Cargo delivery to lysosome‐related organelles universally relies on the recognition of sorting signal by adaptor proteins
Authors: Kook Seunghyi , Harper Dawn C , Franklin Jude , Jaume Alexa , Hanby Hayley A , Meng Shufang , Marks Michael S , Guttentag Susan H 
Published: June 2021

Hermansky‐Pudlak syndrome (HPS) is a group of rare genetic diseases characterized by oculocutaneous albinism and bleeding diathesis, often associated with lung fibrosis. The symptoms of HPS reflect defects in membrane transport processes required to generate tissue‐specific lysosome‐related organelles (LROs), such as melanosomes in pigment cells, lamellar bodies (LB) in type 2 alveolar epithelial cells (AT2) and dense granules in platelets. Although the transport processes that are defective in HPS are shared among distinct LRO‐expressing cells, it is not clear whether the unique cargo proteins that confer distinct morphological and functional characteristics are targeted to their respective LROs by conserved or cell type‐specific recognition mechanisms. To determine whether LB and melanosome cargo proteins contain cell type‐independent LRO sorting information, we exploited the behavior of model cargo proteins, such as melanosomal protein, OCA2 and LB‐specific protein, ATP8A1 in AT2 cells and melanocytes. OCA2 is a pigment cell‐specific membrane protein and localizes to melanosomes via a cytoplasmic dileucine‐based sorting signal that engages both AP‐1 and AP‐3. By overexpression of wild‐type OCA2 in MLE15, we show that wild‐type OCA2 colocalized extensively with the LB marker, mCherry‐ABCA3 in MLE15, an AT2 cell line. OCA2‐AA23N bearing the double E96DP99S mutation that was unable to bind to AP‐1 and AP‐3 localized to the cell surface. To confirm the relative roles of AP‐1 and AP‐3 in transport of OCA2 to LB, we also expressed the AP‐3‐biased OCA2‐AA23‐P99S and AP‐1‐biased OCA2‐AA23‐E96D variants in MLE15. Mutants that preferentially bound either AP‐1 or AP‐3 each partially trafficked toward LB, but AP‐3 binding was necessary for steady‐state LB localization, as it is for delivery of OCA2 to melanosomes. ATP8A1, a member of the P4 subfamily of P‐type ATPases (P4‐ATPases), is highly enriched in LB but expressed at low levels in melanocytes. When expressed in immortalized mouse melan‐Ink4a−/− melanocytes, an EGFP‐tagged, catalytically inactive ATP8A1 partially overlapped with pigmented melanosomes. Overlap was substantially reduced by mutagenesis of a conserved dileucine‐based sorting signal or by expression in AP‐3‐deficient melanocytes. These data provide evidence that cellular machinery in AT2 or melanocytes can recognize a universal LRO targeting signal in a heterologous protein and deliver it to a cell type‐specific LRO. By contrast, two other AT2‐restricted LB cargoes – DC‐LAMP and ABCA3 ‐ failed to localize to pigmented melanosomes upon expression in melan‐Ink4a−/− cells. Together, these data suggest that LRO cargoes employ both universally conserved and cell type‐specific pathways for delivery to nascent LROs.Support or Funding InformationNIH (GM108807/SG and MM). SG is the Julia Carell Stadler Professor of Pediatrics.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.