Paul V. Viitanen

Molecular chaperones and protein folding in plants.

Protein folding in vivo is mediated by an array of proteins that act either as ‘foldases’ or ‘molecular chaperones’. Foldases include protein disulfide isomerase and peptidyl prolyl isomerase, which catalyze the rearrangement of disulfide bonds or isomerization of peptide bonds around Pro residues, respectively. Molecular chaperones are a diverse group of proteins, but they share the property that they bind substrate proteins that are in unstable, non-native structural states. The best understood chaperone systems are HSP70/DnaK and HSP60/GroE, but considerable data support a chaperone role for other proteins, including HSP100, HSP90, small HSPs and calnexin. Recent research indicates that many, if not all, cellular proteins interact with chaperones and/or foldases during their lifetime in the cell. Different chaperone and foldase systems are required for synthesis, targeting, maturation and degradation of proteins in all cellular compartments. Thus, these diverse proteins affect an exceptionally broad array of cellular processes required for both normal cell function and survival of stress conditions. This review summarizes our current understanding of how these proteins function in plants, with a major focus on those systems where the most detailed mechanistic data are available, or where features of the chaperone/foldase system or substrate proteins are unique to plants.

Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone.

Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), beta-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a/b binding protein, chloramphenicol acetyltransferase, and pre-beta-lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subunit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.

Alternate Energy-Dependent Pathways for the Vacuolar Uptake of Glucose and Glutathione Conjugates

Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H+gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H+ gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H+ gradient dependence of glucoside uptake. It is concluded that H+gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H+ antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5′ untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.

Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria

Production of biologically active foreign proteins with correct three-dimensional structures is often difficult in bacteria. Recent advances demonstrate that, for some proteins at least, their correct folding and assembly is facilitated by a class of proteins known as molecular chaperones. An understanding of the function of molecular chaperones may assist in the synthesis in bacteria of functional foreign proteins produced by recombinant techniques.

Purification of mammalian mitochondrial chaperonin 60 through in vitro reconstitution of active oligomers

Publisher Summary The chapter presents a study on purification of mammalian mitochondrial chaperonin 60 through in vitro reconstitution of active oligomers. Because of the technical difficulties associated with the mammalian mt-cpn60, functional characterization of the mammalian mt-cpnl0 has largely been restricted to in vitro interactions with GroEL. In this heterologous test system, GroES and the mammalian mt-cpnl 0 are functionally interchangeable by a number of criteria, including the ability to assist GroEL in the facilitation of protein folding. The resounding conclusion from these experiments is that the fundamental mechanism of the GroE-related chaperonins has been highly conserved from bacteria to mitochondria. The chapter describes the purification of monomeric mammalian mt-cpn60 and its subsequent reassembly into functional oligomers using a general approach that has worked well with other GroEL homologs. The chapter describes the purification of monomeric mammalian mitochondrial chaperonin 60, reconstitution of oligomeric mitochondrial chaperonin 60, properties of in vitro -reconstituted particles and several related concepts.

[17] Oligonucleotide-directed site-specific mutagenesis of the 1ac permease of Escherichia coli

Publisher Summary This chapter illustrates that with the recent advent of site-directed mutagenesis, the goal of determining the role of amino acid residues or sequences in the function of a particular protein or enzyme, can be realized. The chapter describes an application of the technique to the lac permease of Escherichia coli. The lac permease is an intrinsic membrane protein, encoded by the lacY gene, that catalyzes symport (cotransport) of β-galactosides with protons. The lacY gene has been cloned and sequenced, and the permease has been purified to homogeneity in a completely functional state. A putative secondary structure model has been proposed based on the circular dichroic spectrum of the purified protein and on the sequential hydropathic character of the amino acid sequence. Although chemical modification of specific amino acid residues in a protein can provide important information, there are drawbacks to this approach. For this reason, oligonucleotide-directed, sitespecific mutageneses to study the structure and function of the lac permease have been utilized.

