Intracellular Membrane Fusion Mediated by SNARE Proteins

描述(由申请人提供)：对于所有细胞内转运事件，两个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、激素分泌和内吞作用等许多基本功能，因此对囊泡融合的分子机制、如何催化这一反应以及除了SNARs之外还需要哪些蛋白质来完成这一功能的详细了解将对许多与疾病相关的话题产生深远的影响。为了揭示精确的融合机制，我们采用了酵母空泡融合系统。酵母液泡在大规模分离过程中被提纯，并在体外系统中重组。用两个不同的菌株可以很容易地测量液泡的融合效率，一个缺乏液泡碱性磷酸酶PHO8，另一个缺乏液泡蛋白酶PEP4。PHO8是一种非活性的酶原，需要被切割才能激活。融合发生后，PEP4获得未成熟的PHO8酶，将其裂解，从而激活它。使用著名的分析方法，可以很容易地用分光光度法测量磷酸酶的活性。大量的液泡可以被分离出来，便于生化研究。由于这个系统是目前唯一可能解决膜融合的拓扑问题的生理背景下，根据蛋白质如何相互作用(两个融合膜之间)，我们重新调查了当前的教条命名为“拓扑限制模型”。我们利用了酵母空泡融合的一个特性，即融合速度相对较慢，因此很容易在不同的阶段进行解剖。我们发现了一个意想不到的启动后顺式圈套复合体，我们将进一步表征它的稳定性是如何控制的，以及需要哪些其他蛋白质来稳定这个复合体。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，这不是目前的模型通过在两个融合的液泡上使用不同标记的SNARE来预测的。通过使用不同的空泡组合来研究这种反式相互作用的融合相关性，这些空泡的表面含有特定的灭活陷阱。我们将继续产生SNARE突变体，以进一步证实已发现的跨SNARE拓扑结构(AIM2)的生理学相关性。我们还开始阐明，与Sec1/Munc18相关的Hop-Complex和V-ATPase的V0部分等附加因子对膜融合过程具有哪些特定的功能。我们已经确定了V0突变体的特征，它们不能融合，但并没有失去泵浦质子的能力。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都与SNARE功能及其相关蛋白的紊乱有关。此外，某些类型的胰岛素分泌减少是由于圈套功能不足导致的糖尿病样表型。

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.