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A6210 - Escaping the ER Quality Control System: How a Mutation in the ABCA3 Transporter Disrupts Cellular Homeostasis
Author Block: S. Mulugeta, M. Zhao, M. F. Beers; Medicine-Pulmonary, University of Pennsylvania, Philadelphia, PA, United States.
RATIONALE: ATP binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in the biogenesis of alveolar type cell lamellar bodies. A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease. Two functional classes of clinical ABCA3 mutants, designated as Type I and Type II, have been identified where Type I mutations cause aberrant protein trafficking and Type II mutations impair the lipid pump. The proline substitution for leucine at residue 982 (ABCA3L982P) is designated as a Type I mutation in vitro but its specific subcellular routing and the mechanism underlying cellular response to its expression is incompletely defined. APPROACH: In vitro cell line models transiently expressing fluorescent-tagged wild type and mutant ABCA3L982P isoforms were used for co-localization confocal microscopy studies with subcellular organelle markers. In addition, biochemical analyses of the cellular responses to transient and chronic expression of the mutant ABCA3L982P transporter were also performed. RESULTS: Quantitative vesicle counting of double labeled fluorescent images of A549 cells acquired by confocal microscopy revealed that the mutant ABCA3L982P is targeted to both the ER and specific subcellular post-Golgi compartments. Compared to wild type control, ABCA3L982P was predominantly localized in Rab 7- and DC-Lamp-positive compartments as well as within sorting vesicles common to multiple ABCA transporters. Transient expression of ABCA3L982P also induced an ER stress response (upregulation of GRP-78, XBP1, and HDJ2) and proteasome inhibition. The effect of long-term expression of ABCA3L982P on cell phenotype was evaluated using HEK293 cells stably expressing ABCA3L982P. Compared to controls, immunoblots of whole cell lysates from ABCA3L982P expressing cells showed a significant decrease in the autophagosome/amphisome marker Rab 7. In addition, while steady state expression of autophagic markers in ABCA3L982P expressing cells showed a moderate decrease in LC3 and a profound diminution of P62 levels, flux analysis by bafilomycin-induced blockage of autophagy revealed significant upregulation in both LC3 and P62 expression. CONCLUSION: These results indicate that ABCA3L982P mutant transporter escapes cell quality control in a unique fashion. The ER retention and accumulation of the mutant isoform within post-Golgi sorting vesicles represent a unique cellular mistrafficking phenotype which combine to disrupt cellular function via both induction of ER stress and alteration of cellular macroautophagy.