Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes

Authors

  • Nagendran Muthusamy,

    1. Graduate Program in Neuroscience Georgia Health Sciences University, Augusta, GA, USA
    2. Department of Pharmacology and Toxicology Georgia Health Sciences University, Augusta, GA, USA
    Search for more papers by this author
    • Present address: Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

  • Victor Faundez,

    1. Department of Cell Biology and Center for Neurodegenerative Diseases, Emory University, Atlanta, GA, USA
    Search for more papers by this author
  • Clare Bergson

    1. Department of Pharmacology and Toxicology Georgia Health Sciences University, Augusta, GA, USA
    Search for more papers by this author

Address correspondence and reprint requests to Clare Bergson, Ph.D, Department of Pharmacology & Toxicology, Georgia Health Sciences University, 1459 Laney Walker Blvd., Augusta, GA, 30912-2300, USA. E-mail: cbergson@georigahealth.edu

Abstract

Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin-mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ-type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes.

Ancillary