A di-leucine sequence and a cluster of acidic amino acids are required for dynamic, retention in the endosomal recycling compartment of fibroblasts
A di-leucine sequence and a cluster of acidic amino acids are required for dynamic, retention in the endosomal recycling compartment of fibroblasts
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DOI：
10.1091/mbc.12.2.367
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发表时间：
2001-02-01
影响因子：
3.3
通讯作者：
McGraw, TE
中科院分区：
文献类型：
--
作者：
Johnson, AO;Lampson, MA;McGraw, TE
胰岛素调节氨肽酶（IRAP）是一种跨膜氨肽酶，动态地保留在成纤维细胞的内体区室中。这种动态保留的特征是从质膜快速内化并缓慢再循环回到细胞表面。这些专门的运输动力学导致在稳态下细胞表面上的IRAP < 15%，而转铁蛋白受体（另一种在内体和细胞表面之间运输的跨膜蛋白）为35%。在这里，我们证明了IRAP的胞质结构域（残基56-84）的29个氨基酸的区域是必要的，足以促进IRAP的贩运特性。该区域内的二亮氨酸序列和酸性氨基酸簇是减缓IRAP再循环的基序的基本要素。快速内化需要三个不同基序中的任何两个：M-15，M-16，DED 64 -66和LL 76，77。DED和LL序列是调节再循环的基序的一部分，表明该基序是双功能的。在这项研究中，我们使用辣根过氧化物酶淬灭的荧光，以证明IRAP是动态保留在转铁蛋白受体含有一般内体再循环隔室。因此，我们的数据表明，类似于那些确定不同的膜室之间的靶向的基序也可以调节蛋白质从内体室的运输速率。我们提出了一个动态保留模型，其中IRAP从一般的内体再循环区室转运到专门的、缓慢出芽的再循环囊泡中，这些囊泡与介导快速再循环回到表面的囊泡不同（例如，含转铁蛋白受体的转运囊泡）。IRAP的动态保留可能是调节蛋白质在细胞表面和内部之间分布的一般机制的一个例子。
Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in < 15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56-84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M-15,M-16, DED64-66, and LL76,77. The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.