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ATP-binding cassette (ABC) transporters play pivotal physiological roles in substrate transport across membranes, and defective assembly of these proteins can cause severe disease associated with improper drug or ion flux. The yeast protein Yor1p is a useful model to study the biogenesis of ABC transporters; deletion of a phenylalanine residue in the first nucleotide-binding domain (NBD1) causes misassembly and retention in the endoplasmic reticulum (ER) of the resulting protein Yor1p-ΔF670, similar to the predominant disease-causing allele in humans, CFTR-ΔF508. Here we describe two novel Yor1p mutants, G278R and I1084P, which fail to assemble and traffic similar to Yor1p-ΔF670. These mutations are located in the two intracellular loops (ICLs) that interface directly with NBD1, and thus disrupt a functionally important structural module. We isolated 2 second-site mutations, F270S and R1168M, which partially correct the folding injuries associated with the G278R, I1084P, and ΔF670 mutants and reinstate their trafficking. The position of both corrective mutations at the cytoplasmic face of a transmembrane helix suggests that they restore biogenesis by influencing the behavior of the transmembrane domains rather than by direct restoration of the ICL1-ICL4-NBD1 structural module. Given the conserved topology of many ABC transporters, our findings provide new understanding of functionally important inter-domain interactions and suggest new potential avenues for correcting folding defects caused by abrogation of those domain interfaces. [ABSTRACT FROM AUTHOR]

Background & Aims: Normalization of serum alkaline phosphatase (SAP) was recently shown to correlate with better prognosis in Primary Sclerosing Cholangitis (PSC). We aimed at evaluating the impact of SAP improvement to below 1.5 the upper limit of normal (ULN) on the prognosis of this cholestatic liver disease. Methods: Oxford PSC database was screened for cases diagnosed between 1980 and 2004. Cases which met the inclusion criteria were retrospectively examined for clinical parameters, laboratory values, and clinical end points (liver decompensation, liver transplantation, and liver-related deaths including cholangiocarcinoma). Cases were followed-up to 31/12/2010. Results: 139 patients were included, (87 males). Improvement of SAP to below 1.5 ULN was achieved by 55 (40%) patients in a median time of 2years, compared to 84 (60%) who did not. 3/55 (6%) patients with SAP improvement reached an end point compared to 32/84 (38%) patients with no SAP improvement (p <0.0001). 13/84 (15%) patients with no SAP improvement developed cholangiocarcinoma compared to no cholangiocarcinoma in the group with SAP improvement (p =0.002). The end point free survival was significantly longer in patients with SAP improvement (p <0.0001). The significance of SAP improvement as a predictor of prognosis persisted after controlling for other clinical and laboratory variables. Improvement of SAP to below 1.5 ULN was comparable to complete normalization of SAP in terms of prognosis. Conclusions: Improvement in SAP to below 1.5 ULN is associated with better outcome and reduced risk of CCA in PSC. This was comparable to the achievement of complete normalization of SAP. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate substrate translocation across biological membranes. Our goal was to define the intramolecular interactions that contribute to quaternary assembly of a eukaryotic ABC transporter and determine how the architecture of this protein influences its biogenesis within the secretory pathway. We used chemical cross-linking approaches to map interdomain interactions in the yeast ABC transporter, Yor1p, which functions as a pleiotropic drug pump at the plasma membrane. We have defined interactions between the two nucleotide- binding domains (NBDs) and between the NBDs and specific intracellular loops (ICLs) that are consistent with current structural models of bacterial ABC exporters. Furthermore, we detected relatively weak NBD-NBD and ICL-ICL interactions that may correspond to transient sites of cross-talk between domains required for coupling of ATP hydrolysis with substrate translocation. Mutation of a key residue in ICL2 caused misassembly of the altered protein, leading to increased sensitivity to the mitochondrial poison, oligomycin. We identified intragenic suppressing mutations that rescued the oligomycin resistance associated with this aberrant protein and demonstrated that the suppressing mutations restored multiple interdomain interfaces. Together, our biochemical and genetic approaches contribute to a greater understanding of the architecture of this important class of proteins and provide insight into the quality control surveillance that regulates their biogenesis and deployment within the eukaryotic cell. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains. [ABSTRACT FROM AUTHOR]