phosphorylation complexes, densely packed in the IMM are crucial for energy transduction in eukaryotes. We determined three entire II2III₂ IV2 supercomplex (SC) structures with 114 lipids at 2.1-2.4 Å resolution in Perkinsus marinus (P. marinus). The structures show a complete electron transfer pathway from complex II (CII) to complex IV (CIV). These architectures also reveal rotation states of the iron sulfur protein (ISP) in complex III (CIII), from one of which we observed two novel proteins that might impair the electron transfer. We also studied how the salt concentration and detergent affect the electron transfer. We determined the SC III₂ IV-cytochrome c (cyt. c) cryo-EM structure at 20 mM salt concentration condition. Together with kinetic study, these data implicate that multiple cyt. c are involved in electron transfer between CIII and CIV. Our kinetic studies of CIV also indicate a native ligand bound near its K proton pathway which could be removed by detergent, leading to an increase in electron transfer rate and the activity of the enzyme. Most biogenesis of integral membrane proteins in eukaryotes is done in ER, such as the amino acid permeases (AAP), which function as amino acid transporters in the PM. Its synthesis and functional folding in Saccharomyces cerevisiae (S. cerevisiae) requires an ER membrane-localized chaperone, Shr3. We utilized a yeast growth-based genetic assay, in conjunction with a split-ubiquitin yeast two-hybrid assay, to demonstrate the selective interaction between Shr3 and nested C-terminal AAP truncations. This interaction exhibited a distinct pattern, wherein it gradually intensified and then weakened as more transmembrane helices folded. The work presented in this thesis contributions to an increased understanding of the organization and function of SCs, the effects of protein subunits, salt concentrations, and detergents on electron transfer, as well as the mechanism of Shr3 on AAP folding in the ER membrane. Together, these works have shed light on the understanding of the structure and function of several membrane proteins.

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