Stepwise Disassembly Research Articles

Elucidation of Spatial Cooperativity in Chemo-Immunotherapy by a Sequential Dual-pH-Responsive Drug Delivery System.

Combining immune checkpoint blockade with chemotherapy through nanotechnology is promising in terms of safety and efficacy. However, the distinct subcellular distribution of each ingredient's action site makes it challenging to acquire an optimal synergism. Herein, a dual-pH responsive hybrid polymeric micelle system, HNP(αPDL16.9, Dox5.3), is constructed as a proof-of-concept for the spatial cooperativity in chemo-immunotherapy. HNP retains the inherent pH-transition of each polymer, with stepwise disassembly under discrete pH thresholds. Within weakly acidic extracellular tumor environment, αPDL1 is first released to block the checkpoint on cell membranes. The remaining intact Doxorubicin-loaded micelle NP(Dox)5.3 displays significant tropism toward tumor cells and releases Dox upon lysosomal pH for efficient tumor immunogenic cell death without immune toxicity. This sequential-released pattern boosts DC activation and primes CD8+ T cells, leading to enhanced therapeutic performance than single agent or an inverse-ordered combination in multiple murine tumor models. Using HNP, the indispensable role of conventional type 1 DC (cDC1) is identified in chemo-immunotherapy. A co-signature of cDC1 and CD8 correlates with cancer patient survival after neoadjuvant Pembrolizumab plus chemotherapy in clinic. This study highlights spatial cooperativity of chemo- and immuno-agents in immunoregulation and provides insights into the rational design of drug combination for future nanotherapeutics development.

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division.

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA-adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodeling.

Different pKa Shifts of Internal GLU8 in Human β-Endorphin Amyloid Revealing a Coupling of Internal Ionization and Stepwise Fibril Disassembly.

As a functional amyloid, human β-endorphin amyloid fibril features a β-solenoid conformation and store peptide hormones within acidic secretory granules, which would be released into the blood through fibril disassembly when the cellular milieu pH increases from acidic to neutral level on exocytosis. To gain detailed atomic mechanism of β-endorphin amyloid fibrils' pH-responsive disassembly, we conduct constant pH molecular dynamics simulations to investigate the structural and dynamical properties of β-endorphin amyloid fibrils in experiencing the environmental pH changes. Our results demonstrate a clear pKa shift of the internal ionizable residue of GLU8, and this shift becomes even more pronounced when it is buried more deeply in the amyloid fibrils. The unusual pKa of GLU8 reveals that its protonation state changes from the protonated state in the acidic secretory granule to the deprotonated state in the neutral pH conditions in the blood, where the deprotonation of GLU8 leads to unfavorable interactions within the hydrophobic core of the amyloid and subsequent fibril disassembly. The different pKa shifts of GLU8 relative to its positions in the amyloid fibril indicate that the β-endorphin amyloid fibril disassembly is a stepwise process, accounting for the experimental observation that the disassembly always initiates from the outermost layer. This study reveals the critical role of the protonation state of GLU8 in amyloid fibrils' pH-responsive disassembly, and provides clear insights for understanding the structural transitions of amyloids in hormone secretion. This study also provides theoretical basis for designing pH-sensitive biological tools for specific use with precise positioning of ionizable residues into the hydrophobic interior of proteins.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

SUMMARYGABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Insights into the Basal Activity and Activation Mechanism of the β1 Adrenergic Receptor Using Native Mass Spectrometry.

In the absence of orthosteric ligands, most G protein-coupled receptors (GPCRs) exist in an equilibrium of different conformational states. This equilibrium is shifted by an agonist towards the active state or by an inverse agonist towards the inactive state. The basal activity of the receptor, and its ability to activate intracellular signaling pathways, is defined by the probability that a fraction of the receptor adopts the active state in the absence of ligand. Despite breakthroughs in native MS of membrane proteins, GPCR-transducing complexes have not been studied by this approach until very recently. Here, we investigated different conformational states of the turkey β1 adrenergic receptor (tβ1AR) in complex with two transducing partners: a G protein mimicking nanobody, Nb80, and an engineered truncated Gs protein (miniGs), in the presence of the full agonist isoprenaline by native MS. Interestingly, complex formation with both transducing partners was also observed in the absence of agonist, and allowed us to quantify basal activity of tβ1AR. We followed the stepwise disassembly of the transducing complexes by increasing the concentration of the inverse agonist S32212 in the presence of a constant concentration of isoprenaline. This allowed us to determine the relative binding affinity of S32212 in comparison to isoprenaline by native MS. Our approach provides a fast and sensitive way to detect complexes, study their stability in the presence of different ligands, and determine relative ligand affinities. Native mass spectrometry thus has the potential to become a useful tool to screen for orthosteric and allosteric GPCR drugs. Graphical Abstract.

