Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy.

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

GroEL/GroES-mediated folding of a protein too large to be encapsulated.

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.

ClpA mediates directional translocation of substrate proteins into the ClpP protease.

The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease. Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH(2) or COOH terminus with fluorescent probes. Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP. We observed for both substrates that energy transfer occurs 2--4 s sooner with the COOH-terminally labeled molecules than with the NH(2)-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.

ATP-bound states of GroEL captured by cryo-electron microscopy.

The chaperonin GroEL drives its protein-folding cycle by cooperatively binding ATP to one of its two rings, priming that ring to become folding-active upon GroES binding, while simultaneously discharging the previous folding chamber from the opposite ring. The GroEL-ATP structure, determined by cryo-EM and atomic structure fitting, shows that the intermediate domains rotate downward, switching their intersubunit salt bridge contacts from substrate binding to ATP binding domains. These observations, together with the effects of ATP binding to a GroEL-GroES-ADP complex, suggest structural models for the ATP-induced reduction in affinity for polypeptide and for cooperativity. The model for cooperativity, based on switching of intersubunit salt bridge interactions around the GroEL ring, may provide general insight into cooperativity in other ring complexes and molecular machines.

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.

Structure and function in GroEL-mediated protein folding.

Recent structural and biochemical investigations have come together to allow a better understanding of the mechanism of chaperonin (GroEL, Hsp60)-mediated protein folding, the final step in the accurate expression of genetic information. Major, asymmetric conformational changes in the GroEL double toroid accompany binding of ATP and the cochaperonin GroES. When a nonnative polypeptide, bound to one of the GroEL rings, is encapsulated by GroES to form a cis ternary complex, these changes drive the polypeptide into the sequestered cavity and initiate its folding. ATP hydrolysis in the cis ring primes release of the products, and ATP binding in the trans ring then disrupts the cis complex. This process allows the polypeptide to achieve its final native state, if folding was completed, or to recycle to another chaperonin molecule, if the folding process did not result in a form committed to the native state.

Expression and kinetic characterization of methylmalonyl-CoA mutase from patients with the mut- phenotype: evidence for naturally occurring interallelic complementation.

L-Methylmalonyl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requiring mitochondrial matrix enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Inherited defects in the gene encoding this enzyme result in the mut forms of methylmalonic acidemia. Expression of mature human MUT cDNA in Escherichia coli at a post-induction cultivation temperature of 12 degrees C, rather than 37 degrees C, led to the folding of the majority of the synthesized protein to a soluble form, with an activity of 0.2-0.3 U/mg protein in the cell-free extract, 10-15 times higher than that in human liver homogenate. Six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V, were detected in MUT cDNA of patients suffering from the mut- form of methylmalonic acidemia, resulting from defective AdoCbl binding. Two (G623R and G717V) had been reported in other patients. Three (G94V, Y231N and R369H) are the first changes in the NH2-terminal part of the enzyme reported to cause the mut- phenotype. Enzymes with the mutations were individually expressed, and their kinetic parameters were generally in accord with published biochemical data from extracts of fibroblasts from these patients. The mutations increased the K(m) for AdoCbl by 40- to 900-fold, while V(max) values varied from 0.2% to nearly 100% of that of wild-type protein. In one case of a doubly heterozygous cell line, however, neither of the constituent mutant enzymes had a K(m) corresponding to the lower of the two estimated from the extract data. This finding may reflect the natural occurrence of interallelic complementation in vivo in this cell line.

Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.

The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.

Clinical heterogeneity and prognosis in combined methylmalonic aciduria and homocystinuria (cblC).

