No abstract

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na(+)-Cl(-) cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division.

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells whose function is governed by the guanosine diphosphate (GDP) dissociation inhibitor (RabGDI). Using a combination of chemical synthesis and protein engineering, we generated and crystallized the monoprenylated Ypt1:RabGDI complex. The structure of the complex was solved to 1.5 angstrom resolution and provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins. Isoprenoid binding requires a conformational change that opens a cavity in the hydrophobic core of its domain II. Analysis of the structure provides a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.

Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5°rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.T he actin-based molecular motor, myosin, generates force and movement via a series of structural transitions when bound to filamentous actin (F-actin). Among these structural states, the most important contribution to force production comes from the myosin ADP states that bind strongly to actin. Release of ADP from the motor is rapidly followed by MgATP binding, which then leads to myosin dissociation from actin. Once the dissociation occurs, myosin can undergo a structural change that allows it to prime its mechanical element (known as the lever arm; Fig. 1) and hydrolyze ATP. Myosin rebinding to actin triggers release of phosphate and reentry into the force-generating states on actin. Thus, force is produced by MgADP-bound states of myosin bound to actin.In the absence of strain, a strong-binding MgADP state bound to actin can exist for varying durations, depending on the type of myosin. Myosins that are designed to spend the majority of their catalytic cycle bound to actin in force-generating states in the absence of load, such as myosin V a , primarily occupy a state that is characterized by having both a high affinity for MgADP and a high affinity for actin. The myosin motor cycle is summarized in Fig. 1. By not rapidly releasing MgADP, the myosin motors can dwell in a force-generating state on actin that cannot be detached from actin by ATP binding. This is the primary force-generating state for all myosins, and its lifetime on actin is prolonged under load (for a review, see ref. 1).Although the nucleotide-free myosin V X-ray structure (2) (Rigor-like state) provides an atomic level model for the actomyosin state that is formed after ADP is released, and to which ATP can rapidly bind (2), we only have low-resolution EM reconstructions of myosin bound to actin in a MgADP state. Visualization of this state at the EM level was first performed for smooth muscle myosin II (3) and provided the first structural evidence that the myosin lever arm swings as the motor progresses through its actinbound, force-generating states. It was postulated at the time that th...

Generating force and movement is essential for the functions of cells and organisms. A variety of molecular motors that can move on tracks within cells have evolved to serve this role. How these motors interact with their tracks and how that, in turn, leads to the generation of force and movement is key to understanding the cellular roles that these motor-track systems serve. This review is focused on the best understood of these systems, which is the molecular motor myosin that moves on tracks of filamentous (F-) actin. The review highlights both the progress and the limits of our current understanding of how force generation can be controlled by F-actin–myosin interactions. What has emerged are insights they may serve as a framework for understanding the design principles of a number of types of molecular motors and their interactions with their tracks.

Rab molecular switches are key players in defining membrane identity and regulating intracellular trafficking events in eukaryotic cells. In spite of their global structural similarity, Rab-family members acquired particular features that allow them to perform specific cellular functions. The overall fold and local sequence conservations enable them to utilize a common machinery for prenylation and recycling; while individual Rab structural differences determine interactions with specific partners such as GEFs, GAPs and effector proteins. These interactions orchestrate the spatiotemporal regulation of Rab localization and their turning ON and OFF, leading to tightly controlled Rab-specific functionalities such as membrane composition modifications, recruitment of molecular motors for intracellular trafficking, or recruitment of scaffold proteins that mediate interactions with downstream partners, as well as actin cytoskeleton regulation.In this review we summarize structural information on Rab GTPases and their complexes with protein partners in the context of partner binding specificity and functional outcomes of their interactions in the cell.