ATPase Ring Research Articles

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation.

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6.

Mechanism of One-Way Traffic of Hexameric Phi29 DNA Packaging Motor with Four Electropositive Relaying Layers Facilitating Antiparallel Revolution

The importance of nanomotors in nanotechnology is akin to that of mechanical engines to daily life. The AAA+ superfamily is a class of nanomotors performing various functions. Their hexagonal arrangement facilitates bottom-up assembly for stable structures. The bacteriophage phi29 DNA translocation motor contains three coaxial rings: a dodecamer channel, a hexameric ATPase ring, and a hexameric pRNA ring. The viral DNA packaging motor has been believed to be a rotational machine. However, we discovered a revolution mechanism without rotation. By analogy, the earth revolves around the sun while rotating on its own axis. One-way traffic of dsDNA translocation is facilitated by five factors: (1) ATPase changes its conformation to revolve dsDNA within a hexameric channel in one direction; (2) the 30° tilt of the channel subunits causes an antiparallel arrangement between two helices of dsDNA and channel wall to advance one-way translocation; (3) unidirectional flow property of the internal channel loops serves as a ratchet valve to prevent reversal; (4) 5′–3′ single-direction movement of one DNA strand along the channel wall ensures single direction; and (5) four electropositive layers interact with one strand of the electronegative dsDNA phosphate backbone, resulting in four relaying transitional pauses during translocation. The discovery of a riding system along one strand provides a motion nanosystem for cargo transportation and a tool for studying force generation without coiling, friction, and torque. The revolution of dsDNA among 12 subunits offers a series of recognition sites on the DNA backbone to provide additional spatial variables for nucleotide discrimination for sensing applications.

A Force Dependent Gating Mechanism Inhibits ATP Dependent Release of Dynein from Microtubules

Cytoplasmic dynein is a processive minus end directed microtubule motor involved in a wide range of cellular functions. By studying how dynein monomers respond to load in an optical trapping assay, we discovered a force dependent gating mechanism that prevents ATP driven release of dynein from the microtubule. Our results show that tension on dynein's linker domain causes the microtubule release rate of the motor to be insensitive to ATP, while dynein motors with an unloaded linker release at a much faster rate at high ATP concentrations. We found that dynein monomers preferentially release when force is applied towards the minus end of the microtubule rather than the plus end. This strong asymmetry is observed even in the absence of dynein's ATPase ring, implying it is an intrinsic property of the stalk and microtubule binding domain. To test what role these phenomena play in dynein motility, we studied ATPase mutants with altered translocation velocities and measured their force dependent release kinetics. On the basis of these results we developed a minimal model of dynein motility and show how intramolecular tension, powerstroke and force dependent release are main determinants of dynein's motility properties and directionality.

Feeling the Pressure: Dynamics of Viral DNA Packaging are Controlled by the Confined Genome within the Capsid

Double-stranded DNA viruses, including tailed bacteriophages and mammalian herpesviruses, package their genomes into pre-formed protein capsids. The packaging process is driven by a molecular complex known as the packaging motor. This motor is a ring-shaped oligomeric ATPase that utilizes the chemical energy from ATP binding and hydrolysis to perform the mechanical work. We have recently presented a detailed mechanochemical characterization for the bacteriophage phi29 motor, a homo-pentamer that translocates DNA in cycles composed of alternating dwells and bursts (1). We now aim to investigate the behavior of the motor at different stages of packaging, as the motor needs to overcome increasing amounts of internal pressure generated by the compressed dsDNA. We find that the effect of the internal pressure on the motor dynamics is more complex than simply exerting an opposing force to packaging. Through a detailed analysis using ultra-high-resolution DNA translocation data, we find that the internal pressure affects multiple kinetic transitions in the mechanochemical cycle, including events in both the dwell phase and the burst phase. This analysis allows us to make an accurate estimation of the internal force as a function of DNA filling, which is important for the understanding of the DNA organization inside the capsid and the ejection energetics. Remarkably, the motor changes its step size and displays a new class of long-lived pauses towards the completion of packaging. Finally, we determine the structural elements in the packaging complex that are responsible for the signal transduction from the capsid to the motor.(1) G. Chistol∗, S. Liu∗, C. L. Hetherington, J. R. Moffitt, S. Grimes, P. J. Jardine, C. Bustamante, “High Degree of Coordination and Division of Labor Among Subunits in a Homomeric Ring ATPase”, Cell, in press (2012).

