Structure of the HOPS complex and its interactions with SNARE proteins

• Applicant: Dr. Sarah A. Port
• Subject Area: Structural Biology ; Biochemistry
• Term: from 2017 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 352652013

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.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Frederick Hughson