Structure of the HOPS complex and its interactions with SNARE proteins

真核细胞依靠囊泡运输来实现生长、分化、信号传导和许多其他重要的细胞功能。货物通过囊泡与靶细胞器的融合来递送，这是高度特异性的，并由n -乙基马来酰亚胺敏感因子附着蛋白受体（SNAREs）的组装驱动。为了实现膜融合，SNAREs与其他运输辅助因子合作，包括Sec1/Munc18 （SM）蛋白、Rab GTPases和膜系固因子。最重要的膜系扣因子是多亚单位系扣复合物（mtc），被认为是囊泡系扣和融合的主要协调者。与这一假设一致，MTCs与SNAREs和大多数其他贩运辅助因子相互作用，但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表征最好的MTCs之一，同型融合和液泡蛋白分选（HOPS）复合物的功能，这是晚期核内体融合所必需的。HOPS是一种~660 kDa的六聚体复合物，与大多数其他MTCs不同，它包含一个SM蛋白作为一个完整的亚基。了解HOPS复合物的结构及其与SNAREs的相互作用将有助于揭示SNAREs、MTCs和SM蛋白如何协同调节和特异性膜系结和融合。迄今为止，只有不到四分之一的啤酒花络合物的x射线晶体学结构特征。此外，低分辨率负染色电子显微镜（EM）研究产生了相互矛盾的结果。然而，据报道，用于这些研究的酵母啤酒花复合物不是很稳定。因此，为了确定高分辨率的HOPS复合物结构，我们建议使用低温电子显微镜作为一种潜在的高分辨率技术，并推测来自嗜热真核生物嗜热毛藻的更稳定的复合物。SNAREs不仅通过它们的SNARE基序，还通过n端“调控”结构域与HOPS复合物相互作用，其作用尚不清楚。作为Aim 2，我们将通过生化、晶体学和功能方法表征HOPS复合物与其同源SNAREs的不同n端结构域的相互作用。阐明这些相互作用将对我们理解啤酒花复合物（以及潜在的其他MTCs）在snare介导的膜融合中的作用具有决定性的影响。

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.