Regulation of Protein Synthesis Initiation in HeLa Cells Deprived of Single Essential Amino Acids

Abstract

In HeLa cells deprived of valine, histidine, or methionine initiation of protein synthesis decreases rapidly and disaggregation of polyribosomes also occurs. The mechanism of inhibition does not seem to involve the supply of RNA in the cell, and thus it differs from the initiation of inhibition at elevated temperatures. Polyribosomes rapidly form again if the missing amino acid is restored, even in the presence of actinomycin D.Studies of HeLa cells deprived of single essential amino acids -have shown that protein synthesis continues in this situation at a reduced rate determined by the rate of endogenous protein turnover (1). Such cells are in a condition of suspended growth and offer a unique opportunity for the study of metabolic regulation. It has previously been established that the rate of synthesis of ribosomal precursor RNA is reduced in these cells (2, 3), and maturation into complete ribosomal subunits is restricted (3), though synthesis of ribosomal proteins is uninterrupted (4).In several recent studies (5, 6), it was found that ascites tumor cells deprived of a complete mixture of essential -amino acids had extensive disaggregation of polyribosomes into inactive monoribosomes. The disaggregation was reversed if the set of essential amino acids was restored. However, since all of the essential amino acids were withheld or supplied at the same time, it is uncertain whether the apparent -regulation of protein synthesis that was observed involved the availability of amino acids per se, or amino acids in the role of donors of amide or methyl groups.We have found that HeLa cells, deprived of any one of several essential amino acids, promptly exhibit a marked inhibition of translational initiation. This inhibition and its -reversal have been studied and compared to previous demonstrations of control of translational initiation in cells undergoing mitosis (7) and in cells exposed to elevated temperatures (8). MATERIALS AND METHODS Cell cultureSuspension cultures of HeLa cells were maintained in Eagle's medium with 5% calf serum, as described (9, 10), at a con-* To whom reprint requests should be sent. Reprints will be available only outside of North America. Amino-acid Depriation, Cell Labeling, and Fractionation. Cells were collected by centrifugation and rinsed in Eagle's medium without valine, histidine, or methionine, but with leucine at a concentration of 0.02 mM (one-twentieth of the normal concentration), and 5% dialyzed calf serum. They were then resuspended in a medium of the same composition at a concentration of 3 X 106 cells/ml. Cells acting as growing controls were resuspended in medium to which the missing amino acid had been added to its normal concentration. All cultures were incubated at 370C, except where noted Suspension cultures deprived of an amino acid for specified times, or growing control cultures, were incubated for 45 min, and then labeled for various times with ['4C]leucine (0.25 pCi/ml, 12.5 Ci/mol final specific activity). 8-ml samples were then rapidly cooled and fract...

A Specific Inhibitor of Polypeptide-Chain Initiation in Escherichia coli

“…The inhibition of polypeptide-chain initiation reported here, and its release under conditions of active aminoacylation of tRNA, may be related to the observation (21,22) …”

Hormonal Regulation of Protein Synthesis in Rat Ventral Prostate

“…However, there has been considerable evidence to suggest that the protein synthesis is regulated at the posttranscriptional level (Thompson et al, 1970;Tomkins et al, 1972). It has also been claimed by several investigators that the rate determining step in the protein synthesis occurs during the initiation phase of the translation (Fleck et al, 1965;Stirewalt et al, 1967;Vaughan et al, 1971 ;Van Venrooji et al,1972;Pain, 1973).…”

Stimulation of Polypeptide Initiation In Vitro After Protein Synthesis Inhibition In Vivo in HeLa Cells

“…These include in vivo studies of cells during mitosis (12), at elevated temperatures (ref. 13; Goldstein, E., & Penman, S., manuscript in press), after poliovirus infection (14), and in cells starved for an essential amino acid (15,16). The experiments in this report are a first step in studying these control mechanisms in vitro.…”

“…Fig. 2 (15). In contrast to the total incorporated radioactivity, the number of radioactive N-terminal amino-acids incorporated by lysates rises sharply during the first 90 min of amino-acid starvation.…”

“…The response to methionine starvation appears initially to be similar to the response to inhibition of protein synthesis by cycloheximide. Vaughan (15) has shown that prolonged amino-acid starvation appears to interfere with the initiation process, and this may account for the subsequent decline in N-terminal formation by the lysates.…”