RESUMEN
ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.
Asunto(s)
Proteínas AAA , Adenosina Trifosfato , Proteínas AAA/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/genética , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismoRESUMEN
Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.
Asunto(s)
Adenosina Trifosfatasas/genética , Fusión de Membrana , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Proteínas de Transporte Vesicular/genética , Adenosina Trifosfatasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Two proteins called Sec17 and Sec18 may have a larger role in membrane fusion than is commonly assumed in textbook models.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas , Fusión de Membrana , Proteínas SNARE , Saccharomyces cerevisiae , Proteínas de Transporte VesicularRESUMEN
Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/αSNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. We now report that Sec17 and Sec18 can drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE that face the center of the 4-SNARE coiled coils with polar residues. These blocks, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Fusión de Membrana/genética , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/genética , Comunicación Celular , Fusión de Membrana/fisiología , Dominios Proteicos , Proteínas SNARE/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
Asunto(s)
Adenosina Trifosfatasas/genética , Etilmaleimida/metabolismo , Ácidos Fosfatidicos/química , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequeñas/química , Proteínas de Transporte Vesicular/genética , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/genética , Adenosina Trifosfatasas/química , Fusión de Membrana/efectos de los fármacos , Fusión de Membrana/genética , Lípidos de la Membrana/química , Lípidos de la Membrana/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Simulación de Dinámica Molecular , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/genética , Ácidos Fosfatidicos/antagonistas & inhibidores , Proteínas SNARE/química , Proteínas SNARE/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/genética , Vacuolas/genética , Proteínas de Transporte Vesicular/químicaRESUMEN
Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Ácidos Fosfatidicos/farmacología , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfato/metabolismo , Simulación de Dinámica Molecular , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosforilación , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína/efectos de los fármacos , Proteínas SNARE/química , Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
In eukaryotes, organelles and vesicles modulate their contents and identities through highly regulated membrane fusion events. Membrane trafficking and fusion are carried out through a series of stages that lead to the formation of SNARE complexes between cellular compartment membranes to trigger fusion. Although the protein catalysts of membrane fusion are well characterized, their response to their surrounding microenvironment, provided by the lipid composition of the membrane, remains to be fully understood. Membranes are composed of bulk lipids (e.g., phosphatidylcholine), as well as regulatory lipids that undergo constant modifications by kinases, phosphatases, and lipases. These lipids include phosphoinositides, diacylglycerol, phosphatidic acid, and cholesterol/ergosterol. Here we describe the roles of these lipids throughout the stages of yeast vacuole homotypic fusion.
Asunto(s)
Colesterol/metabolismo , Ergosterol/metabolismo , Glicéridos/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositoles/metabolismo , Vacuolas/metabolismo , Colesterol/química , Ergosterol/química , Glicéridos/química , Humanos , Fusión de Membrana , Ácidos Fosfatidicos/química , Fosfatidilinositoles/química , Vacuolas/químicaRESUMEN
Microscale thermophoresis is a relatively new technique used by an increasing number of academic laboratories to estimate relative binding affinities between ligand (analyte) that is titrated and a target (generally protein) that is either fluorescently labeled exogenously in the red or blue channel (labeled thermophoresis) or endogenously labeled via the presence of sufficient aromatic amino acid residues such as tryptophan (label-free thermophoresis). There are advantages and disadvantages to each technique; however, one major disadvantage of label-free thermophoresis is that protein-protein interactions cannot be measured, as generally most proteins have enough aromatic residues to generate an interfering signal. Thermophoresis can be used to determine steady-state binding affinities as between SNAREs and relevant binding partners of SNAREs and labeled thermophoresis is increasingly becoming a reliable technique to screen binding partners of fusion machinery to determine relevance in terms of direct biochemical interactions.
Asunto(s)
Unión Proteica , Proteínas SNARE/metabolismo , Difusión Térmica , Sitios de Unión , Colorantes Fluorescentes/química , Liposomas/química , Liposomas/metabolismo , Fusión de Membrana , Nanoestructuras/química , Rodaminas/química , Proteínas SNARE/químicaRESUMEN
Surface plasmon resonance (SPR) can be used to analyze both binding affinities and kinetic parameters between a ligand and an analyte. SPR can be performed by either cross-linking a given ligand to a sensor chip covalently or utilizing high-affinity non-covalent interactions to secure a ligand in a particular conformation to a chip, both of which have their potential advantages. SPR measurements are mass based and reflect the proportional amount of analyte bound to a given ligand at a given concentration when flowed at a set rate in order to determine the binding parameters of a given biochemical interaction. The resultant sensorgram can indicate different types of binding events as well as provide both ka and kd, which can be used to determine an equilibrium dissociation constant KD. SPR can be used to measure binding affinity of proteins involved in fusion such as between SNAREs, SNAREs, and proteins that interact with them such as Sec18 (NSF) or Sec17 (alpha-SNAP), or to measure the binding of any fusion-related protein to a specific lipid or other small molecules; however, KDs are determined by SPR using a titration of concentrations of analyte and a maximum point on the sensorgram signifying saturation of the protein in order to determine a steady-state KD.
Asunto(s)
Unión Proteica , Proteínas SNARE/metabolismo , Resonancia por Plasmón de Superficie/métodos , Cinética , Ligandos , Lípidos , Fusión de Membrana , Proteínas Sensibles a N-Etilmaleimida/química , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Nanoestructuras/química , Proteínas SNARE/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Resonancia por Plasmón de Superficie/instrumentaciónRESUMEN
Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Liposomas/metabolismo , Fosfolípidos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Liposomas/química , Fusión de Membrana , Fosfolípidos/química , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/aislamiento & purificaciónRESUMEN
At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Fusión de Membrana , Proteolípidos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatasas/química , Transporte Biológico , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Unión Proteica , Proteolípidos/química , Proteínas SNARE/química , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/química , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/químicaRESUMEN
Sequestration of aggregates into specialized deposition sites occurs in many species across all kingdoms of life ranging from bacteria to mammals and is commonly believed to have a cytoprotective function. Yeast cells possess at least 3 different spatially separated deposition sites, one of which is termed "Insoluble Protein Deposit (IPOD)" and harbors amyloid aggregates. We have recently discovered that recruitment of amyloid aggregates to the IPOD uses an actin cable based recruitment machinery that also involves vesicular transport. 1 Here we discuss how different proteins known to be involved in vesicular transport processes to the vacuole might act to guide amyloid aggregates to the IPOD. These factors include the Myosin V motor protein Myo2 involved in transporting vacuolar vesicles along actin cables, the transmembrane protein Atg9 involved in the recruitment of large precursor hydrolase complexes to the vacuole, the phosphatidylinositol/ phosphatidylcholine (PI/PC) transfer protein Sec 14 and the SNARE chaperone Sec 18. Furthermore, we present new data suggesting that the yeast dynamin homolog Vps1 is also crucial for faithful delivery of the amyloid model protein PrD-GFP to the IPOD. This is in agreement with a previously identified role for Vps1 in recruitment of heat-denatured aggregates to a perivacuolar deposition site. 2.