Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.
Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.
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DOI：
10.1091/mbc.e11-06-0509
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发表时间：
2012-08
影响因子：
3.3
通讯作者：
Marks MS
中科院分区：
文献类型：
--
作者：
Sitaram A;Dennis MK;Chaudhuri R;De Jesus-Rojas W;Tenza D;Setty SR;Wood CS;Sviderskaya EV;Bennett DC;Raposo G;Bonifacino JS;Marks MS
OCA 2被用作模型黑素体货物蛋白，以定义酸性二亮氨酸基序与衔接子AP-1和AP-3结合所需的一级序列元件。OCA 2必须与AP-3结合以进行黑素体定位。BLOC-1也是必需的，因此可以与任一适配器合作，将货物递送到溶酶体相关的细胞器。产生独特的溶酶体相关细胞器（LRO）的细胞类型，如黑素细胞中的黑素体，用从内体转移的货物填充新生的LRO。向黑素体的货物分选与经由细胞质暴露的分选信号与异四聚体衔接子AP-1或AP-3的结合相关。一些货物结合两个适配器，但每个适配器的货物识别的相对贡献和运输过程中与其他效应黑素体的功能相互作用尚不清楚。在这里，我们利用色素细胞特异性蛋白OCA 2中基于酸性二亮氨酸的分选信号的靶向诱变来剖析AP-1和AP-3在运输到黑素体中的相对作用。我们发现，AP-1或AP-3的结合取决于信号的主要序列，而不是其在胞质结构域中的位置。优先结合AP-1或AP-3的突变体各自向黑素体运输，并在功能上补充OCA 2缺陷，但AP-3结合是稳态黑素体定位所必需的。与酪氨酸酶不同，酪氨酸酶也参与AP-3的最佳黑素体递送，AP-1和AP-3都有利于OCA 2变体需要BLOC-1进行黑素体转运。这些数据为AP-1和AP-3在OCA 2转运至黑素体中的不同作用提供了证据，并表明BLOC-1可以在货物分选至LRO期间与任一适配器合作。
OCA2 is used as a model melanosome cargo protein to define primary sequence elements required for acidic dileucine–motif binding to adaptors AP-1 and AP-3. OCA2 must bind to AP-3 for melanosome localization. BLOC-1 is also required and thus can cooperate with either adaptor for cargo delivery to lysosome-related organelles. Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.