Biogenesis of the Regulated Secretory Pathway

描述（由申请人提供）：包括单胺类神经递质和神经肽在内的神经调节剂控制着广泛的行为现象，它们的失调会导致神经精神疾病，但我们对它们被调节释放背后的细胞机制知之甚少。特别是，我们不了解蛋白质是如何向介导调节释放的大致密核心囊泡（LDCVs）排序的，而不是向介导构成分泌的囊泡排序的。以前的工作甚至提出了管腔和膜的相互作用，而不是细胞质的相互作用，这种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，我使用了水疱单胺转运蛋白（VMAT2），这是一种多位膜蛋白，它用单胺类神经递质填充分泌囊泡，用于胞外释放。VMAT2有效且直接地分选进入调节分泌通路（RSP），并且在一个保守的细胞质二亮氨酸样基序中的突变将VMAT2转移到构成通路，增加细胞表面递送。该基序对于VMAT2直接分选到ldcv中很重要，这表明，与之前的研究相反，细胞质因子介导了VMAT2向RSP的分选，从而促进了ldcv的生物发生。我们的目标是在果蝇S2细胞中使用无偏RNAi筛选来确定参与RSP形成的细胞机制。此外，我们将描述已鉴定的细胞因子在哺乳动物PC12细胞中的作用。特别是，我们将确定这些因素是否专门负责VMAT2的分选，或者更普遍地负责该途径的生物发生。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