Isoforms of ankyrin-3 that lack the NH2-terminal repeats associate with mouse macrophage lysosomes.
Isoforms of ankyrin-3 that lack the NH2-terminal repeats associate with mouse macrophage lysosomes.
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DOI：
10.1083/jcb.136.5.1059
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发表时间：
1997-03-10
期刊：
影响因子：
--
通讯作者：
Lux SE
中科院分区：
文献类型：
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作者：
Hoock TC;Peters LL;Lux SE
我们最近克隆并表征了ankyrin-3（也称为ankyrinG），这是一种广泛分布的新锚蛋白，特别是在上皮组织、肌肉和神经元轴突中（Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995。细胞生物学杂志 130：313-330）。在这里，我们表明，在小鼠巨噬细胞中，ankyrin-3 仅表达为两种缺少 NH2 末端重复的小亚型（120 和 100 kD）。通过反转录和扩增小鼠巨噬细胞 RNA（GenBank 编号 U89274 和 U89275）获得的分离 Ank3 cDNA 克隆的序列分析显示，血影蛋白结合和调节结构域与肾 ankyrin-3（GenBank 编号 L40631）中的相同，前面是膜的 29 个氨基酸片段（“重复”）结构域，从最后一个重复的末端附近开始。对锚蛋白 3 的调节域和血影蛋白结合域具有特异性的抗体可将蛋白质定位于整个巨噬细胞胞质中的细胞内囊泡表面。在质膜上未发现它。此外，在 COS 细胞中瞬时表达的表位标记的小鼠巨噬细胞 ankyrin-3 与细胞内膜而不是血浆膜相关。相比之下，也在小鼠巨噬细胞中表达的ankyrin-1（红细胞锚蛋白，ankyrinR）仅位于质膜上。 Ankyrin-3 阳性囊泡在相差显微镜下呈黑色。两项观察结果表明它们是溶酶体。首先，它们是内吞途径中的晚期区室。它们只有在孵育 24 小时后才能被荧光内吞示踪剂 (FITC-葡聚糖) 检测到，此时所有含有 FITC-葡聚糖的囊泡都含有锚蛋白 3，反之亦然。其次，ankyrin-3 阳性囊泡含有溶酶体相关膜糖蛋白 (LAMP-1)，这是一种公认的溶酶体标记物。这是锚蛋白与溶酶体关联的第一个证据，也是同一细胞中存在两个锚蛋白分离到不同位置的例子。
We have recently cloned and characterized ankyrin-3 (also called ankyrinG), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313–330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29–amino acid segment of the membrane (“repeat”) domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrinR), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3–positive vesicles appear dark on phasecontrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran– containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3–positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.