Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast.

Members of the Hsp60 chaperonin family, such as Escherichia coli GroEL/S and the eukaryotic cytosolic chaperonin complex, TRiC (TCP ring complex), are double toroid complexes capable of assisting the folding of proteins in vitro in an ATP-dependent fashion. TRiC differs from the GroEL chaperonin in that it has a hetero rather than homo-oligomeric subunit composition and lacks a GroES-like regulatory cofactor. We have established greater than 57% identity between a protein encoded by the TCP20 gene from a human cDNA library and the newly identified protein encoded by the TCP20 gene located on the right arm of chromosome IV of the yeast Saccharomyces cerevisiae. These Tcp20 proteins showed approximately 30% identity to Tcp1, a known subunit of TRiC. Gel filtration, followed by Western analysis of purified bovine testis TRiC with a Tcp20-specific antibody, indicated that Tcp20 is a subunit of the hetero-oligomeric TRiC. Gene disruption experiments showed that TCP20, like TCP1, is an essential gene in yeast, consistent with the view that TRiC is required for folding of key proteins. The amino acid sequence similarities and the derived evolutionary relationships established that the human and yeast Tcp20 proteins represent members of a new family of subunits of TRiC chaperonins.

Phorbol ester-induced membrane proteins in chronic leukemic B-lymphocytes. Candidate proteins for the L-system amino acid transporter.

Chronic lymphocytic leukemia (CLL) B-lymphocytes have markedly diminished membrane L-system amino acid transport as compared with normal mature B- and T-lymphocytes. L-system functional recovery is induced in CLL B-cells by the maturational agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). The studies reported here extend the analysis of CLL B-cell maturation by comparing membrane protein expression in untreated and TPA-treated CLL B-cells, with the identification of candidate proteins for the L-system transporter. Cell membrane proteins of resting and TPA-treated CLL B-lymphocytes were studied using ultrahigh resolution giant two-dimensional gel electrophoresis. Cellular proteins were metabolically labeled with [35S]methionine, and, in separate experiments, membrane proteins were photoaffinity labeled with [125I] iodoazidophenylalanine, an amino acid transporter by the L-system and which binds at or near the L-system transport carrier. In a partially purified membrane preparation, approximately 1400 proteins were identified by metabolic labeling. Following TPA treatment for 17 h, 14 new metabolically labeled membrane proteins were identified, and five of these also were labeled by the L-system photoprobe. Photolabeling of four of these proteins was inhibited by an excess of the L-system prototype amino acid, 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid. Given these labeling characteristics, one or more of these four proteins may be related to the L-system amino acid transport carrier.

A difference in the rate of ribosomal elongation balances the synthesis of eukaryotic translation initiation factor (eIF)-2 alpha and eIF-2 beta.

Eukaryotic translation initiation factor 2 (eIF-2) is a heterotrimer composed of three subunits designated alpha, beta, and gamma. These proteins exist in equimolar amounts in the cell and have not been detected as isolated subunits. Our research examines the basis of their balanced synthesis. Northern analysis of K562 cell mRNA revealed that eIF-2 beta was five times more abundant than eIF-2 alpha. However, immunoprecipitation of pulse-labeled K562 cells showed an equimolar rate of synthesis of eIF-2 alpha and -beta despite the 5-fold difference in the size of their mRNA pools. Addition of equal amounts of synthetic capped mRNA for eIF-2 alpha and eIF-2 beta to an in vitro translation reaction produced five times more eIF-2 alpha protein than eIF-2 beta. Determination of the polysome profile for alpha and beta mRNA in K562 cells indicated eIF-2 alpha was translated more efficiently than eIF-2 beta. Substitution of either the initiation codon context or the leader of the beta mRNA for that of alpha had only a minor effect on the translational efficiency of beta. Comparison of the rate of ribosomal elongation for the two mRNAs indicated that ribosomes associated with the beta mRNA elongate at a rate 4-fold less than that of eIF-2 alpha. Thus, the balanced translation of alpha and beta mRNA is primarily the result of a 4-fold difference in the rate of ribosomal elongation.

A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast.

