MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEIN

ADP-核糖基化因子结构域蛋白1（ARD 1）是鸟嘌呤核苷酸结合蛋白的ADP-核糖基化因子（ARF）家族的成员，其与其他ARF的不同之处在于存在46-kDa氨基末端延伸，其充当其ARF结构域的GTP酶激活蛋白（GAP）。与ARF GAP类似，ARD 1差距域包含对活性至关重要的锌指基序和精氨酸残基。它与其他ARF GAP的不同之处在于其与GTP结合结构域的共价结合及其对ARD 1的ARF结构域的特异性。ARF被认为在细胞内运输囊泡的形成和它们从一个隔室到另一个隔室的运动中起关键作用。我们在这里报告，ARD 1在细胞中过度表达，作为一种融合或非融合蛋白，定位于囊泡结构，主要集中在核周区域，但也发现在整个胞质溶胶。显微镜共定位和亚细胞分级分离研究表明，ARD 1与高尔基体和溶酶体结构的元素。ARD 1表达为绿色荧光融合蛋白，首先检测到与高尔基体网络相关，随后发现定位于溶酶体。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ARD 1，因此定位于这些细胞器，似乎不是过表达的假象。这些观察结果表明，ARF相关蛋白ARD 1可能在溶酶体的形成或功能以及高尔基体和溶酶体之间的蛋白质运输中发挥作用。对ARD 1基因的持续研究表明，转录调控元件可能位于距离近端5-UTR相当远的位置。- ARF，ARD，GTP酶激活蛋白，溶酶体，高尔基体

ADP-ribosylation factor domain protein 1 (ARD1) is a member of the ADP- ribosylation factor (ARF) family of guanine nucleotide-binding proteins that differs from other ARFs by the presence of a 46-kDa amino-terminal extension, which acts as a GTPase-activating protein (GAP) for its ARF domain. Similar to ARF GAPs, the GAP domain of ARD1 contains a zinc finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP- binding domain and its specificity for the ARF domain of ARD1. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and in their movement from one compartment to another. We report here that ARD1 overexpressed in cells, as a fusion or nonfusion protein, is localized in vesicular structures that are concentrated mainly in the perinuclear region, but are found also throughout the cytosol. Microscopic colocalization and subcellular fractionation studies showed that ARD1 was associated with elements of Golgi and lysosomal structures. ARD1, expressed as a green fluorescent fusion protein, was first detected in association with the Golgi network and subsequently seen localized to lysosomes. Lysosomal and Golgi membranes isolated from human liver by immunoaffinity contained native ARD1, thus localization to these organelles, did not appear to be an artefact of overexpression. These observations suggest that the ARF-related protein ARD1 may play a role in the formation or function of lysosomes and in protein trafficking between Golgi and lysosomes. Continuing work on the ARD1 gene suggests that transcription regulatory elements may reside at a considerable distance from the proximal 5-UTR. - ARF, ARD, GTPase- activating protein, lysosomes, Golgi