Biogenesis of the Regulated Secretory Pathway

描述(申请人提供)：神经调节剂，包括单胺类神经递质和神经肽，控制着广泛的行为现象，它们的失调导致神经精神疾病，但我们仍然对它们调节释放的细胞机制知之甚少。特别是，我们不知道蛋白质是如何分类到介导调节释放的大而致密的核心小泡(LDCV)，而不是介导结构性分泌的小泡。以前的工作甚至提出了腔和膜的作用，而不是细胞质的相互作用，这种相互作用有助于其他运输活动，如内吞作用。为了确定与LDCV形成有关的因素，我使用了囊泡单胺转运体(VMAT2)，这是一种多角体膜蛋白，它用单胺类神经递质填充分泌囊泡以进行胞吐释放。VMAT2有效地直接进入受调控的分泌途径(RSP)，而保守的细胞质二亮氨酸样基序的突变将VMAT2转移到构成途径，增加细胞表面的递送。这个基序对于将VMAT2直接分选到LDCVs中似乎很重要，这表明与以前的工作不同，细胞质因素介导了VMAT2到RSP的分选，从而有助于LDCVs的生物发生。我们的目标是通过在果蝇S2细胞中进行无偏见的RNAi筛选，确定参与RSP形成的细胞机制。此外，我们还将描述已确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专门负责VMAT2的分选，还是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素。这些信息将使我们能够操纵受调节的和结构性的分泌，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