RESUMEN
His Domain Protein Tyrosine Phosphatase (HD-PTP) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, PI3K/AKT and RTKs such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. ESCRT components Vps4 and HRS have previously been implicated in cholesterol homeostasis, thus these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.
RESUMEN
The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition.
Asunto(s)
Cuerpos Multivesiculares , Proteínas de Saccharomyces cerevisiae , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Endosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central "V" domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/química , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Ubiquitina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Sitios de Unión , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Humanos , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/genética , Dispersión del Ángulo Pequeño , Técnicas del Sistema de Dos Híbridos , ATPasas de Translocación de Protón Vacuolares/genética , Difracción de Rayos XRESUMEN
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.
Asunto(s)
Cilios , Ciliopatías , Animales , Ciliopatías/genética , Ciliopatías/metabolismo , Femenino , Deformidades Congénitas de la Mano , Cardiopatías Congénitas , Proteínas Hedgehog/metabolismo , Hidrocefalia , Mamíferos/metabolismo , Embarazo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Cuerpos Multivesiculares/enzimología , Biogénesis de Organelos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/genética , Sitios de Unión , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Activación Enzimática , Microscopía Fluorescente , Modelos Moleculares , Cuerpos Multivesiculares/genética , Mutación , Dominios Proteicos , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
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.
Asunto(s)
Conductos Biliares/metabolismo , Células Epiteliales/metabolismo , Vesículas Extracelulares/metabolismo , Transporte Biológico , Línea Celular , Movimiento Celular , Polaridad Celular/fisiología , Endocitosis , Endosomas/metabolismo , Epitelio/metabolismo , Exosomas/metabolismo , Humanos , Transducción de SeñalRESUMEN
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.
Asunto(s)
Actinas , Proteína Forkhead Box M1/metabolismo , Neoplasias , Actinas/metabolismo , Animales , GTP Fosfohidrolasas , Ratones , Neoplasias/genética , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Trastornos de los Cromosomas/metabolismo , Mutación del Sistema de Lectura , Haploinsuficiencia , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Centrosoma/metabolismo , Centrosoma/patología , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/patología , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Ratones , Ratones Mutantes , Mosaicismo , Neoplasias/genética , Neoplasias/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
HIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner.IMPORTANCE The Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die.
Asunto(s)
Apoptosis , Caspasa 8/metabolismo , Infecciones por VIH/metabolismo , Proteasa del VIH/metabolismo , VIH-1/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Caspasa 8/genética , Infecciones por VIH/virología , Proteasa del VIH/genética , Humanos , Células Jurkat , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Replicación ViralRESUMEN
TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Microambiente Celular/inmunología , Infecciones por VIH/inmunología , Isoformas de Proteínas/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Empalme Alternativo/genética , Apoptosis , Efecto Espectador/inmunología , Linfocitos T CD4-Positivos/virología , Línea Celular Tumoral , Membrana Celular/inmunología , Células HEK293 , Infecciones por VIH/patología , Infecciones por VIH/virología , Células HeLa , Humanos , Células Jurkat , Isoformas de Proteínas/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesisRESUMEN
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.
Asunto(s)
Inestabilidad Cromosómica , Ciclina A2/metabolismo , Enzimas Reparadoras del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Centrosoma/metabolismo , Ciclina A2/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Cinesinas/metabolismo , Proteína Homóloga de MRE11 , Meiosis/genética , Ratones , Ratones Mutantes , Mitosis/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Fase S/genéticaRESUMEN
Ubiquitinated membrane proteins are sorted into intralumenal endosomal vesicles on their way for degradation in lysosomes. Here we summarize the discovery of the Cos proteins, which work to organize and segregate ubiquitinated cargo prior to its incorporation into intralumenal vesicles of the multivesicular body (MVB). Importantly, cargoes such as GPI-anchored proteins (GPI-APs) that cannot undergo ubiquitination, rely entirely on Cos proteins for sorting into intralumenal vesicles using the same pathway that depends on ESCRTs and ubiquitin ligases that typical polytopic membrane proteins do. Here we show Cos proteins provide functions as not only adaptor proteins for ubiquitin ligases, but also as cargo carriers that can physically usher a variety of other proteins into the MVB pathway. We then discuss the significance of this new sorting model and the broader implications for this cargo adaptor mechanism, whereby yeast Cos proteins, and their likely animal analogs, provide a ubiquitin sorting signal in trans to enable sorting of a membrane protein network into intralumenal vesicles.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Cuerpos Multivesiculares/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Tetraspaninas/fisiología , Modelos Biológicos , Transporte de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Tetraspaninas/química , Tetraspaninas/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pliegue de Proteína , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/químicaRESUMEN
The abundance of cell-surface membrane proteins is regulated by internalization and delivery into intralumenal vesicles (ILVs) of multivesicular bodies (MVBs). Many cargoes are ubiquitinated, allowing access to an ESCRT-dependent pathway into MVBs. Yet how nonubiquitinated proteins, such as glycosylphosphatidylinositol-anchored proteins, enter MVBs is unclear, supporting the possibility of mechanistically distinct ILV biogenesis pathways. Here we show that a family of highly ubiquitinated tetraspan Cos proteins provides a Ub signal in trans, allowing sorting of nonubiquitinated MVB cargo into the canonical ESCRT- and Ub-dependent pathway. Cos proteins create discrete endosomal subdomains that concentrate Ub cargo prior to their envelopment into ILVs, and the activity of Cos proteins is required not only for efficient sorting of canonical Ub cargo but also for sorting nonubiquitinated cargo into MVBs. Expression of these proteins increases during nutrient stress through an NAD(+)/Sir2-dependent mechanism that in turn accelerates the downregulation of a broad range of cell-surface proteins.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Animales , Proteínas de la Membrana/metabolismo , Unión Proteica/fisiología , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/fisiología , Caspasa 8/metabolismo , Proteasa del VIH/fisiología , VIH-1/enzimología , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/virología , Caspasa 8/química , Células HEK293 , Humanos , Células Jurkat , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteolisis , Proteína Destructora del Antagonista Homólogo bcl-2/químicaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Coenzimas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Aminoácidos , Animales , Coenzimas/química , Coenzimas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación de la Expresión Génica , Humanos , Hidrólisis , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Citocinesis , Análisis Mutacional de ADN , Endosomas/metabolismo , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
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.