Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean

Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.     

Phosphorylation in vivo of Ribosomes in Tetrahymena pyriformis.

Phosphorylation of ribosomal proteins in vivo was studied in exponentially growing and starved cells of the ciliated protozoan, Tetrahymena pyriformis. No phosphorylation of ribosomal proteins could be demonstrated in cells growing exponentially in complex nutrient media. However, when Tetrahymena cells were transferred into a non-nutrient medium, pronounced phosphorylation of a single ribosomal protein was observed. During two-dimensional polyacrylamide gel electrophoresis the phosphorylated ribosomal protein migrated in a manner virtually identical to that of the phosphorylated ribosomal protein S6 of rat liver. The phosphorylated ribosomal protein has a molecular weight of 38000 as estimated by dodecylsulfate polyacrylamide gel electrophoresis. Thus, the phosphorylated ribosomal protein found in starved Tetrahymena is apparently homologous with the ribosomal protein which is predominantly phosphorylated in higher eukaryotes. When phosphorylated ribosomes were dissociated by treatment with high concentration of KCl, the phosphorylated protein was found only on the small subunit. If dissociation was achieved by dialysis against a buffer low in MgCl2, the phosphorylated protein was distributed almost equally between the two subunits. This indicates that the phosphorylated ribosomal protein is located at the interface between the two subunits.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Cytosolic chaperonin is up-regulated during cell growth. Preferential expression and binding to tubulin at G(1)/S transition through early S phase

The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone assisting in the folding of actin, tubulin, and other cytosolic proteins. The expression levels of CCT subunits varied among seven mouse cell lines tested but showed a close correlation with growth rate. Both the CCT protein and mRNA levels in the human promyelolytic cell HL60 decreased concomitant with growth arrest during differentiation. More rapid decrease in CCT level occurred when the mouse interleukin (IL)-3-dependent myeloid DA3 cells were starved for IL-3. Readdition of IL-3 caused rapid resumption of CCT synthesis during synchronous growth: the maximum CCT protein and mRNA levels were observed at G(1)/S transition through early S phase. The turnover rate of CCT was nearly constant regardless of growth. Gel filtration and immunoprecipitation analyses indicated that CCT in vivo is associated with tubulin at early S phase, but not at G(0)/G(1) phase. These results demonstrated that CCT expression is strongly up-regulated during cell growth especially from G(1)/S transition to early S phase and is primarily controlled at the mRNA level. CCT appears to play important roles for cell growth by assisting in the folding of tubulin and other proteins.     

Growth factor regulated expression of poly(A) binding protein messenger RNA

A 72,000 mol wt protein designated PABP binds to the poly(A)+ track of messenger RNAs with high affinity and has been suggested to play an important role in mRNA metabolism in eucaryotic cells. We have employed a human PABP cDNA probe to study the expression of this gene at the mRNA level in BALB/c3T3 mouse cells under different growth conditions and in exponentially growing HeLa cells throughout the cell division cycle. We describe experiments which establish that in BALB/c3T3 cells the expression of this gene is growth factor regulated. Moreover, the gene behaves like a primary response gene in that its induction in quiescent cells does not require the prior synthesis of other growth factor-regulated proteins. In exponentially growing HeLa cells PABP mRNA is expressed throughout the cell division cycle indicating that the expression of this gene is not limited to a specific phase of the cell cycle.     

Subunits of the eukaryotic cytosolic chaperonin CCT do not always behave as components of a uniform hetero-oligomeric particle

The chaperonin CCT is an hetero-oligomeric molecular chaperone complex. Studies in yeast suggest each of its eight gene products are required for its major identified functions in producing native tubulins and actins. However, it is unclear whether these eight components always form a single particle, covering all functions, or else can also exist as heterogeneous mixtures and/or free subunits in cells. Using mouse P19 embryonal carcinoma cells, which divide rapidly, yet in retinoic acid adopt a neuronal phenotype, admixed with occasional (approximately 10%) fibroblast-like cells, together with a panel of peptide-specific antibodies raised to 7 of the 8 CCT subunits we show that; (1) adoption of a post mitotic phenotype is accompanied by reduced CCT protein expression, significantly more so for CCTbeta, CCTdelta, CCTepsilon, and CCTtheta than for CCTalpha (TCP-1), CCTgamma and CCTzeta; (2) CCTalpha is detected preferentially over other subunits in neurites of P19 neurons; (3) small amounts of CCTalpha and gamma are localised in nuclei (i.e. are not exclusively cytoplasmic), selectively so compared with other subunits; (4) numerous cytosolic foci exist in the cytoplasm which, when detected by double immunofluorescence can contain only one of the subunits probed for; (5) while a "core" chaperonin particle can be immunoprecipitated under native conditions, epitope access is modified both by nucleotides and by non-CCT co-precipitating proteins. Collectively, these findings indicate that CCT subunits are not only components of the hetero-oligomeric chaperonin particle but exist as significant populations of free subunits or smaller oligomers in cells.     

Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of alpha, delta and zeta-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the alpha and delta subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower alpha and delta subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the alpha and delta subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of alpha and delta subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells.     

Novel gene hKCNE4 slows the activation of the KCNQ1 channel

The KCNE genes encode small, single transmembrane domain peptides that associate with pore-forming potassium channel subunits to form mixed complexes with unique characteristics. We have identified a novel member of the human KCNE gene family, hKCNE4. The hKCNE4 gene encodes 170 amino acid protein and is localized to chromosome 2q35-36. The protein sequence shows 90% homology to mouse KCNE4 and 38% identity to human KCNE1. Northern blot analysis revealed that hKCNE4 is expressed strongly in heart, skeletal muscle, and kidney, less in placenta, lung, and liver, and weakly in brain and blood cells. Electrophysiological study showed that hKCNE4 modulates the activation of the KCNQ1 channel.     

Activation of the Bacillus subtilis hut operon at the onset of stationary growth phase in nutrient sporulation medium results primarily from the relief of amino acid repression of histidine transport.

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.     

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCT? in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.     

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.