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，两个膜相互融合导致脂质和含量混合是必不可少的。这一基本过程被认为是由称为SNAREs（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生物系统中都是保守的，从酵母开始，到人类结束。由于囊泡融合涉及突触传递、激素分泌和内吞等许多基本功能，因此详细了解分子机制、该反应如何催化以及除了SNAREs之外还需要哪些蛋白质来完成该功能将对许多疾病相关的主题产生深远的影响。为了揭示其确切的融合机制，采用酵母液泡融合系统。酵母液泡在大规模分离过程中纯化，并在体外系统中重组。用两种不同的菌株，一种缺乏液泡碱性磷酸酶PHO8，另一种缺乏液泡蛋白酶PEP4，可以很容易地测量液泡的融合效率。PHO8作为一种无活性的前酶存在，它需要被裂解才能具有活性。融合发生后，PEP4进入未成熟的PHO8酶，将其切割并激活它。磷酸酶活性可以很容易地测量分光光度法使用一个众所周知的化验。液泡可以大量分离，便于生化研究。由于该系统是在生理背景下解决膜融合拓扑问题的唯一现有可能性，即蛋白质如何在反式中相互作用（在两个融合膜之间），我们重新研究了当前称为“拓扑限制模型”的教条。我们利用了酵母液泡融合的特性，即融合速度相对较慢，因此在不同的阶段很容易被解剖。我们发现了一个意想不到的启动后顺式snare复合物，我们将进一步描述其稳定性是如何控制的，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SNARE蛋白的另一种拓扑反式相互作用，目前的模型没有通过在两个融合液泡上使用不同标记的SNARE来预测。通过在液泡表面使用含有特异性失活SNAREs的不同液泡组合，研究了这种反式相互作用的融合相关性。我们将继续生成SNARE突变体，以进一步确认所发现的跨SNARE拓扑结构（Aim2）的生理相关性。我们也开始阐明，其他因子如SEC1/MUNC18相关的hops复合物和v - atp酶的V0部分对膜融合过程有什么特定的功能。我们已经描述了V0突变体，它们不能融合，但没有失去泵送质子的能力。本提案的目标3涉及这一主题。

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.