Intracellular Membrane Fusion Mediated by SNARE Proteins

描述（由申请人提供）：对于所有细胞内运输事件，两种膜相互融合导致脂质和内容物混合是必不可少的。这一基本过程被认为是由称为SNARE（可溶性NSF附着蛋白受体）的特定蛋白质催化的。它们的序列和结构在所有真核系统中是保守的，从酵母开始，到人类结束。由于囊泡融合涉及许多基本功能，如突触传递，激素分泌和内吞作用，有关分子机制的详细知识，该反应是如何催化的，以及除了SNARE之外还需要哪些蛋白质来实现这一功能将对许多疾病相关的主题产生深远的影响。为了阐明精确的融合机制，采用了酵母液泡融合系统。在大规模分离程序中纯化酵母空泡，并在体外系统中重建。液泡的融合效率可以通过使用两种不同的菌株容易地测量，一种缺乏液泡碱性磷酸酶PHO 8，另一种缺乏液泡蛋白酶PEP 4。PHO 8是一种无活性的前酶，需要被切割才能具有活性。融合发生后，PEP 4接近未成熟的PHO 8酶，将其裂解并因此激活它。可以大量分离出异戊醇，便于进行生物化学研究。由于该系统是唯一存在的可能性，以解决拓扑问题的膜融合在生理背景下的蛋白质如何相互作用的反式（两个融合膜之间），我们重新研究了目前的教条，题为“拓扑限制模型”。我们利用了酵母液泡融合的某些特性，即融合速率相对较慢，因此可以很容易地在不同阶段进行解剖。我们发现了一种意想不到的引发后顺式SNARE复合物，我们将进一步表征其稳定性如何控制以及需要哪些其他蛋白质来稳定该复合物。目标1将解决这个问题。此外，我们还发现了另一种拓扑反式相互作用的陷阱蛋白，而不是预测目前的模型，通过使用不同的标签陷阱的两个融合的液泡。这种反式相互作用的融合相关性进行了研究，通过采用不同的组合的空泡含有特定的失活SNARE在其表面上。我们将继续产生SNARE突变体，以进一步确认发现的反SNARE拓扑结构（Aim 2）的生理相关性。我们也开始阐明，什么样的特定功能的额外的因素，如SEC 1/MUC 18相关的HOPS复合物和V-ATP酶的V0部分的膜融合过程。我们已经描述了V0突变体的特征，它们不能融合，但没有失去泵质子的能力。本提案的目标3涉及这一主题。 公共卫生相关性：SNARE（可溶性NSF附着蛋白受体）介导的细胞内融合对于突触传递、激素分泌和内吞作用是必不可少的。突触传递中的许多疾病与SNARE功能及其相关蛋白质的紊乱有关。此外，某些种类的胰岛素分泌减少是由SNARE功能不足引起的，从而导致糖尿病样表型。

DESCRIPTION (provided by applicant): For all intracellular trafficking events it is indispensable, that two membranes fuse with each other resulting in lipid and content mixing. This fundamental process is supposed to be catalyzed by specific proteins termed SNAREs (soluble NSF attachment protein receptors). Their sequence and structure is conserved in all eukaryotic systems, beginning with yeast and ending in humans. Since vesicle fusion is involved in many essential functions like synaptic transmission, hormone secretion and endocytosis, detailed knowledge about the molecular mechanism, how this reaction is catalyzed and what proteins besides of SNAREs are needed to fulfill this function will have deep impact on many disease-relevant topics. In order to unravel the precise fusion mechanism the yeast vacuolar fusion system is employed. Yeast vacuoles are purified in a large-scale isolation procedure and reconstituted in an in vitro system. Fusion efficiency of vacuoles can be easily measured by using two different strains, one lacking the vacuolar alkaline phosphatase PHO8, the other lacking the vacuolar protease PEP4. PHO8 is present as an inactive pro-enzyme, which needs to be cleaved in order to be active. After fusion has occurred, PEP4 gains access to the immature PHO8 enzyme, cleaves it and therefore activates it. Phosphatase activity can easily measure spectrophotometrically using a well-known assay. Vacuoles can be isolated in large quantities facilitating biochemical investigations. Since this system is the only existing possibility to address topological issues for membrane fusion in a physiological context in terms of how proteins interact in trans (between two fusing membranes), we reinvestigated a current dogma entitled as "topological restriction model". We took advantage of a certain property of yeast vacuolar fusion, namely that fusion rate is relatively slow and therefore can easily dissected in different stages. We have discovered an unexpected post-priming cis-SNARE complex, which we will characterize further in terms of how its stability is controlled and what other proteins are needed to stabilize this complex. Aim 1 will address this issue. Furthermore, we have discovered another topological trans-interaction of SNARE proteins, not predicted by the current model by using differently tagged SNAREs on the two fusing vacuoles. The fusion relevance of this trans-interaction was investigated by employing different combinations of vacuoles containing specifically inactivated SNAREs on their surface. We will continue to generate SNARE mutants to further confirm the physiological relevance of the discovered trans-SNARE topology (Aim2). We also started to elucidate, what specific function additional factors like the SEC1/MUNC18 related HOPS-complex and the V0 part of the V-ATPase have for the membrane fusion process. We have characterized V0 mutants, which are unable to fuse but have not lost their ability to pump protons. Aim 3 of this proposal addresses this topic. PUBLIC HEALTH RELEVANCE: SNARE (soluble NSF attachment protein receptors)-mediated intracellular fusion is essential for synaptic transmission, hormone secretion and endocytosis. Many diseases in synaptic transmission are linked to a disorder of SNARE-function and their associated proteins. Also some sorts of decreased insulin secretion are caused by insufficient SNARE-function resulting in Diabetes-like phenotypes.