Mitotic entry drives replisome disassembly at stalled replication forks

The disassembly of eukaryotic replisome during replication termination is mediated by CRL-dependent poly-ubiquitylation of Mcm7 and p97 segregase. The replisome also disassembles at stalled or collapsed replication forks under certain stress conditions, but the underlying mechanism is poorly understood. Here, we discovered a novel pathway driving stepwise disassembly of the replisome at stalled replication forks after forced entry into M-phase using Xenopus egg extracts. This pathway was dependent on M-CDK activity and K48- and K63-linked poly-ubiquitylation but not on CRL and p97, which is different from known pathways. Furthermore, this pathway could not disassemble converged replisomes whose Mcm7 subunit had been poly-ubiquitylated without p97. These results suggest that there is a distinctive pathway for replisome disassembly when stalled replication forks persist into M-phase.

The Loop Formed by Residues 340 to 343 of Reovirus μ1 Controls Entry-Related Conformational Changes.

Reovirus particles are covered with 200 μ1/σ3 heterohexamers. Following attachment to cell surface receptors, reovirus is internalized by receptor-mediated endocytosis. Within the endosome, particles undergo a series of stepwise disassembly events. First, the σ3 protector protein is degraded by cellular proteases to generate infectious subviral particles (ISVPs). Second, the μ1 protein rearranges into a protease-sensitive conformation to generate ISVP*s and releases two virus-encoded peptides, μ1N and Φ. The released peptides promote delivery of the genome-containing core by perforating the endosomal membrane. Thus, to establish a productive infection, virions must be stable in the environment but flexible to disassemble in response to the appropriate cellular cue. The reovirus outer capsid is stabilized by μ1 intratrimer, intertrimer, and trimer-core interactions. As a consequence of ISVP-to-ISVP* conversion, neighboring μ1 trimers unwind and separate. Located within the μ1 jelly roll β barrel domain, which is a known regulator of ISVP* formation, residues 340 to 343 form a loop and have been proposed to facilitate viral entry. To test this idea, we generated recombinant reoviruses that encoded deletions within this loop (Δ341 and Δ342). Both deletions destabilized the outer capsid. Notably, Δ342 impaired the viral life cycle; however, replicative fitness was restored by an additional change (V403A) within the μ1 jelly roll β barrel domain. In the Δ341 and Δ342 backgrounds, V403A also rescued defects in ISVP-to-ISVP* conversion. Together, these findings reveal a new region that regulates reovirus disassembly and how perturbing a metastable capsid can compromise replicative fitness.IMPORTANCE Capsids of nonenveloped viruses are composed of protein complexes that encapsulate, or form a shell around, nucleic acid. The protein-protein interactions that form this shell must be stable to protect the viral genome but also sufficiently flexible to disassemble during cell entry. Thus, capsids adopt conformations that undergo rapid disassembly in response to a specific cellular cue. In this work, we identify a new region within the mammalian orthoreovirus outer capsid that regulates particle stability. Amino acid deletions that destabilize this region impair the viral replication cycle. Nonetheless, replicative fitness is restored by a compensatory mutation that restores particle stability. Together, this work demonstrates the critical balance between assembling virions that are stable and maintaining conformational flexibility. Any factor that perturbs this balance has the potential to block a productive infection.

Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut.

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.

Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus.

Tight confinement of naked genomes within some viruses results in high internal pressure that facilitates their translocation into the host. Adenovirus, however, encodes histone-like proteins that associate with its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation decreases core condensation and primes the virion for proper uncoating via unidentified mechanisms. Here we open individual, mature and immature adenovirus cages to directly probe the mechanics of their chromatin-like cores. We find that immature cores are more rigid than the mature ones, unveiling a mechanical signature of their condensation level. Conversely, intact mature particles demonstrate more rigidity than immature or empty ones. DNA-condensing polyamines revert the mechanics of mature capsid and cores to near-immature values. The combination of these experiments reveals the pressurization of adenovirus particles induced by maturation. We estimate a pressure of ∼30 atm by continuous elasticity, which is corroborated by modeling the adenovirus mini-chromosome as a confined compact polymer. We propose this pressurization as a mechanism that facilitates initiating the stepwise disassembly of the mature particle, enabling its escape from the endosome and final genome release at the nuclear pore.

Structures of intermediates during RES complex assembly.

The action of the spliceosome depends on the stepwise cooperative assembly and disassembly of its components. Very strong cooperativity was observed for the RES (Retention and Splicing) hetero-trimeric complex where the affinity from binary to tertiary interactions changes more than 100-fold and affects RNA binding. The RES complex is involved in splicing regulation and retention of not properly spliced pre-mRNA with its three components—Snu17p, Pml1p and Bud13p—giving rise to the two possible intermediate dimeric complexes Pml1p-Snu17p and Bud13p-Snu17p. Here we determined the three-dimensional structure and dynamics of the Pml1p-Snu17p and Bud13p-Snu17p dimers using liquid state NMR. We demonstrate that localized as well as global changes occur along the RES trimer assembly pathway. The stepwise rigidification of the Snu17p structure following the binding of Pml1p and Bud13p provides a basis for the strong cooperative nature of RES complex assembly.

