Formation of the Regulated Secretory Pathway

描述（由申请人提供）：肽激素，神经肽，生长因子和单胺的调节释放取决于它们在能够调节胞吐的大致密核心囊泡（ldcv）中的储存。然而，我们仍然对蛋白质如何进入这种调节分泌途径（RSP）而不是构成分泌途径知之甚少，而构成分泌途径赋予大多数新合成的蛋白质立即释放。在反式高尔基网络（TGN）中，用于LDCV的蛋白质聚集形成密集的核心，表明管腔或可能是膜的相互作用驱动了LDCV的生物发生，在LDCV成熟的后续过程中，用于其他细胞器的蛋白质被移除。然而，我们之前已经确定了一个细胞质基序，该基序需要将囊泡单胺转运蛋白VMAT2分选为ldcv，这表明细胞质机制的作用。该基序的突变增加了转运蛋白的细胞表面表达，显然是通过将转运蛋白从调控途径转移到构成途径。考虑到LDCV生物发生的缺陷应该在VMAT2中表型化这些突变的影响，我们在果蝇S2细胞中筛选了转运体的细胞表面表达增加，这些细胞对RNAi高度敏感。我们发现S2细胞表达RSP，值得注意的是，果蝇VMAT （dVMAT）含有与哺乳动物转运蛋白相同的分选基序，该基序的突变也增加了S2细胞中细胞表面的表达。通过流式细胞术筛选保存到哺乳动物的7000个果蝇序列，寻找野生型dVMAT表达增加的基因，我们发现了少数影响受调节蛋白分泌的基因。我们将重点放在异四聚体接头蛋白AP-3上，因为其中两个亚基在筛选中呈阳性，我们发现AP-3的缺失也会失调哺乳动物细胞的分泌。尽管在没有AP-3的情况下LDCVs仍能形成，但我们发现它们缺乏调节释放所需的蛋白质，如synaptotagmin。在前两个目标中，我们将通过测试AP-3分离运往RSP的货物的假设来确定AP-3如何促进RSP的形成，并且在没有AP-3的情况下，两种分泌途径混合。我们还发现，AP-3相互作用蛋白VPS41的下调会导致蛋白质分泌失调，在第三个目标中，我们将测试VPS41作为AP-3适配器的外壳蛋白的假设。我们还将把分析扩展到缺乏AP-3和VPS41的小鼠。这些结果将为今后研究LDCV形成的分子机制及其对生理、发育和疾病的影响奠定基础。

DESCRIPTION (provided by applicant): The regulated release of peptide hormones, neural peptides, growth factors and monoamines depends on their storage inside large dense core vesicles (LDCVs) capable of regulated exocytosis. However, we still understand remarkably little about how proteins sort into this regulated secretory pathway (RSP) rather than the constitutive secretory pathway that confers the immediate release of most newly synthesized proteins. In the trans-Golgi network (TGN), proteins destined for LDCVs aggregate to form a dense core, suggesting that lumenal or possibly membrane interactions drive LDCV biogenesis, with proteins destined for other organelles removed during the subsequent process of LDCV maturation. However, we have previously identified a cytoplasmic motif required for the sorting of vesicular monoamine transporter VMAT2 into LDCVs, suggesting a role for cytosolic machinery. Mutations in this motif increase cell surface expression of the transporter, apparently by diverting it from the regulated to the constitutive pathway. Reasoning that a defect in LDCV biogenesis should phenocopy the effect of these mutations in VMAT2, we screened for increased cell surface expression of the transporter in Drosophila S2 cells, which are highly susceptible to RNAi. We find that S2 cells express an RSP and remarkably, Drosophila VMAT (dVMAT) contains the same sorting motif as the mammalian transporter, with mutations in this motif also increasing cell surface expression in S2 cells. Screening 7000 Drosophila sequences conserved to mammals by flow cytometry for increased expression of wild type dVMAT, we identified a small number of genes that affect regulated protein secretion. Focusing on the heterotetrameric adaptor protein AP-3 because two of the subunits scored positive in the screen, we found that loss of AP-3 also dysregulates secretion in mammalian cells. Although LDCVs still form in the absence of AP-3, we find that they lack the proteins such as synaptotagmin required for regulated release. In the first two aims, we will determine how AP-3 contributes to formation of the RSP by testing the hypothesis that AP-3 functions to segregate cargo destined for the RSP, and in its absence, the two secretory pathways mix. We have also found that knockdown of the AP-3-interacting protein VPS41 dysregulates protein secretion, and in the third aim, will test the hypothesis that VPS41 functions as a coat protein for the AP-3 adaptor. We will also extend the analysis to mice lacking AP-3 and VPS41. The results will provide a foundation for future work on the molecular mechanisms involved in LDCV formation and the consequences for physiology, development and disease.