Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes.
Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes.
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DOI：
10.1111/j.1471-4159.2012.07814.x
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发表时间：
2012-10
影响因子：
4.7
通讯作者：
Bergson C
中科院分区：
文献类型：
--
作者：
Muthusamy N;Faundez V;Bergson C
Calcyon是一种富含神经的单一跨膜蛋白，可与胞内蛋白轻链(CLC)相互作用，刺激胞内蛋白组装和胞内吞噬作用。异四聚体适配器蛋白(AP)复合体AP-1、AP-2和AP-3具有类似的性质，它们招募货物以插入到笼状蛋白涂层的运输小泡中。在这里，我们报道了AP Medium(µ)亚基与位于钙蛋白细胞质结构域133-136残基的Yxx型酪氨酸基序相互作用。关键的酪氨酸和大型疏水残基酪氨酸133和蛋氨酸136的定点突变优先取消了普遍存在的和神经元亚型u3的结合，也影响了u1和u2的结合，但程度较小。在体内用携带钙蛋白零等位基因的小鼠探索了这些相互作用的相关性。诱变研究表明，小鼠钙离子缺失优先改变了AP-3的亚细胞分布，提示钙离子可以调节AP-3和AP-3的膜结合池的功能。为了验证这一假设，我们将注意力集中在海马门区，那里钙蛋白、AP-3和AP-3含量丰富。我们分析了对照组和钙蛋白缺失小鼠的脑冰冻切片中锌转运蛋白3(Znt3)和磷脂酰肌醇-4-激酶II型α(PI4KIIα)，这两种蛋白是明确定义的AP-3蛋白。共聚焦显微镜显示，在钙调素基因敲除脑的海马区苔藓纤维中，ZnT3型和PI4KI型α显著减少，这是以前在AP-3缺陷中描述的表型。综上所述，我们的数据表明Calcyon直接与u3A和u3B相互作用，并调节AP-3的亚细胞分布和AP-3货物的靶向。
Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain (CLC) and stimulates clathrin assembly and clathrin mediated endocytosis (CME). 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 but 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.
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