Serotonin Receptor Agonist 5-Nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry.

Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development. Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.

Heat- and light-induced detachment of the light-harvesting antenna complexes of photosystem I in isolated stroma thylakoid membranes

The multisubunit pigment–protein complex of photosystem I (PSI) consists of a core and peripheral light-harvesting antenna (LHCI). PSI is thought to be a rather rigid system and very little is known about its structural and functional flexibility. Recent data, however, suggest LHCI detachment from the PSI supercomplex upon heat and light treatments. Furthermore, it was suggested that the splitting off of LHCI acts as a safety valve for PSI core upon photoinhibition (Alboresi et al., 2009).In this work we analyzed the heat- and light-induced reorganizations in isolated PSI vesicles (stroma membrane vesicles enriched in PSI). Using differential scanning calorimetry we revealed a stepwise disassembly of PSI supercomplex above 50°C. Circular dichroism, sucrose gradient centrifugation and 77K fluorescence experiments identified the sequence of events of PSI destabilization: 3min heating at 60°C or 40min white light illumination at 25°C resulted in pronounced Lhca1/4 detachment from the PSI supercomplex, which is then followed by the degradation of Lhca2/3.The similarity of the main structural effects due to heat and light treatments supports the notion that thermo-optic mechanism, structural changes induced by ultrafast local thermal transients, which has earlier been shown to be responsible for structural changes in the antenna system of photosystem II, can also regulate the assembly and functioning of PSI antenna.

Janus Nanodisc of Diblock Copolymers

Janus nanodiscs of diblock copolymers are prepared by stepwise disassembly of PS-b-P4VP disc-stacked particles. The Janus nanodiscs are uniform in thickness and regular in contour. By preferential growth of functional materials at the positively charged P4VP side, the composition, microstructure, and performance of the Janus nanodiscs are tunable.

Acid-induced disassemblable nanoparticles based on cyclic benzylidene acetal-functionalized graft copolymer via sequential RAFT and ATRP polymerization

Polymeric nanoparticles assembled from the amphiphilic graft copolymer with cyclic benzylidene acetal-functionalized backbone and short poly(2-hydroxyethyl methacrylate) side chains can remain structurally stable at pH 7.4, but undergo stepwise disassembly in mildly acidic conditions due to the acid-triggered cleavage of the acetals, providing a promising nanocarrier for cancer therapy.

Reovirus Cell Entry Requires Functional Microtubules

ABSTRACTMammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells.

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

Transport to Late Endosomes Is Required for Efficient Reovirus Infection

Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.

PH-Dependent Structures of Ferritin and Apoferritin in Solution: Disassembly and Reassembly

The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40-10.0, and the ferrihydrite core was stable over the pH range 2.10-10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.

Src Kinase Mediates Productive Endocytic Sorting of Reovirus during Cell Entry

Reovirus cell entry is initiated by viral attachment to cell surface glycans and junctional adhesion molecule A. Following receptor engagement, reovirus is internalized into cells by receptor-mediated endocytosis using a process dependent on β1 integrin. Endocytosed virions undergo stepwise disassembly catalyzed by cathepsin proteases, followed by endosomal membrane penetration and delivery of transcriptionally active core particles into the cytoplasm. Cellular factors that mediate reovirus endocytosis are poorly defined. We found that both genistein, a broad-spectrum tyrosine kinase inhibitor, and PP2, a specific Src-family kinase inhibitor, diminish reovirus infectivity by blocking a cell entry step. Although neither inhibitor impedes internalization of reovirus virions, both inhibitors target virions to lysosomes. Reovirus colocalizes with Src during cell entry, and reovirus infection induces phosphorylation of Src at the activation residue, tyrosine 416. Diminished Src expression by RNA interference reduces reovirus infectivity, suggesting that Src is required for efficient reovirus entry. Collectively, these data provide evidence that Src kinase is an important mediator of signaling events that regulate the appropriate sorting of reovirus particles in the endocytic pathway for disassembly and cell entry.

Disassembly intermediates of RbsD protein remain oligomeric despite the loss of an intact secondary structure

Many proteins exist as homo-oligomers in living organisms wherein the change of oligomeric status apparently serves as an effective means for modulating their biological activities. We have previously reported that the homo-decameric RbsD from Escherichia coli undergoes stepwise disassembly and non-stepwise reassembly. Here the structural status of the urea-induced RbsD disassembly intermediates was examined, mainly using urea-containing polyacrylamide gel electrophoresis and chemical cross-linking. Such intermediates were found to remain oligomeric while losing their intact secondary structures. Such disassembly intermediates were able to effectively refold when the concentration of the urea denaturant was reduced to a lower level, or to refold/reassemble into the native decamers when urea was completely removed, as detected by non-denaturing polyacrylamide gel electrophoresis. These novel observations strongly suggest that the assembly of oligomeric proteins may occur before the completion of subunit folding.