The ATP-binding cassette transporter ABCA3 is a member of the ABC superfamily of transporters that function in the translocation of substrates across cell membranes. Predominantly localized in the limiting membrane of the lamellar bodies of lung alveolar type II cells, ABCA3 is believed to function as a lipid and phospholipid transporter. Recently, ABCA3 has received considerable attention because mutations in the gene are associated with various lung disorders including fatal surfactant deficiency and respiratory distress syndrome (RDS) in newborns and interstitial lung disease (ILD) in older children and adults. While many of the studies thus far have focused primarily on the functional aspects of ABCA3 as a transporter, the cellular responses and consequences in cellular and pulmonary homeostasis as a result of expressing mutant isoforms of ABCA3 are largely undefined. The overall objective of this project is to use a reductionist approach aimed at understanding the molecular mechanisms underlying ABCA3 biosynthesis, and to elucidate the consequences of expression of mutant isoforms of ABCA3 proteins associated with RDS and ILD. Specific Aim 1 will test the hypothesis that the N-terminal domain of ABCA3 is comprised of a short 21 amino acid sequence that harbors signal motifs for insertion of the nascent protein into the ER membrane and for targeting of the protein to post-Golgi distal compartments and the cell surface. Using well established in vitro systems including cell free systems, two epithelial cell lines, human alveolar type II cells, and various molecular and biochemical techniques, we will experimentally elucidate the transmembrane topology and functional motifs of the N-terminal domain of the ABCA3 transporter. In Specific Aim 2, we will investigate cellular responses and molecular mechanisms underlying protein dysfunction and cell injury caused by the expression of misfolded mutant isoforms of ABCA3 using both in vitro studies and in vivo mouse transgenic strategies. We will extend the in vitro studies to include the evaluation of chemical chaperones. These chaperones have the potential to prevent or ameliorate cellular damage caused by promoting proper protein folding and trafficking and by restoring function of the mutant transporter. PROJECT NARRATIVE: In recent times, a considerable attention has been given to the ABCA3 transporter because mutation in the gene is believed to cause various lung diseases. We are taking logical steps to understand the mechanisms underlying the cause of these diseases by focusing on two major aspects of the ABCA3 transporter that have been largely undefined. These include: 1) In vitro examination of ABCA3 protein make up in terms of its targeting motifs and N-terminal domain topology; and 2) In vitro and in vivo elucidation of cellular response as well as effects on cellular homeostasis in response to the expression of its mutant isoforms. We believe the knowledge gained from this project will have a broad application not only toward a better understanding of lung pathogenesis but will offer insights for targeted therapeutic intervention.