Biogenesis of the Regulated Secretory Pathway

描述（由申请人提供）：包括单胺神经递质和神经肽的神经调节剂控制广泛的行为现象，并且它们的失调有助于神经精神疾病，但是我们仍然对它们调节释放的细胞机制知之甚少。特别是，我们不明白蛋白质如何分类到介导调节释放的大致密核心囊泡（LDCV），而不是介导组成性分泌的囊泡。以前的工作甚至提出了内腔和膜的作用，而不是细胞质的相互作用，有助于其他运输事件，如内吞作用。为了确定参与LDCV形成的因素，我使用囊泡单胺转运蛋白（VMAT 2），一种多位膜蛋白，它用单胺神经递质填充分泌囊泡进行胞吐释放。VMAT 2有效地直接分选到调节分泌途径（RSP）中，并且保守的细胞质双亮氨酸样基序中的突变将VMAT 2转移到组成性途径，增加细胞表面递送。该基序对于将VMAT 2直接分选到LDCV中显得很重要，这表明，与以前的工作相反，细胞质因子介导VMAT 2分选到RSP，从而有助于LDCV的生物发生。我们的目标是确定参与RSP形成的细胞机制，在果蝇S2细胞中使用无偏RNAi筛选。此外，我们将鉴定哺乳动物PC12细胞中细胞因子的作用。特别是，我们将确定这些因素是否专门负责VMAT 2的分选，或更普遍的途径的生物发生。我们的研究将使我们能够确定参与调节分泌途径形成的细胞因子。这些信息将使我们能够操纵调节和组成性分泌，并确定它们在正常行为和精神疾病中的相对作用

DESCRIPTION (provided by applicant): Neuromodulators including monoamine neurotransmitters and neural peptides control a wide range of behavioral phenomena, and their dysregulation contributes to neuropsychiatric disease, but we still understand little about the cellular mechanisms that underlie their regulated release. In particular, we do not understand how proteins sort to the large dense core vesicles (LDCVs) that mediate regulated release, rather than to the vesicles that mediate constitutive secretion. Previous work has even suggested a role for lumenal and membrane rather than cytoplasmic interactions of the kind that contribute to other trafficking events such as endocytosis. To identify factors involved in the formation of LDCVs, I am using the vesicular monoamine transporter (VMAT2), a polytopic membrane protein which fills secretory vesicles with monoamine neurotransmitters for exocytotic release. VMAT2 sorts efficiently and directly into the regulated secretory pathway (RSP), and mutations in a conserved, cytoplasmic dileucine-like motif divert VMAT2 to the constitutive pathway, increasing cell surface delivery. This motif appears important for sorting VMAT2 directly into LDCVs, suggesting that, in contrast to previous work, cytoplasmic factors mediate the sorting of VMAT2to the RSP and thus contribute to the biogenesis of LDCVs. We aim to identify the cellular machinery involved in formation of the RSP, using an unbiased RNAi screen in Drosophila S2 cells. Further, we will characterize the role of identified cellular factors in mammalian PC12 cells. In particular, we will determine whether these factors are responsible specifically for the sorting of VMAT2,or more generally for biogenesis of the pathway. Our studies will enable us to identify cellular factors involved in formation of the regulated secretory pathway. This information will allow us to manipulate regulated and constitutive secretion and determine their relative role in normal behavior and psychiatric illness