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AP‐3‐dependent Mechanisms Regulate the Trafficking of ATP8a1 to Lamellar Bodies in Alveolar Type 2 Cells
Author(s) -
Kook Seunghyi,
Wang Ping,
Meng Shufang,
Hanby Hayley A.,
Jaume Alexa,
Goetzl Laura,
Marks Michael S.,
Guttentag Susan H.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.542.12
Subject(s) - lamellar granule , microbiology and biotechnology , secretion , signal transducing adaptor protein , endosome , transport protein , biology , protein targeting , crispr , chemistry , membrane protein , signal transduction , gene , pulmonary surfactant , biochemistry , membrane , intracellular
Alveolar type 2 cells (AT2) are involved in the maintenance of epithelial homeostasis and repair after injury. Lamellar bodies (LB) in AT2 play a critical role in lung health and disease as the site of surfactant synthesis, packaging and secretion. LB biogenesis is poorly understood, especially mechanisms that foster membrane and protein trafficking to LB. In this study, we found that ATP8a1, a member of the P4 subfamily of P‐;type ATPases (P 4 ‐;ATPases), was highly enriched in the ABCA3‐positive LB fraction derived from human fetal lung explants. In AT2 from mice lacking the adaptor protein 3 complex (AP‐3), the pearl mouse model of Hermansky Pudlak syndrome type 2 (HPS2), ATP8a1 was largely depleted from ABCA3‐positive LB and accumulated in Rab11‐positive recycling endosomes (REs). Using targeted mutagenesis, we identified a di‐leucine‐based sorting signal (ExxxLL) present in the C‐terminal cytoplasmic domain of ATP8a1 that is necessary for its proper targeting to LB of AT2. To dissect the relative roles of AP‐1 and AP‐3 in the transport of ATP8a1 to LB, we have developed MLE‐15 cell lines in which a subunit of AP‐1 (Ap1γ1) and/or AP‐3 (Ap3β1) has been inactivated using CRISPR/Cas9 gene editing and tested the targeting of ATP8a1 in these mutated cell lines. The lack of AP‐3 in MLE‐15 reduced efficient LB delivery of ATP8a1, while deletion of the AP‐1 subunit had no effect on ATP8a1 delivery to LB. P 4 ‐;ATPases typically flip aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of organelle membranes. PS is highly enriched in REs and is essential for endosomal membrane traffic. Here, we show that the cytoplasmic leaflet of LB in MLE‐15 and human AT2 are highly enriched in PS using a GFP‐LactC2 probe; in contrast, AP‐3‐deficient cells showed impaired distribution of PS, favoring RE locazliation. These data provide evidence that ATP8a1 is dependent on AP‐3 for trafficking from RE to LB in AT2, and suggest that loss of ATP8a1 targeting to LB in HPS2 impairs PS localization to LB in AT2 cells. Support or Funding Information National Institutes of Health (GM108807/SG, MM and LG). SG is the Julia Carell Stadler Professor of Pediatrics. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .