Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis.

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

Ciliopathy protein HYLS1 coordinates the biogenesis and signaling of primary cilia by activating the ciliary lipid kinase PIPKIγ.

Mutation of ciliopathy protein HYLS1 causes the perinatal lethal hydrolethalus syndrome (HLS), yet the underlying molecular etiology and pathogenesis remain elusive. Here, we reveal unexpected mechanistic insights into the role of mammalian HYLS1 in regulating primary cilia. HYLS1 is recruited to the ciliary base via a direct interaction with the type Iγ phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIPKIγ). HYLS1 activates PIPKIγ by interrupting the autoinhibitory dimerization of PIPKIγ, which thereby expedites depletion of centrosomal PI(4)P to allow axoneme nucleation. HYLS1 deficiency interrupts the assembly of ciliary NPHP module and agonist-induced ciliary exit of ß-arrestin, which, in turn, disturbs the removal of ciliary Gpr161 and activation of hedgehog (Hh) signaling. Consistent with this model of pathogenesis, the HLS mutant HYLS1D211G supports ciliogenesis but not activation of Hh signaling. These results implicate mammalian HYLS1 as a multitasking protein that facilitates ciliogenesis and ciliary signaling by coordinating with the ciliary lipid kinase PIPKIγ.

Polarized human cholangiocytes release distinct populations of apical and basolateral small extracellular vesicles.

Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.

FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling to drive cancer.

FoxM1 activates genes that regulate S-G2-M cell-cycle progression and, when overexpressed, is associated with poor clinical outcome in multiple cancers. Here we identify FoxM1 as a tumor suppressor in mice that, through its N-terminal domain, binds to and inhibits Ect2 to limit the activity of RhoA GTPase and its effector mDia1, a catalyst of cortical actin nucleation. FoxM1 insufficiency impedes centrosome movement through excessive cortical actin polymerization, thereby causing the formation of non-perpendicular mitotic spindles that missegregate chromosomes and drive tumorigenesis in mice. Importantly, low FOXM1 expression correlates with RhoA GTPase hyperactivity in multiple human cancer types, indicating that suppression of the newly discovered Ect2-RhoAmDia1 oncogenic axis by FoxM1 is clinically relevant. Furthermore, by dissecting the domain requirements through which FoxM1 inhibits Ect2 GEF activity, we provide mechanistic insight for the development of pharmacological approaches that target protumorigenic RhoA activity.

Mosaic-variegated aneuploidy syndrome mutation or haploinsufficiency in Cep57 impairs tumor suppression.

A homozygous truncating frameshift mutation in CEP57 (CEP57T/T) has been identified in a subset of mosaic-variegated aneuploidy (MVA) patients; however, the physiological roles of the centrosome-associated protein CEP57 that contribute to disease are unknown. To investigate these, we have generated a mouse model mimicking this disease mutation. Cep57T/T mice died within 24 hours after birth with short, curly tails and severely impaired vertebral ossification. Osteoblasts in lumbosacral vertebrae of Cep57T/T mice were deficient for Fgf2, a Cep57 binding partner implicated in diverse biological processes, including bone formation. Furthermore, a broad spectrum of tissues of Cep57T/T mice had severe aneuploidy at birth, consistent with the MVA patient phenotype. Cep57T/T mouse embryonic fibroblasts and patient-derived skin fibroblasts failed to undergo centrosome maturation in G2 phase, causing premature centriole disjunction, centrosome amplification, aberrant spindle formation, and high rates of chromosome missegregation. Mice heterozygous for the truncating frameshift mutation or a Cep57-null allele were overtly indistinguishable from WT mice despite reduced Cep57 protein levels, yet prone to aneuploidization and cancer, with tumors lacking evidence for loss of heterozygosity. This study identifies Cep57 as a haploinsufficient tumor suppressor with biologically diverse roles in centrosome maturation and Fgf2-mediated bone formation.

Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding-dependent role that ensures adequate repair of common replication errors.

Conformational Changes in the Endosomal Sorting Complex Required for the Transport III Subunit Ist1 Lead to Distinct Modes of ATPase Vps4 Regulation.

Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.

Vps4 stimulatory element of the cofactor Vta1 contacts the ATPase Vps4 α7 and α9 to stimulate ATP hydrolysis.

The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 ß-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically α-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation.

The linker region plays a regulatory role in assembly and activity of the Vps4 AAA ATPase.

The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.

Relief of autoinhibition enhances Vta1 activation of Vps4 via the Vps4 stimulatory element.

The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.

Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106.

Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)(2) tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)(2). We show that the Rtt106-(H3-H4)(2) interaction is important for gene silencing and the DNA damage response.

Regulation of Vps4 during MVB sorting and cytokinesis.

Multivesicular body (MVB) formation is the result of invagination and budding of the endosomal limiting membrane into its intralumenal space. These intralumenal vesicles (ILVs) contain a subset of endosomal transmembrane cargoes destined for degradation within the lysosome, the result of active selection during MVB sorting. Membrane bending and scission during ILV formation is topologically similar to cytokinesis in that both events require the abscission of a membrane neck that is oriented away from the cytoplasm. The endosomal sorting complexes required for transport (ESCRTs) represent cellular machinery whose function makes essential contributions to both of these processes. In particular, the AAA-ATPase Vps4 and its substrate ESCRT-III are key components that seem to execute the membrane abscission reaction. This review summarizes current knowledge about the Vps4-ESCRT-III system and discusses a model for how the recruitment of Vps4 to the different sites of function might be regulated.

Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly.

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

Assembly of the AAA ATPase Vps4 on ESCRT-III.

Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Ist1, Vta1, and Did2. The order of events leading to active, ESCRT-III-associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Ist1-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in turn is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer.

Structural basis of Ist1 function and Ist1-Did2 interaction in the multivesicular body pathway and cytokinesis.

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.

Regulation of Vps4 ATPase activity by ESCRT-III.

MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

Structural basis of Vta1 function in the multivesicular body sorting pathway.

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.

ESCRT-III family members stimulate Vps4 ATPase activity directly or via Vta1.

The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.