Regulation of cellular functions by chaperone-like AAA-ATPases, VCP and NVL

VCP是一种AAA-ATPase，在内质网相关降解(ERAD)和细胞器生物发生等细胞功能中发挥重要作用。SVIP是一种与VCP相互作用的蛋白，它的表达通过内质网的扩张导致细胞空泡化。为了阐明SVIP的细胞功能，我们首先通过体内和体内实验研究了SVIP在ERAD中的作用。结果表明，过量的SVIP抑制ERAD，这可能是内质网空泡化的原因之一。接下来，为了鉴定VCP-SVIP复合体中的其他蛋白质组分，以整个SVIP为诱饵进行酵母双杂交筛选，获得了几个编码SVIP相关蛋白的cDNA。NVL2是一个与VCP具有很高氨基酸相似性的蛋白质。与主要分布在胞浆中的VCP相反，NVL2的定位仅限于核仁，提示NVL2在这个核亚室中具有特殊的作用。在这项研究中，我们证明了NVL2与RNA解旋酶相互作用，并参与了60S核糖体的生物发生。虽然NVL2显性负向突变体的表达没有影响rRNA的加工，但它导致解旋酶在一个大的复合体中积累，可能是一个核糖体前颗粒。此外，突变研究发现了NVL2中的核仁定位信号。这一信号是NVL2与核糖体蛋白L5结合所必需的，这表明NVL2通过与L5结合而被招募到核仁中。

VCP is an AAA-ATPase, which plays critical roles for cellular functions such as endoplasmic reticulum associated degradation(ERAD) and organella biogenesis. SVIP has been identified as a VCP-interacting protein, whose expression causes cell vacuolation by expansion of ER. To clarify a cellular function of SVIP, we first investigated a role of SVIP for ERAD by employing assays in virto and in vivo. The results from both the assays showed that extra amount of SVIP inhibits ERAD and this may be a cause of the ER vacuolation. Next, to identify other protein components involved in the VCP-SVIP complex, yeast two-hybrid screening was performed using entire SVIP as a bait As a result, several cDNAs encoding putative SVIP-associated proteins were obtained.NVL2 is a protein that shows high amino acid similarity with VCP. In contrast to mainly cytosolic distribution of VCP, localization of NVL2 is restricted to the nucleolus, suggesting a specialized role of NVL2 in this nuclear subcompartment. In this research, we showed that NVL2 interacts with a RNA helicase and is involved in biogenesis of 60S ribosomes. Although expression of a dominant-negative mutant of NVL2 did not affect rRNA processing, it caused accumulation of the helicase in a large complex, probably a pre-ribosomal particle. Additionally, mutagenesis studies identified a nucleolar localization signal in NVL2. This signal is required for the binding of NVL2 with ribosomal protein L5,suggesting that NVL2 is recruited to the nucleolus by associating with L5.