Interaction between FliJ and FlhA, Components of the Bacterial Flagellar Type III Export Apparatus

A soluble protein, FliJ, along with a membrane protein, FlhA, plays a role in the energy coupling mechanism for bacterial flagellar protein export. The water-soluble FliH(X)-FliI(6) ATPase ring complex allows FliJ to efficiently interact with FlhA. However, the FlhA binding site of FliJ remains unknown. Here, we carried out genetic analysis of a region formed by well-conserved residues-Gln38, Leu42, Tyr45, Tyr49, Phe72, Leu76, Ala79, and His83-of FliJ. A structural model of the FliI(6)-FliJ ring complex suggests that they extend out of the FliI(6) ring. Glutathione S-transferase (GST)-FliJ inhibited the motility of and flagellar protein export by both wild-type cells and a fliH-fliI flhB(P28T) bypass mutant. Pulldown assays revealed that the reduced export activity of the export apparatus results from the binding of GST-FliJ to FlhA. The F72A and L76A mutations of FliJ significantly reduced the binding affinity of FliJ for FlhA, thereby suppressing the inhibitory effect of GST-FliJ on the protein export. The F72A and L76A mutations were tolerated in the presence of FliH and FliI but considerably reduced motility in their absence. These two mutations affected neither the interaction with FliI nor the FliI ATPase activity. These results suggest that FliJ(F72A) and FliJ(L76A) require the support of FliH and FliI to exert their export function. Therefore, we propose that the well-conserved surface of FliJ is involved in the interaction with FlhA.

In vivo three-dimensional structural analysis of cilia by cryo-electron tomography

Ciliary bending motion is driven by sliding of microtubule doublets with dynein molecular motors. Dyneins form two complexes: inner and outer dynein arms. The inner arm is supposed to determine the waveform, while the outer arm is likely an accelerator. However, the mechanism to integrate linear motion of dynein into well-orchestrated axonemal bending is still to be investigated. We are analyzing 3D structure of cilia from Tetrahymena, Chlamydomonas, sea urchin sperm and mouse trachea, to reveal the novel architecture of cilia. By comparing 3D structure of Chlamydomonas mutant axonemes, we located all the three outer arm dyneins and the eight inner arm dyneins (Bui et al. (2008) J. Cell Biol. 183, 923). Dyneins, which consist of the N-terminal tail, the ATPase ring, the stalk and the microtubule binding domain, undergo conformational change during the phosphate release: the ATPase ring shifts 8nm toward the distal end, which can explain the minus-end driven motor activity of dynein. Interestingly, in vivo, some dyneins change their conformations, while the other stay in the apo form, suggesting negative cooperativity among dyneins and bending mechanism caused by torsion at the boundary between dynein in the apo form and in the nucleotide-binding form (Movassagh et al. (2010) Nat. Struct. Mol. Biol. 17, 761). We also reconstructed 3D structure of the radial spoke, regulatory complex of 23 proteins (Pigino et al. (2011) J. Cell Biol. 195, 673). Each radial spoke has pseudo-two fold symmetry, indicating dimeric nature. Our 3D structure implies the assembly pathway of the radial spoke. http://www.psi.ch/lbr/takashi-ishikawa

High Degree of Coordination and Division of Labor among Subunits in a Homomeric Ring ATPase

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

Structural Basis for Specific Recognition of Rpt1p, an ATPase Subunit of 26 S Proteasome, by Proteasome-dedicated Chaperone Hsm3p

The 26 S proteasome is a 2.5-MDa molecular machine that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of 6 ATPase (Rpt) and 13 non-ATPase (Rpn) subunits. Multiple proteasome-dedicated chaperones facilitate the assembly of the proteasome, but little is known about the detailed mechanisms. Hsm3, a 19 S RP dedicated chaperone, transiently binds to the C-terminal domain of the Rpt1 subunit and forms a tetrameric complex, Hsm3-Rpt1-Rpt2-Rpn1, during maturation of the ATPase ring of 19 S RP. To elucidate the structural basis of Hsm3 function, we determined the crystal structures of Hsm3 and its complex with the C-terminal domain of the Rpt1 subunit (Rpt1C). Hsm3 has a C-shaped structure that consists of 11 HEAT repeats. The structure of the Hsm3-Rpt1C complex revealed that the interacting surface between Hsm3 and Rpt1 is a hydrophobic core and a complementary charged surface. Mutations in the Hsm3-Rpt1 surface resulted in the assembly defect of the 26 S proteasome. Furthermore, a structural model of the Hsm3-Rpt ring complex and an in vitro binding assay suggest that Hsm3 can bind Rpt2 in addition to Rpt1. Collectively, our results provide the structural basis of the molecular functions of Hsm3 for the RP assembly.

