Identification of lysosomal and Golgi localization signals in GAP and ARF domains of ARF domain protein 1.
Identification of lysosomal and Golgi localization signals in GAP and ARF domains of ARF domain protein 1.
复制标题
ARF 结构域蛋白 1 的 GAP 和 ARF 结构域中溶酶体和高尔基体定位信号的鉴定。
DOI：
10.1128/mcb.20.19.7342-7352.2000
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发表时间：
2000
影响因子：
5.3
通讯作者：
Vaughan,M
中科院分区：
文献类型：
--
作者：
Vitale,N;Ferrans,VJ;Moss,J;Vaughan,M
ADP核糖基化因子（ARFs）是一种约20 kda的鸟嘌呤核苷酸结合蛋白，可激活霍乱毒素和磷脂酶D，是囊泡运输途径的关键组成部分。ARF结构域蛋白1 （ARF domain protein 1, ARD1）是ARF超家族的成员之一，其氨基末端延伸长度为46 kda，作为gtpase激活蛋白（GAP），对其ARF结构域具有活性。当过表达时，ARD1与溶酶体和高尔基体相关。与这一发现一致的是，通过免疫亲和从人肝脏分离的溶酶体和高尔基体膜含有天然ARD1。ARD1作为一种绿色荧光融合蛋白表达，最初与高尔基网络相关，随后出现在溶酶体上，表明ARD1可能在两个细胞器之间进行了载体运输。在这里，我们通过显微镜共定位显示，GAP和ARF结构域分别决定溶酶体和高尔基体定位，这与多个信号基序的存在一致。利用截断的ARD1分子，表达为绿色荧光融合蛋白，发现溶酶体定位信号存在于GAP结构域的301 ~ 402残基上。位点特异性突变表明，GAP结构域的369kxxxq373序列负责溶酶体定位。ARD1与高尔基体的关联需要酪氨酸基序。含有QKQQQQF基序的绿色荧光融合蛋白与溶酶体部分相关，表明该基序包含溶酶体靶向的足够信息。这些结果表明，ARD1是一个具有ARF和GAP区域的多结构域蛋白，它们分别包含高尔基体和溶酶体定位信号，可能在囊泡运输中起作用。
ADP ribosylation factors (ARFs) are ∼20-kDa guanine nucleotide-binding proteins that activate cholera toxin and phospholipase D and are critical components of vesicular trafficking pathways. ARF domain protein 1 (ARD1), a member of the ARF superfamily, contains a 46-kDa amino-terminal extension, which acts as a GTPase-activating protein (GAP) with activity towards its ARF domain. When overexpressed, ARD1 was associated with lysosomes and the Golgi apparatus. In agreement with this finding, lysosomal and Golgi membranes isolated from human liver by immunoaffinity contained native ARD1. ARD1, expressed as a green fluorescent fusion protein, was initially associated with the Golgi network and subsequently appeared on lysosomes, suggesting that ARD1 might undergo vectorial transport between the two organelles. Here we show by microscopic colocalization that GAP and ARF domains determine lysosomal and Golgi localization, respectively, consistent with the presence of more than one signal motif. Using truncated ARD1 molecules, expressed as green fluorescent fusion proteins, it was found that the signal for lysosomal localization was present in residues 301 to 402 of the GAP domain. Site-specific mutagenesis demonstrated that the sequence369KXXXQ373in the GAP domain was responsible for lysosomal localization. Association of ARD1 with the Golgi apparatus required tyrosine-based motifs. A green fluorescent fusion protein containing the QKQQQQF motif was partially associated with lysosomes, suggesting that this motif contains the information sufficient for lysosomal targeting. These results suggest that ARD1 is a multidomain protein with ARF and GAP regions, which contain Golgi and lysosomal localization signals, respectively, that could function in vesicular trafficking.