William Skach, MD PhD - US grants

Biogenesis and assembly of eukaryotic multispanning integral membrane proteins is proposed to occur cotranslationally through the action of independent topogenic sequences which direct sequential translocation initiation, termination and membrane integration events at the ER membrane. Recent studies, however, have demonstrated new complexities in this biosynthetic pathway. For a subset of native multispanning proteins, cooperative interactions rather than sequential independent activities may transiently delay integration of the chain into the membrane until synthesis of multiple transmembrane segments has been completed. Remarkably, novel topogenic determinants have also been identified which direct biogenesis of multiple topologic isoforms from a single polypeptide chain. These findings have important implications for the structure and function of diverse membrane proteins and require a detailed reexamination of the molecular mechanisms involved in their assembly. To better understand different biogenesis pathways utilized by polytopic proteins, the proposed studies will: i) identify the nature of information encoded within sequence determinants in the nascent chain which direct different translocation and membrane integration events, ii) determine how this information is interpreted by translocation machinery and/or chaperones at the ER membrane to effect different assembly mechanisms and, iii) investigate the effects of different biogenesis pathways on final protein structure and function. Three different proteins, human MDR1, CHIP28, AND MIWC, each of which demonstrate different biogenesis pathways will be studied using cell-free translation systems, Xenopus oocytes and cultured mammalian cells. Topogenic sequence determinants will be characterized in defined protein chimeras and in native contexts to identify key information responsible for translocation specificity and membrane integration. Interactions between these determinants and components of the translocation machinery will be identified by crosslinking ER proteins to nascent chains at specific stages of biogenesis. Finally, the role of different topological isoforms in protein maturation and function will be investigated. These studies will accomplish three important goals. First, they systematically define determinants which direct distinct events of topological maturation. Second, they identify translocation machinery and cellular chaperones through which these determinants act. Third, they correlate different biogenesis mechanisms with requirements for protein function. This work will provide a detailed understanding of cellular mechanisms which regulate polytopic protein assembly and allow further investigation into how these pathways might be disrupted in acquired or inherited human diseases.

Cystic Fibrosis (CF) is a prototype for inherited disorders of protein folding. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein expressed in the apical membrane of human epithelial cells. CFTR biogenesis occurs in the endoplasmic reticulum and is facilitated by a complex set of cellular machinery. Greater than 70% of wild type and up to 99% of common mutant forms of CFTR fail to fold properly and are recognized by cellular quality control machinery and degraded by the ubiquitin- proteasome pathway. These observations raise several questions central to CF pathogenesis and treatment. How does cellular machinery coordinate CFTR folding in different cellular compartments? How is misfolded CFTR protein identified by the cell? How is this recognition event coupled to degradation? And how might the efficiency of CFTR folding be improved in patients? The long term goal of this proposal is to characterize the composition, recruitment and function of cellular folding and quality control machinery that regulates the fate of newly synthesized CFTR in the endoplasmic reticulum. The specific aims of this study will use complimentary heterologous expression systems to: l) define the role of the Sec61 translocation machinery in directing early events of CFTR assembly into the ER membrane, 2) identify the dynamic nature of cellular chaperone complexes that govern the fate of newly synthesized CFTR, and 3) examine the role of ER machinery in CFTR degradation by the 26S proteasome complex. Proposed experiments will incorporate photoactive crosslinking probes at precise locations within CFTR to characterize cellular machinery that orients and assembles the nascent chain into the lipid bilayer. Additional studies will use in vitro and in vivo systems to analyze the changing composition of cellular chaperones associated wild type and mutant CFTR during sequential stages of maturation and degradation. Finally lumenal and membrane-bound ER quality control machinery will be identified by biochemical complementation. Together these studies will generate a comprehensive picture of how cellular machinery coordinates and monitors folding events in multiple compartments and ultimately governs the balance between productive and non-productive pathways. Identification of key components that regulate this decision process will be a major step in the development of pharmacologic strategies aimed at improving folding and trafficking of mutant proteins in patients with inherited disorders such as CF.

DESCRIPTION (provided by applicant): The long-term goal of this project is to define general principles and molecular mechanisms of Aquaporin (AQP) integration, folding and tetrameric assembly in the endoplasmic reticulum (ER) membrane. Aquaporins comprise a conserved family of 6-spanning, homotetrameric membrane proteins that play critical roles in water homeostasis in the kidney, lung, brain and other tissues. The molecular basis of water transport is achieved by the precise arrangement of six transmembrane helices and two half helices in a two-fold inverted symmetry around a monomeric pore. This structure is generated by the coordinated actions of the ribosome and Sec61 translocation machinery. A major unanswered question in this field is how subtle changes in nascent peptide structure influence this machinery to direct unique, and often pathological folding events. Interestingly, closely related AQPs exhibit different native folding pathways that are specified by just three variant residues. In addition, inherited point mutations in AQP2 disrupt folding and thereby cause nephrogenic diabetes insipidus (NDI), a life threatening disease of impaired urinary concentration. AQPs are therefore ideal model substrates for investigating normal and pathological mechanisms of membrane protein biogenesis that are implicated in a growing number of human protein folding disorders. The current proposal will use modified aminoacyl tRNAs to cotranslationally insert photocrosslinking and fluorescent probes into nascent AQP integration intermediates that are kinetically trapped at defined stages of synthesis. This approach provide a powerful new method to determine how the ER translocation machinery coordinates nascent chain folding in lumenal, cytosolic and membrane environments that closely mimic conditions in the cell. With these techniques we will: 1) define the molecular basis responsible for different AQP folding pathways, 2) define precisely how AQP2 folding is disrupted in nephrogenic diabetes insipidus, 3) define the functional and structural basis of AQP tetramerization required for intracellular trafficking. Results of these studies will significantly advance our understanding of AQP biology and improve our general ability to understand folding properties of complex integral membrane proteins. They will also establish a useful platform to investigate how folding is corrupted by inherited mutations, and thereby ultimately facilitate new strategies to treat diverse protein- folding disorders.