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.

Engineered Dynein Mutants Reveal Minimal Structural and Catalytic Requirements for Processive Motility

Cytoplasmic dynein is a molecular motor responsible for minus-end directed transport along microtubules. In contrast to kinesins and myosins, the detailed mechanism of dynein processivity and force generation remains unclear. Dynein’s structure and evolutionary origin are different from these motors, suggesting unique mechanistic features. In this work, we engineered novel dynein constructs with altered mechanical, chemical and geometric properties to test the roles of rigid linkage between monomers, interaction between the ATPase rings and the proposed linker swing mechanism in maintaining dynein processivity. We found that a rigid linkage between monomers and dimerization through the N-terminal tail domains are not essential for dynein processivity. Instead, processivity minimally requires the linker domain of one monomer with an active ATPase ring to be attached to a partner monomer, which can be replaced by an inert protein retaining the microtubule-binding domain. To understand how one active head could provide motility to an inactive partner, we quantified the force-dependent microtubule release rates of dynein monomers. A clear directional asymmetry for detachment was observed, with significantly faster release towards the minus-end, providing insight into dynein’s directionality. These results led us to a detailed mechanistic model of dynein processivity, directionality and force generation.

The Structure of the Dynein Motor Domain

Dyneins are microtubule-based motor proteins that power ciliary beating, transport intracellular cargos, and help to construct the mitotic spindle. Evolved from ring-shaped hexameric AAA-family adenosine triphosphatases (ATPases), dynein’s large size and complexity have posed challenges for understanding its structure and mechanism. Here, we present a 6 angstrom crystal structure of a functional dimer of two ∼300-kilodalton motor domains of yeast cytoplasmic dynein. The structure reveals an unusual asymmetric arrangement of ATPase domains in the ring-shaped motor domain, the manner in which the mechanical element interacts with the ATPase ring, and an unexpected interaction between two coiled coils that create a base for the microtubule binding domain. The arrangement of these elements provides clues as to how adenosine triphosphate-driven conformational changes might be transmitted across the motor domain.

Complete Characterization of the Mechanochemical Cycle of a Homomeric Ring ATPase

Understanding how multimeric ring ATPases couple chemical transitions to mechanical motion and coordinate their subunits is essential for elucidating the functions of these important molecular machines. Here we used high-resolution optical tweezers to study one such ATPase - the bacteriophage phi29 DNA packaging motor. This pentameric motor is known to translocate DNA by cycling through a dwell phase, in which subunits load ATPs, and a burst phase, in which 10 base pairs of DNA are packaged in four 2.5-base-pair steps. By monitoring packaging in buffers containing various nucleotides and nucleotide analogs, we determined the exact timing of chemical transitions (nucleotide hydrolysis and product release) with respect to the mechanical phases of the motor's cycle. Our results reveal an intricate coordination mechanism with multiple levels of communication between neighboring subunits. ADP release and ATP binding occur in a concerted alternating fashion, with ATP binding to one subunit facilitating the release of ADP in the following subunit. ATP hydrolysis also appears to occur sequentially around the ring through an intersubunit allosteric effect. Moreover, our data suggest that all five subunits of the ring bind and hydrolyze ATP. Only four out of the five hydrolysis events are coupled to DNA translocation, while the fifth subunit consumes ATP to fulfill a regulatory role, resetting the motor for the next mechanochemical cycle. The phi29 motor is able to tolerate a single inactive subunit and still function, albeit at a much slower pace. These new findings allow us to present the most complete mechanochemical model of a homomeric ring ATPase to date, which should provide insight into the operating principles of other ring-shaped ATPases.

