Evidence for a Direct Role of tRNA in an Amino Acid Transport System*

SUMMARY The transport of phenylalanine by the general aro- matic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylala- nine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. preparations


SUMMARY
The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNA'lh" and tRNA""' were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNA"'" and tRNA""' gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNA""' was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNA'lh' was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNA'l'"'in stimulation of phenylalanine transport.
Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNA'lh" rather than the major component tRNA'lh", and the slight activity of preparations of tRNA'l'r' is probably due to a contamination of the active tRNA'lh'.
Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA. Escherichia coli is known to have at least four aromatic amino acid transport systems: a general aromatic amino acid transport system that utilizes phenylalanine, tyrosine, and tryptophan which compete for a common receptor, and three systems, each of which transport specifically one of the three amino acids (1, 2). An inducible system transporting tryptophan has been reported (3, 4) in addition to the above four systems.
On the basis of studies of mutants of E. coli, repression of branched chain amino acid transport has been proposed to involve the interaction of leucine with its aminoacyl-tRNA synthetase and its cognate leucyl-tRNA (5-8). A significant enhancement in the initial rate of uptake of leucine and other L system (9) amino acids was observed in Chinese hamster ovary cell lines when the temperature-sensitive leucyl-tRNA synthetase mutant tsH1 was shifted from a normal growth temperature of 34°C to a marginally permissive temperature for growth of 38°C (10). In a similar manner, a temperaturesensitive asparaginyl-tRNA synthetase mutant exhibited a significant enhancement of the transport activity of A system amino acids (glycine, alanine, proline, serine) when the growth temperature was shifted from 33°C to 39.5"C (10). The product of the regulator gene, tyrK, which has been shown to be essential in the regulation of a number of enzymes involved in aromatic amino acid biosynthesis (ll-14), has also been shown to be involved in the repression regulation of the aromatic amino acid transport systems (15).
In this investigation, it has been found that tltNA directly stimulates the general aromatic amino acid transport in E. coli spheroplasts, and there is a high degree of specificity in enhancing the transport of the amino acid by its corresponding tRNA species. were approximately 40% as active as intact cells in the uptake of phenylalanine or tyrosine by the general aromatic transport system under the conditions described below. Amino Acid Uptak-'l'he total uptake of amino acids (phenylalanine or tyrosine) was determined by a technique adapted from that of Weiner and Heppel (17). The uptake medium contained 1 mg of glucose, 2.2 nmol of L-["Clphenylalanine, or ~["Cltyrosine and appropriate amounts of tKNA in 0.1 ml. The spheroplast suspension and the substrates were kept at room temperature and 0°C to 4"C, respectively, until used. Just prior to the assa,y, the tubes containing assay substrates were incubated for 2 min at 27°C. The uptake was initiated by addition of 0.1 ml of the spheroplast suspension to the assay, tubes. After 2 min at 2i"C, the uptake was terminated by the addltlon of 2 ml of the phosphate buffer described above, and followed by rapid filtration of the reaction mixture on a 25-mm, 0.8-p pore size, Millipore filter. The filters were dried and placed in vials containing 10 ml of toluene with 0.5'); (w/v) 2,5-diphenyl-l,3-oxazole for counting. I'henylalanine-specific transport was determined as described above except that supplements of 125 nmol each of r,-tyrosine and L-tryptophan were added. Higher levels of tyrosine and tryptophan did not further reduce the uptake of phenylalanine in spheroplasts, and the remaining activity is presumed to result primarily from the phenyl-1013 alanine-specific transport system. Phenylalanine uptake by the GAT' system was calculated by the difference in the specific and total uptake. Tyrosine-specific transport was similarly determined in the presence of 125 nmol of L-phenylalanine and L-tryptophan, and the tyrosine uptake by the GAT system was calculated by the difference between total and specific tyrosine uptake. The uptake of amino acids was linear under these conditions for more than 10 min, and the uptake, for experiments in the absence of chloramphenicol, represents amino acid accumulated in the amino acid pool and incorporated into protein.
Also, the kinetics of transport of phenylalanine in spheroplasts of E. coli is similar to that in intact cells. The Km of the general aromatic transport determined by this assay method is 1.25 PM which compares with 0.27 pM observed with intact E. coli cells' and the V,,, is 40 pmol/min/mg dry weight of spheroplasts and is comparable to that observed in intact cells. For the uptake of phenylalanine not inhibited by tyrosine and tryptophan and ascribed to phenylalanine-specific transport, kinetic data indicate a K,,, of 6.3 PM for the above spheroplast technique compared with 3.1 pM for intact cells.'  (21). Upon dissolution, the tRNA solution was aspirated for 30 min at room temperature in order to remove traces of ethanol.