The clbC form of methylmalonic acidaemia is a rare and poorly understood condition which results from impaired biosynthesis of methylcobalamin and adenosylcobalamin. The consequent functional deficiencies of methylmalonyl-CoA mutase and methionine synthase produce both methylmalonic aciduria and homocystinuria. Systemic symptoms and neurological decompensation comprise the clinical phenotype. In an effort to clarify the phenotype and prognosis, we obtained clinical information on 50 patients with methylmalonic acidaemia whose cells had been assigned to the cblC complementation group. We identified two distinct phenotypes; they differed in age of onset, presence of systemic symptoms, type of neurological symptoms, and outcome after diagnosis and treatment. Forty-four patients presented in the first year of life. Feeding difficulties, neurological dysfunction (hypotonia, seizures, developmental delay), and ophthalmological and haematological abnormalities characterized their clinical picture. About one-quarter of those patients died. Survival was associated with neurological impairment; only one infant was neurologically intact at follow-up. Onset in childhood, in contrast, was associated with less severe haematological abnormalities, largely involving the red cell series. Extrapyramidal signs, dementia, delirium or psychosis characterized the neurological findings. Survival, with mild to moderate disability in some, was typical in patients with later onset. Treatment in both groups included hydroxycobalamin, betaine and carnitine; complete normalization of biochemical parameters was rare.

GroEL-mediated protein folding.

I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks.

Release of both native and non-native proteins from a cis-only GroEL ternary complex.

Protein folding by the double-ring chaperonin GroEL is initiated in cis ternary complexes, in which polypeptide is sequestered in the central channel of a GroEL ring, capped by the co-chaperonin GroES. The cis ternary complex is dissociated (half-life of approximately 15 s) by trans-sided ATP hydrolysis, which triggers release of GroES. For the substrate protein rhodanese, only approximately 15% of cis-localized molecules attain their native form before hydrolysis. A major question concerning the GroEL mechanism is whether both native and non-native forms are released from the cis complex. Here we address this question using a 'cis-only' mixed-ring GroEL complex that binds polypeptide and GroES on only one of its two rings. This complex mediates refolding of rhodanese but, as with wild-type GroEL, renaturation is quenched by addition of mutant GroEL 'traps', which bind but do not release polypeptide substrate. This indicates that non-native forms are released from the cis complex. Quenching of refolding by traps was also observed under physiological conditions, both in undiluted Xenopus oocyte extract and in intact oocytes. We conclude that release of non-native forms from GroEL in vivo allows a kinetic partitioning among various chaperones and proteolytic components, which determines both the conformation and lifetime of a protein.

Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase.

Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.

Putting a lid on protein folding: structure and function of the co-chaperonin, GroES.

The co-chaperonin GroES is an essential partner in protein folding mediated by the chaperonin, GroEL. Two recent crystal structures of GroES provide a structural basis to understand how GroES forms the lid on the folding-active cis ternary complex, and how the GroEL-GroES complex enhances folding.

Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction.

Recent studies of GroE-mediated protein folding indicate that substrate proteins are productively released from a cis ternary complex in which the nonnative substrate is sequestered within the GroEL channel underneath GroES. Here, we examine whether protein folding can occur in this space. Stopped-flow fluorescence anisotropy of a pyrene-rhodanese-GroEl complex indicates that addition of GroES and ATP (but not ADP) leads to a rapid change in substrate flexibility at GroEL. Strikingly, when GroES release is blocked by the use of either a nonhydrolyzable ATP analog or a single-ring GroEL mutant, substrates complete folding while remaining associated with chaperonin. We conclude that the cis ternary complex, in the presence of ATP, is the active state intermediate in the GroE-mediated folding reaction: folding is initiated in this state and for some substrates may be completed prior to the timed release of GroES triggered by ATP hydrolysis.

Mechanism of GroEL action: productive release of polypeptide from a sequestered position under GroES.

The chaperonin GroEL is a large, double-ring structure that, together with ATP and the cochaperonin GroES, assists protein folding in vivo. GroES forms an asymmetric complex with GroEL in which a single GroES ring binds one end of the GroEL cylinder. Cross-linking studies reveal that polypeptide binding occurs exclusively to the GroEL ring not occupied by GroES (trans). During the folding reaction, however, released GroES can rebind to the GroEL ring containing polypeptide (cis). The polypeptide is held tightly in a proteolytically protected environment in cis complexes, in the presence of ADP. Single turnover experiments with ornithine transcarbamylase reveal that polypeptide is productively released from the cis but not the trans complex. These observations suggest a two-step mechanism for GroEL-mediated folding. First, GroES displaces the polypeptide from its initial binding sites, sequestering it in the GroEL central cavity. Second, ATP hydrolysis induces release of GroES and productive release of polypeptide.