On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20.

Type I chaperonins are fundamental protein folding machineries that function in eubacteria, mitochondria and chloroplasts. Eubacteria and mitochondria contain chaperonin systems comprised of homo-oligomeric chaperonin 60 tetradecamers and co-chaperonin 10 heptamers. In contrast, the chloroplast chaperonins are heterooligomeric tetradecamers that are composed of two subunit types, alpha and beta. Additionally, chloroplasts contain two structurally distinct co-chaperonins. One, ch-cpn10, is probably similar to the mitochondrial and bacterial co-chaperonins, and is composed of 10 kDa subunits. The other, termed ch-cpn20 is composed of two cpn10-like domains that are held together by a short linker. While the oligomeric structure of ch-cpn10 remains to be elucidated, it was previously suggested that ch-cpn20 forms tetramers in solution, and that this is the functional oligomer. In the present study, we investigated the properties of purified ch-cpn10 and ch-cpn20. Using bifunctional cross-linking reagents, gel filtration chromatography and analytical ultracentrifugation, we show that ch-cpn10 is a heptamer in solution. In contrast, ch-cpn20 forms multiple oligomers that are in dynamic equilibrium with each other and cover a broad spectrum of molecular weights in a concentration-dependent manner. However, upon association with GroEL, only one type of co-chaperonin-GroEL complex is formed.

Purification of recombinant plant and animal GroES homologs: chloroplast and mitochondrial chaperonin 10.

Publisher Summary The chapter presents a study on the purification of recombinant plant and animal GroES homologs focusing on chloroplast and mitochondrial chaperonin 10. The chapter describes large-scale purification schemes for spinach chloroplast cpnl0 (ch-cpnl0) and mouse mitochondrial cpnl0 (mt-cpnl0), two eukaryotic GroES homologs that are available as functional recombinant proteins. Although both proteins can be purified from their natural sources, the yields obtained are insufficient for biochemical and structural studies. In contrast, the two recombinant proteins are readily abundant and easily purified to homogeneity. Purification schemes for recombinant human and rat mt-cpnl0 are also available, and an affinity purification procedure, potentially applicable to all cpnl0 homologs, has been published. Cpn60 and cpnl0 are also both required for protein folding in these organelles. The higher plant ch-cpn10and mammalian mt-cpn10 was initially identified through their abilities to form stable, isolatable complexes with GroEL. The recombinant spinach ch-cpnl0 and mouse mt-cpnl0 are both well expressed in Escherichia coli , at levels exceeding 10% of the total soluble protein. The first step in the purification of the spinach ch-epnl0 uses the cation exchange resin S-Sepharose Fast Flow. The purified eukaryotic GroES homologs are both able to assist GroEL in the in vitro refolding of prokaryotic Rubisco.

Activation and detection of (Pro)mutagenic chemicals using recombinant strains ofStreptomyces griseus

Two recombinant strains ofStreptomyces griseus have been developed to report on the activation of promutagenic° chemicals. This activation is monitored by reversion of the bacterial test strains to a kana-mycin-resistant phenotype. Strain H69 detects point mutations and was reverted at an increased frequency by acetonitrile, 2-aminoanthracene, 1,2-benzanthracene, benzidine, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene, and glycine. The second strain, FS2, detects frame shift mutations and was reverted at an increased frequency by 1,2-benzanthracene, benzidine, and glycine. Compounds such as butylated hydroxytoluene, catechol, chlorobenzene, hydroquinone, potassium chloride, phenol,cis-stilbene,trans-stilbene, and toluene did not elicit positive responses in either strain. In addition, these strains are capable of detecting direct-acting mutagens such asN-methyl-N’-nitrosoguanidine and ICR-191, providing further evidence of their promise for detecting a wider range of mutagens. To our knowledge, this is the first report of bacterial strains capable of activating promutagenic compounds and detecting their mutagenic metabolites without the benefit of an exogenous activation system such as the rodent liver homogenate (S9).