期刊
JOURNAL OF NEUROCHEMISTRY
卷 123, 期 1, 页码 60-72出版社
WILEY-BLACKWELL
DOI: 10.1111/j.1471-4159.2012.07814.x
关键词
clathrin mediated endocytosis; PI4KIIa; synaptic vesicle; tyrosine motif; YXXO; ZnT3
资金
- National Institutes of Health (NIH) [NS42599, GM077569]
- GHSU Brain and Behavior Discovery Institute Collaborative Neuroscience Grant Initiative
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 (mu) subunits interact with a YXX phi-type tyrosine motif located at residues 133136 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 mu 3, and also impacted mu 1 and mu 2 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 (PI4KIIa), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIa 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 mu 3A and mu 3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes.
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