Structure of the HOPS complex and its interactions with SNARE proteins

真核细胞的生长、分化、信号传递和许多其他重要的细胞功能都依赖于囊泡运输。货物是通过囊泡与目标细胞器的融合来运送的，目标细胞器具有高度特异性，并由N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARs)的组装驱动。对于膜融合，SNARS与其他运输辅助因子合作，包括Sec1/Munc18(SM)蛋白、Rab GTP酶和膜系留因子。最重要的膜系留因子是多亚单位系留复合体(MTCs)，被认为是囊泡系留和融合的主要协调者。与这一假设一致，MTCs与圈套和大多数其他贩运辅助因素相互作用，但其潜在的分子机制尚不清楚。我们现在建议使用生化和结构方法来研究最具特征的MTCs之一-同型融合和空泡蛋白分类(HOPS)复合体的功能，它对于晚期内体的融合是必不可少的。啤酒花是一个约660 kDa的六聚体复合体，与大多数其他MTCs不同，它含有一个SM蛋白作为一个完整的亚基。了解啤酒花复合体的结构及其与SNARS的相互作用将有助于阐明SNARES、MTCs和SM蛋白如何协作调节膜系留和融合的特异性。到目前为止，只有不到四分之一的啤酒花复合体被X射线结晶学表征。此外，低分辨率负染色电子显微镜(EM)研究得出了相互不一致的结果。然而，据报道，用于这些研究的酵母啤酒花复合体并不是很稳定。因此，为了确定啤酒花复合体的高分辨率结构，我们建议将冷冻电子显微镜作为一种潜在的高分辨率技术，并推测更稳定的来自嗜热真核生物Chaetomium thermophilum的复合体。SNARE不仅通过它们的陷阱基序与啤酒花复合体相互作用，而且通过N-末端的“调节”结构域与之相互作用，其作用尚不清楚。作为目标2，我们将用生化、结晶学和功能方法表征啤酒花复合体与其同源SNARs的不同N-末端结构域的相互作用。阐明这些相互作用应该对我们对啤酒花复合体--以及潜在的其他MTCs--在SNARE介导的膜融合中的作用的分子理解有决定性的影响。

Eukaryotic cells rely on vesicle trafficking for growth, differentiation, signaling and many other crucial cellular functions. Cargoes are delivered by fusion of the vesicles with the target organelle, which is highly specific and driven by the assembly of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). For membrane fusion, SNAREs collaborate with other trafficking co-factors including Sec1/Munc18 (SM) proteins, Rab GTPases, and membrane tethering factors. The most important membrane tethering factors are the multisubunit tethering complexes (MTCs), thought to be the major orchestrators of vesicle tethering and fusion. Consistent with this hypothesis, MTCs interact with SNAREs and most of the other trafficking co-factors, but the underlying molecular mechanisms are poorly understood. We now propose to use biochemical and structural methods to study the function of one of the best characterized MTCs, the homotypic fusion and vacuolar protein sorting (HOPS) complex, which is essential for the fusion of late endosomes. HOPS is a ~660 kDa, hexameric complex and, unlike most of the other MTCs, contains a SM protein as an integral subunit. Understanding the structure of the HOPS complex and its interactions with the SNAREs will shed light on how SNAREs, MTCs and SM proteins collaborate for the regulation and specificity of membrane tethering and fusion.To date, less than a quarter of the HOPS complex has been structurally characterized by X-ray crystallography. Furthermore, low-resolution negative-stain electron microscopy (EM) studies have yielded mutually inconsistent results. However, the yeast HOPS complex used for those studies is reportedly not very stable. To determine a higher-resolution structure of the HOPS complex, we therefore propose as Aim 1 to use cryo-electron microscopy as a potentially high-resolution technology and the presumably more stable complex derived from the thermophilic eukaryote Chaetomium thermophilum.SNAREs interact with the HOPS complex not only via their SNARE motifs but also via N-terminal "regulatory" domains, the role of which are not well understood yet. As Aim 2, we will characterize the interactions of the HOPS complex with the different N-terminal domains of its cognate SNAREs by biochemical, crystallographic, and functional methods. Elucidating these interactions should have a decisive impact on our molecular understanding of the role of the HOPS complex - and potentially other MTCs - in SNARE-mediated membrane fusion.