Allosteric Regulation of Nucleotide Binding to the Proteasomal ATPases

Protein degradation by the eukaryotic 26S proteasome or the homologous archaeal PAN-20S proteasome complex is a multistep process that requires ATP hydrolysis by the proteasome-associated AAA ATPase complex. However, the mechanisms by which these hexameric ATPases bind and hydrolyze ATP to promote protein breakdown are poorly understood. Although PAN contains six identical ATPase subunits, we found that it exhibits three types of ATP binding sites: 2 high affinity conformations (ATP-binding sites), 2 with lower affinity (ADP-binding sites), and 2 with conformations that do not bind nucleotides. Correlation of ADP off rates with rates of ATP hydrolysis suggests that ADP leaving may be the rate limiting step in hydrolysis. ATP binding to the high and lower affinity sites has distinct functional consequences on the proteasome. With two ATPγS molecules bound, PAN maximally stimulates opening of the gated channel for substrate entry into the 20S proteasome and has a high affinity for the 20S. However, the binding of 4 ATPγS reduces PAN’s affinity for the 20S, which can be explained by steric hindrances in the PAN-20S interface. Because ATP binding drives the association of the C-termini of the ATPase with the 20S and only two ATPase subunits bind ATP for maximal function it’s likely that only two ATPases’ C-termini dock into the 20S at any time and in a predictable pattern mirroring the cyclical pattern of ATP hydrolysis. This observation suggests how the symmetrically mismatched hexameric ATPase ring associates with the heptameric 20S proteasome to regulate substrate degradation.

The Mechanism of Cytoplasmic Dynein Processivity

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. It is currently unknown how the two catalytic head domains interact and move relative to each other during processive movement. We have tracked the relative positions of both heads with nanometer precision and directly observed that the heads move independently along the microtubule. The heads remain widely separated and the stepping behavior of the heads varies as a function of interhead separation. Consistent with a lack of tight coordination, only one active head is sufficient for processive movement and the active head drags its inactive partner head forward. Only a single active ATPase ring is sufficient for processivity, and the linker swing provides required force to drive minus end directed motion. These results show that dynein is the first dimeric motor that moves processively without interhead coordination, a mechanism fundamentally distinct from hand-over-hand motion of kinesin and myosin.

Stalled Proteasomes Are Directly Relieved by P97 Recruitment

The 26 S proteasome is the eukaryotic protease responsible for the degradation of most cellular proteins. As such it accommodates the ability to function under diverse conditions that the cell may encounter. This function is supported by various adaptors that modulate various aspects in protein degradation, these include regulation of substrate delivery, deubiquitination, unfolding, and 20 S gate dilation. Here we show a new functional complex between the P97 and the proteasome that is assembled in response to proteasomal impairment. This entails P97 binding to the 26 S proteasome via the 19 S particle thereby forming an additional hexameric ATPase ring to relieve repression. P97-bound proteasomes showed selective binding toward the Npl4-ufd1 P97 co-factors, indicating a unique cellular role for P97 binding to proteasomes. P97-bound proteasomes display enhanced activity, showing a relief in proteolysis impairment. Our findings place P97 directly in non-ERAD proteasomal functions and establish a new checkpoint in UPS impairment. The ability to modulate proteasome activity and properly respond to protein misfolding, is of great importance in cellular regulation.

How Chromatin Is Remodelled during DNA Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae

Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation–induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin.

Conjugal plasmid transfer inStreptomycesresembles bacterial chromosome segregation by FtsK/SpoIIIE

Conjugation is a major route of horizontal gene transfer, the driving force in the evolution of bacterial genomes. Antibiotic producing soil bacteria of the genus Streptomyces transfer DNA in a unique process involving a single plasmid-encoded protein TraB and a double-stranded DNA molecule. However, the molecular function of TraB in directing DNA transfer from a donor into a recipient cell is unknown. Here, we show that TraB constitutes a novel conjugation system that is clearly distinguished from DNA transfer by a type IV secretion system. We demonstrate that TraB specifically recognizes and binds to repeated 8 bp motifs on the conjugative plasmid. The specific DNA recognition is mediated by helix α3 of the C-terminal winged-helix-turn-helix domain of TraB. We show that TraB assembles to a hexameric ring structure with a central ∼3.1 nm channel and forms pores in lipid bilayers. Structure, sequence similarity and DNA binding characteristics of TraB indicate that TraB is derived from an FtsK-like ancestor protein, suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells.

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA.

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.

Modular assembly of yeast mitochondrial ATP synthase

The mitochondrial ATP synthase (F(1)-F(0) complex) of Saccharomces cerevisiae is a composite of different structural and functional units that jointly couple ATP synthesis and hydrolysis to proton transfer across the inner membrane. In organello, pulse labelling and pulse-chase experiments have enabled us to track the mitochondrially encoded Atp6p, Atp8p and Atp9p subunits of F(0) and to identify different assembly intermediates into which they are assimilated. Surprisingly, these core subunits of F(0) segregated into two different assembly intermediates one of which is composed of Atp6p, Atp8p, at least two stator subunits, and the Atp10p chaperone while the second consists of the F(1) ATPase and Atp9p ring. These studies show that assembly of the ATP synthase is not a single linear process, as previously thought, but rather involves two separate but coordinately regulated pathways that converge at the end stage.