Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis.

Saturating concentrations of the initiation-specific inhibitors poly(I) and T-2 toxin inhibit protein synthesis by over 35% and cause ribosome 'run-off' from the polyribosomes. The elongation-specific inhibitor cycloheximide totally prevents protein synthesis and 'freezes' the ribosomes in the pattern of unincubated controls. These results prove that our Tetrahymena extracts are capable of protein-synthesis initiation, a conclusion which is confirmed by a 30% inhibition of synthesis by the mRNA cap analogue, 7-methylguanosine 5'-monophosphate.     

Mysteries of mitochondrial protein synthesis

Mitochondria possess an endogenous system of translation, in which all constituent components are unique. An electrophoretic analysis of mitochondrial translation products revealed that the content of polypeptides in mitochondria is two times as high as that of mitochondrial DNA genes. The electrophoretically determined molecular mass of proteins synthesized in mitochondria is much less than that calculated from gene sequencing data. The average amino acid composition of the proteins synthesized in mitochondria differs significantly from that encoded by the nucleotide sequence of corresponding mitochondrial genes. These enigmas of mitochondrial protein synthesis await further solution.     

Induction of acquired thermotolerance in Tetrahymena thermophila: effects of protein synthesis inhibitors.

When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable. However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h. Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca. 70% survival after 1 h). This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs. However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized. The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs. But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis.     

Stimulation of mitochondrial protein synthesis by metal ions.

1--10 muM Cu2+, Ag+, and Au3+ were found to stimulate rat liver mitochondrial protein synthesis in vitro. Cu2+ and Ag+ also produced an increase in mitochondrial volume ("swelling"). Thus, thyroid hormones and their analogs are not unique, as suggested previously (Buchanan, J.L., Primack, M.P. and Tapley, D.F. (1970) Endocrinology 87, 993--999), in stimulating both mitochondrial protein synthesis and swelling. Furthermore, the data suggest a role for Cu2+ in the regulation of mitochondrial protein synthesis.     

A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in Tetrahymena thermophila.

We have identified and purified a 58-kilodalton protein of Tetrahymena thermophila whose synthesis during heat shock parallels that of the major heat shock proteins. This protein, hsp58, was found in both non-heat-shocked as well as heat-shocked cells; however, its concentration in the cell increased approximately two- to threefold during heat shock. The majority of hsp58 in both non-heat-shocked and heat-shocked cells was found by both cell fractionation studies and immunocytochemical techniques to be mitochondrially associated. During heat shock, the additional hsp58 was found to selectively accumulate in mitochondria. Nondenatured hsp58 released from mitochondria of non-heat-shocked or heat-shocked cells sedimented in sucrose gradients as a 20S to 25S complex. We suggest that this protein may play a role in mitochondria analogous to the role the major heat shock proteins play in the nucleus and cytosol.     

Poly(A)+ RNA from Tetrahymena: stimulation of protein synthesis in vitro.

Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at approximately 17S and 25S. After heat denaturation all the 25S RNA was converted to 17S, indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A) +/- RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15-fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28,000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

RNA synthesis in starved deciliated Tetrahymena pyriformis.

Tetrahymena pyriformis which has been starved for 20 h by incubation in buffer, and then deciliated, can regenerate its cilia in about 90 min while still in suspension in non-nutrient medium. The process of reciliation is accompanied by protein synthesis which begins a few minutes after deciliation and by synthesis of ribosomal and messenger RNAs during a period extending from about 1 h to about 3 h after deciliation. Although net synthesis of RNA remains at a very low level until 1 h after deciliation, a qualitative change in the translatable poly(A)-containing messenger RNA content of deciliated cells, and in particular, formation of beta-tubulin mRNA can be detected almost immediately after deciliation.     

Regulation of mitochondrial protein synthesis at the polyribosomal level.

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.