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

VCP是一种aaa - atp酶，在细胞功能如内质网相关降解（ERAD）和细胞器生物发生中起关键作用。SVIP是一种与vcp相互作用的蛋白，其表达通过内质网扩增引起细胞空泡形成。为了阐明SVIP的细胞功能，我们首先通过体内和体外实验研究了SVIP在ERAD中的作用。两种分析的结果都表明，额外的SVIP抑制ERAD，这可能是内质网空泡化的原因。接下来，为了鉴定VCP-SVIP复合物中涉及的其他蛋白质成分，我们以整个SVIP为诱饵进行酵母双杂交筛选，结果获得了几个编码SVIP相关蛋白的cdna。NVL2是一种与VCP氨基酸相似性较高的蛋白。与VCP的主要细胞质分布相反，NVL2的定位仅限于核核，表明NVL2在该核亚室中的特殊作用。在这项研究中，我们发现NVL2与RNA解旋酶相互作用，并参与60S核糖体的生物发生。尽管nnvl2的显性阴性突变体的表达不影响rRNA加工，但它导致解旋酶在一个大复合体中积累，可能是核糖体前颗粒。此外，诱变研究在NVL2中发现了核仁定位信号。该信号是NVL2与核糖体蛋白L5结合所必需的，这表明NVL2是通过与L5结合而被招募到核仁的。

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.