PERK‐ and ATF6‐dependent cell quality control mechanisms in response to chronic expression of an aberrantly trafficked mutant ABCA3 lipid transporter
Authors: Zhao Ming , Mulugeta Surafel 
Published: April 2020

RATIONALEATP binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in the pulmonary surfactant function. 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 mutations at residues 101 (ABCA3L101P) and 982 (ABCA3L982P) are designated as a Type I mutations. We previously reported that whereas ABCA3L101P is retained within the ER, ABCA3L982P partially escapes the ER quality control system and is localized in Rab 7‐positive, endosome/amphisome compartments, both mutations disrupting cellular homeostasis. The mechanism underlying cellular responses to chronic expression of these aberrantly trafficked transporters is incompletely defined.APPROACHIn vitro cell line models transiently or stably expressing fluorescent‐tagged ABCA3 negative controls (wild type and a functional mutant (ABCA3E292V)), positive control (ABCA3L101P) and experimental ABCA3L982P mutant isoforms were used for co‐localization confocal microscopy studies with subcellular organelle markers. Additionally, biochemical analyses of the cellular responses to acute and chronic expression of these mutant transporters were evaluated.RESULTSA549 and HEK293 cells transiently expressing ABCA3 isoforms showed clearance of overexpressed transporters via both the proteasome and autophagic pathways regardless of isoform (WT or mutants) or cell type. However, ER stress response (GRP78 and HDJ2) and activation of one of the three arms of the unfolded proteins response, IRE1 (upregulation of XBP1), were observed only in cells transiently expressing the mistargeted transporters (ABCA3L101P or ABCA3L982P) while ATF6 and PERK remained unaffected. The effect of long‐term expression of mistargeted mutant transporters on cell phenotype was evaluated using HEK293 cells stably expressing ABCA3L101P or ABCA3L982P. Compared to controls, chronic expression of the misstrafficked transporters elicited considerable PERK upregulation but normalization of IRE1 signaling. Likewise, significant ATF6 upregulation was observed only in cells expressing ABCA3L982P. Moreover, upregulation of autophagic flux was noted in cells expressing either trafficking mutant marked by substantial increases in LC3 and P62 levels during bafilomycin‐induced autophagic block. However, while steady state levels of the autophagic markers were increased in ABCA3L101P expressing cells, they were decreased in Rab7‐routed ABCA3L982P expressing cells (including Rab7).CONCLUSIONThese results indicate that cellular response and survival from long‐term expression of aberrantly trafficked ABCA3 mutant transporters is partially dependent on their subcellular routing but requires the adaptive and descriminatory use of overlapping proteostatic repertoires including upregulation of selective arms of the unfolded protein response and cellular macroautophagy.Support or Funding InformationNIH HL 129150 (SM)