Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery.

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and pathogenic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our data suggest a model for how HOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part of the membrane fusion machinery.

Our cells break down the nutrients that they receive from the body to create the building blocks needed to keep us alive. This is done by compartments called lysosomes that are filled with a cocktail of proteins called enzymes, which speed up the breakdown process. Lysosomes are surrounded by a membrane, a barrier of fatty molecules that protects the rest of the cell from being digested. When new nutrients reach the cell, they travel to the lysosome packaged in vesicles, which have their own fatty membrane. To allow the nutrients to enter the lysosome without creating a leak, the membranes of the vesicles and the lysosome must fuse. The mechanism through which these membranes fuse is not fully clear. It is known that both fusing membranes must contain proteins called SNAREs, which wind around each other when they interact. However, this alone is not enough. Other proteins are also required to tether the membranes together before they fuse. To understand how these tethers play a role, Shvarev, Schoppe, König et al. studied the structure of the HOPS complex from yeast. This assembly of six proteins is vital for lysosomal fusion and, has a composition similar to the equivalent complex in humans. Using cryo-electron microscopy, a technique that relies on freezing purified proteins to image them with an electron microscope and reveal their structure, allowed Shvarev, Schoppe, König et al. to provide a model for how HOPS interacts with SNAREs and membranes. In addition to HOPS acting as a tether to bring the membranes together, it can also bind directly to SNAREs. This creates a bridge that allows the proteins to wrap around each other, driving the membranes to fuse. HOPS is a crucial component in the cellular machinery, and mutations in the complex can cause devastating neurological defects. The complex is also targeted by viruses ­ such as SARS-CoV-2 ­ that manipulate HOPS to reduce its activity. Shvarev, Schoppe, König et al.'s findings could help researchers to develop drugs to maintain or recover the activity of HOPS. However, this will require additional information about its structure and how the complex acts in the biological environment of the cell.

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

AP-3 vesicle uncoating occurs after HOPS-dependent vacuole tethering.

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.