Mechanism of how the universal module XMAP215 γ-TuRC nucleates microtubules.

It has become increasingly evident in recent years that nucleation of microtubules from a diverse set of MTOCs requires both the γ-tubulin ring complex (γ-TuRC) and the microtubule polymerase XMAP215. Despite their essentiality, little is known about how these nucleation factors interact and work together to generate microtubules. Using biochemical domain analysis of XMAP215 and structural approaches, we find that a sixth TOG domain in XMAP215 binds γ-TuRC via γ-tubulin as part of a broader interaction involving the C-terminal region. Moreover, TOG6 is required for XMAP215 to promote nucleation from γ-TuRC to its full extent. Interestingly, we find that XMAP215 also depends strongly on TOG5 for microtubule lattice binding and nucleation. Accordingly, we report a cryo-EM structure of TOG5 bound to the microtubule lattice that reveals promotion of lateral interactions between tubulin dimers. Finally, we find that while XMAP215 constructs' effects on nucleation are generally proportional to their effects on polymerization, formation of a direct complex with γ-TuRC allows cooperative nucleation activity. Thus, we propose that XMAP215's C-terminal TOGs 5 and 6 play key roles in promoting nucleation by promoting formation of longitudinal and lateral bonds in γ-TuRC templated nascent microtubules at cellular MTOCs.

Conformational states of the microtubule nucleator, the γ-tubulin ring complex.

Microtubules (MTs) perform essential functions in the cell, and it is critical that they are made at the correct cellular location and cell cycle stage. This nucleation process is catalyzed by the γ-tubulin ring complex (γ-TuRC), a cone-shaped protein complex composed of over 30 subunits. Despite recent insight into the structure of vertebrate γ-TuRC, which shows that its diameter is wider than that of a MT, and that it exhibits little of the symmetry expected for an ideal MT template, the question of how γ-TuRC achieves MT nucleation remains open. Here, we utilized single particle cryo-EM to identify two conformations of γ-TuRC. The helix composed of 14 γ-tubulins at the top of the γ-TuRC cone undergoes substantial deformation, which is predominantly driven by bending of the hinge between the GRIP1 and GRIP2 domains of the γ-tubulin complex proteins. However, surprisingly, this deformation does not remove the inherent asymmetry of γ-TuRC. To further investigate the role of γ-TuRC conformational change, we used cryo electron-tomography (cryo-ET) to obtain a 3D reconstruction of γ-TuRC bound to a nucleated MT, providing insight into the post-nucleation state. Rigid-body fitting of our cryo-EM structures into this reconstruction suggests that the MT lattice is nucleated by spokes 2 through 14 of the γ-tubulin helix, which entails spokes 13 and 14 becoming more structured than what is observed in apo γ-TuRC. Together, our results allow us to propose a model for conformational changes in γ-TuRC and how these may facilitate MT formation in a cell.

Mutagenesis and structural modeling implicate RME-8 IWN domains as conformational control points.

After endocytosis, transmembrane cargo is differentially sorted into degradative or recycling pathways. This process is facilitated by recruitment into physically distinct degradative or recycling microdomains on the limiting membrane of individual endosomes. Endosomal sorting complexes required for transport (ESCRT) mark the degradative microdomain, while the recycling domain is marked by the retromer complex and associated proteins RME-8 and SNX-1. The separation of endosomal microdomains is also controlled by RME-8 and SNX-1, at least in part via removal of degradative component HRS/HGRS-1 from the recycling microdomain. This activity is likely due to recruitment and activation of chaperone Hsc70 on the endosome by the RME-8 DNAJ domain. To better understand the mechanism of RME-8 function we performed a new phylogenetic analysis of RME-8 and identified new conserved sequence features. In a complementary approach, we performed structure-function analysis that identified the C-terminus as important for microdomain localization and likely substrate binding, while N-terminal sequences beyond the known single N-terminal PH-like domain are important for endosome recruitment. Random mutagenesis identified IWN4, and by analogy IWN3, to be important for the autoinhibitory DNAJ domain binding, with IWN3 playing a critical role in HRS uncoating activity. Combining AlphaFold structural predictions with in vivo mutation analysis of RME-8, we propose a model whereby SNX-1 and the IWN domains control the conformation of RME-8 and hence the productive exposure of the DNAJ domain. Furthermore, we propose that the activation of RME-8 is cyclical, with SNX-1 acting as an activator and a target of RME-8 uncoating activity.

LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope.

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide, which acts as a barrier and contributes to antibiotic resistance. The systems that mediate phospholipid trafficking across the periplasm, such as MCE (Mammalian Cell Entry) transporters, have not been well characterized. Our ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Our results support a model in which LetB establishes a physical link between the two membranes and creates a hydrophobic pathway for the translocation of lipids across the periplasm.