In Saccharomyces cerevisiae, phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2) by protein kinase GCN2 stimulates translation of GCN4 mRNA. In mammalian cells, phosphorylation of eIF-2 alpha inhibits the activity of eIF-2B, the GDP-GTP exchange factor for eIF-2. We present biochemical evidence that five translational regulators of GCN4 encoded by GCD1, GCD2, GCD6, GCD7, and GCN3 are components of a protein complex that stably interacts with eIF-2 and represents the yeast equivalent of eIF-2B. In vitro, this complex catalyzes guanine nucleotide exchange on eIF-2 and overcomes the inhibitory effect of GDP on formation of eIF-2.GTP.Met-initiator tRNA(Met) ternary complexes. This finding suggests that mutations in GCD-encoded subunits of the complex derepress GCN4 translation because they mimic eIF-2 alpha phosphorylation in decreasing eIF-2B activity. Our results indicate that translational control of GCN4 involves a reduction in eIF-2B function, a mechanism used in mammalian cells to regulate total protein synthesis in response to stress.

Regulation of eukaryotic translation initiation factor expression during T-cell activation.

Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. Upon mitogenic stimulation the rate of protein synthesis increases 10-fold. We have studied the role of eIF-2 and eIF-4 alpha (eIF-4E) expression in the mechanism of translational activation. During this period, the levels of eIF-2 alpha and eIF-4 alpha mRNA increase some 50-fold. Similar to the increase in ribosomes and mRNA, the number of eIF-2 alpha, eIF-2 beta, and eIF-4 alpha molecules per cell also increase 2-3-fold. This suggests that in addition to an increase in the pool size of translational components, an additional mechanism exists which results in an increased efficiency of factor utilization. We have looked at initiation factor phosphorylation. We find that eIF-2 alpha does not undergo significant changes in its phosphorylation state nor is there a change in the efficiency of eIF-2 utilization. However, there is a rapid increase in the phosphorylation state of eIF-4 alpha which correlates with the rapid increase in translational activity. It thus appears there are 2 distinct components responsible for the translational activation of quiescent T-cells during mitogenic stimulation. The first is the phosphorylation of eIF-4 alpha, with a concomitant increase in the efficiency of eIF-4 alpha utilization. The second is an increase in the pool sizes of eIF-2 and eIF-4 alpha.

Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.

Increased eIF-2 alpha expression in mitogen-activated primary T lymphocytes.

G0 human T cells synthesize protein at low rates and contain very low levels of eIF-2 alpha mRNA. eIF-2 alpha plays a pivotal role in the earliest regulated steps of translation initiation. We examined eIF-2 alpha gene expression in normal human T cells stimulated with PHA. Nuclear run-on assays indicate low rates of eIF-2 alpha gene transcription in G0 cells and these change 2-fold with PHA treatment. Actinomycin D chase experiments show that the t1/2 of eIF-2 alpha mRNA is similar in G0 and PHA-treated T cells. Analysis of nuclear RNA with probes specific for eIF-2 alpha intron sequences shows that increased eIF-2 alpha expression after PHA treatment is largely due to intranuclear stabilization of the primary transcript. The increase in eIF-2 alpha mRNA does not require new protein synthesis. Hence, expression of this gene appears to be a part of the primary response program of T cells when they are exposed to mitogen.

Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA.

DNA binding of nonreactive metabolite models derived from benz(a)anthracene was studied. The molecules investigated include 1,2,3,4-tetrahydrobenz(a)anthracene (1), 5,6-dihydrobenz(a)anthracene (2), and 8,9,10,11-tetrahydrobenz(a)anthracene (3), as well as anthracene and phenanthrene. Measurements of the effects of DNA binding upon fluorescence intensities and fluorescence lifetimes indicate that molecules 1 and 3 (KA = 1.5 - 2.5 x 10(3) M-1) bind more strongly to native DNA than does molecule 2 (KA congruent to 0.5 x 10(3) M-1). Furthermore, molecules 1 and 3 bind to DNA much more effectively than do the two less sterically hindered pi electron metabolite models, anthracene and phenanthrene. Photoelectron data suggests that the enhanced binding of molecules 1 and 3 is due to increases in polarizability. Experiments carried out with denatured DNA indicate that the binding of molecule 1 entails the greatest intercalation.