RESULTS AND DISCUSSION
In a study of the transport of phenylalanine in spheroplasts of E. coli, a slight stimulation by tRNA on the total transport was noted. The observed stimulation of total transport was found to be primarily associated with the GAT system ( Table  I). The stimulation did not occur with periodate-treated tRNA, and a significant inhibition of uptake rather than stimulation was found to occur with tRNA acylated with phenylalanine (data not shown). These results' indicated the possibility of specific tRNA effects on the general aromatic amino acid transport system. Accordingly, the effects of different tRNA species on the transport of phenylalanine by the GAT system were determined.
The uptake of ["Clphenylalanine into E. coli spheroplasts from a concentration of 11 PM amounted to 57.2 pmol/min/mg dry weight of spheroplast in the 2-min incubation period, and the stimulatory effects of preparations of various species of tRNA were determined as shown in Fig. 1. Of the various preparations of amino acidspecific tRNAs, tRNAG'" and tRNAVa' showed no detectable activity, while the preparation of tRNAPhe was at least 3.5 times as active as the mixture of tRNAs (E. coli B tRNA).
Since the preparation of tRNATy' used in this experiment contained 9.7% tRNAPh", the slightly greater activity of the tRNATP preparation than that of the unfractionated tRNA is probably the result of the impurity rather than the major species, tRNATy'. Since the activity of the preparation of tRNAPhe does not account for the total activity of E. coli B tRNA, the possibility of isoacceptor species accounting for the activity appeared likely. Preliminary work of the fractionation of tRNA acylated with ['*C]phenylalanine into minor and major isoacceptors followed by hydrolysis of the phenylalanine group has provided samples which show activity for only the minor isoacceptor(s). Thus, it appears likely that the slight activity of tRNATy' over that of the crude tRNA represents a contamination with the active tRNAPh" isoacceptor(s). In order to investigate further the specificity of tRNA species for stimulation of uptake by the general aromatic amino acid transport system, the effect of tRNA species on ' The abbreviation used is: GAT, general aromatic amino acid transport.
' M. L. Pratt (1977) Ph.D. dissertation, The University of Texas at Austin, Austin, Tex.  1. Stimulatory effects of tRNAs on the phenylalanine uptake by the GAT system of E. coli spheroplasts. Substrate concentration was 11 PM and the uptake assays were done in the presence of tRNAPh' (O), tRNATY' (O), total tRNA (O), tRNAG'" (+), or tRNA""' (A). Total tRNA means unfractionated E. coli B tRNA. Enhanced uptake is expressed as per cent stimulation over control (57.2 pmol/ min/mg dry weight of spheroplasts) with respect to tRNA concentration. All methods are described under "Experimental Procedures." the uptake of tyrosine by the system was determined.
The preparation of tRNATy' was found to be 10 times as active as the unfractionated tRNA and stimulated the uptake of tyrosine 100% at a concentration of 0.16 mg/ml and 167% at a concentration of 0.35 mg/ml as shown in Fig. 2. tRNAGIU and tRNA""' showed no detectable stimulation, but the tRNAPh' sample was approximately twice as active as the unfractionated tRNA.
These results indicate a specificity of each tRNA species for stimulation of the general aromatic transport system for its specific amino acid, but the possibility cannot be excluded that there may be a cross-stimulation to some degree of tRNAPh" and tRNATV with each being far more effective for stimulation of transport of its corresponding amino acid. Whether the low activity of the tRNAPh' preparation in stimulating tyrosine uptake can be ascribed to its content of tRNAT" impurity (0.8%) requires further investigation of the stimulation by isoacceptor species. The stimulation of uptake of aromatic amino acids by tRNA was not affected by chloramphenicol which indicates that these effects do not require protein synthesis and apparently 160 Preliminary investigation of glutamic acid and valine uptake have shown tRNA stimulations which appear to have specificity. Preparations of tRNAG1" are more active than crude tRNA, and the tRNAPhe preparation is inactive in stimulating glutamic acid uptake. Whether the exposed cell membrane in the absence of the cell wall allows tRNAs to act externally on the membrane receptors or to penetrate into the spheroplasts requires further investigation.
However, it is apparent that the spheroplast technique offers an unusual system for further investigation of roles of tRNA, particularly of isoacceptor species. FIG. 2. Stimulatory effects of tRNAs on the tyrosine uptake by 9. the general aromatic amino acid transport system of E. coli spheroplasts. Enhanced uptake is expressed as per cent stimulation over control (7.7 pmol/min/mg dry weight of spheroplasts). The other 10.
conditions are the same as Fig. 1. 11.
represent direct effects upon the transport system. Although several papers have been published suggesting that aminoacyl-tRNA or aminoacyl-tRNA synthetase, or both, are involved in the repression control of some amino acid transports in E. coli and mammalian cell lines (8, lo), the stimulation of transport by specific tRNA of the GAT system is the fist indication of a direct involvement of tRNA in amino acid 18. transport.

20.
regulator gene, tyrR, appears to be involved not only in the 21 22