Eglena gracilis Chloroplast EF-Ts EVIDENCE THAT IT IS A NUCLEAR-CODED GENE PRODUCT*

Extracts of Euglena gracilis cells contain high levels of elongction factor (EF)-Ts (EF"I'schl) activity which can be assayed by measuring the rate of exchange of GDP with Escherichia coli EF-Tu-GDP. The appear-ance of EF-Ts activity in E. gracilis cells is light-stim- ulated, suggesting that the EF-Ts is required for chloroplast function. However, based on experiments with a mutant of E. gracilis lacking chloroplast DNA, as well as studies on the effect of antibiotics on EF-Ts synthesis, it is concluded that the EF-Tschl gene is nuclear- coded. synthesis M e t tRNA initiation factors active elongation factors and G from spinach chloroplasts (EF-Tuchl,' EF-GCM) (6) and EF-GcM from Euglena gracilis 8) can function with E. coli ribosomes. Detailed studies remain to be done comparing the physical and chemical properties of purified chloroplast and E. coli elongation factors. Of consid-erable interest is the location of the genes for the chloroplast

In chloroplasts, many of the protein synthesis components are thought to be similar to those of prokaryotes. This conclusion has come mainly from studies showing that (i) antibiotics active against bacterial ribosomes inhibit chloroplast protein synthesis (1-4); (ii) initiation of protein synthesis uses M e t -tRNA (4); (iii) initiation factors from Escherichia coli are active with spinach ribosomes (5); and (iv) highly purified elongation factors Tu and G from spinach chloroplasts (EF-Tuchl,' EF-GCM) (6) and EF-GcM from Euglena gracilis (7,8 ) can function with E. coli ribosomes. Detailed studies remain to be done comparing the physical and chemical properties of purified chloroplast and E. coli elongation factors. Of considerable interest also is the location of the genes for the chloroplast elongation factors. In spinach, EF-Tu,hr and EF-Gct,l are coded by chloroplast DNA (9), whereas E. gracilis EF-G is nuclear-coded (8). To better understand the nature of the chloroplast elongation factors and the regulation of their synthesis, we have initiated studies on the purification of chloroplast elongation factors from E. gracilis. The present report describes the presence of EF-Tschl in E. gracilis extracts and presents evidence that it is a nuclear-coded gene product. Klebs' var. bacillaris Cori (Euglena B) and the mutant strain W,BUL were obtained from W. E. Barnett and J. Schiff, respectively. EF-Tu was purified from extracts of E. coli as described previously (IO).
* The costs of publication of this article were defrayed in p a t by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. For experiments to examine the effect of light on EF-Ts synthesis, cells were grown in a volume of 3 liters, in the dark, to a density of 0.5 to 1.0 X IO6 cells/ml in a Difco Euglena broth medium. The cells were centrifuged at 2500 rpm for 15 min and then gently suspended in 3 liters of phototrophic medium, pH 3.5 (11). The suspension was divided and a portion was incubated in the dark, whereas a second aliquot was exposed to white light (400 fc) provided by fluorescent lights and a spotlight. Both incubations were carried out at 27°C. At various times, the cells were removed and harvested by centrifugation (15 min at 3000 X g ) . The cells were washed with 40 ml of a buffer containing 10 mM each of Tris/C1, pH 7.4, MgC12, and NH,CI (Buffer A) and then suspended in 2 ml of a buffer containing 50 mM Tris/Cl, pH 7.8,O.l m~ EDTA, 50 mM NH4Cl, 5 mM MgClp, 6 mM P-mercaptoethanol, and 10% glycerol (Buffer B). The cells were disrupted by sonicating for 2 X 30 s in a Bronwill sonicator. The suspension was centrifuged for 10 min at 30,000 X g and the supernatant was centrifuged at 150,000 x g for 2 h.
EF-Ts activity was measured by the ability of this factor to cata- The reaction mixture for EF-Tu activity was similar to that for the EF-Ts assay except that the incubations were done at 37OC for 10 min in the absence of E. coli EF-Tu. GDP.

EF-Ts Activity in E. gracilis Extract-
The most convenient procedure to assay for EF-Ts activity takes advantage of the ability of EF-Ts to catalyze an exchange of GDP with GDP bound to EF-Tu (EF-Tu.GDP). The EF-Ts activity in Euglena extracts could be assayed using E. coli EF-Tu.
GDP. As seen in Fig. 1, a cell-free extract had high levels of EF-Ts activity which was proportional to the amount of extract added. The activity in these extracts was often greater than 20,000 units/ml with a specific activity of 1500 to 2000. This is comparable to the specific activity of EF-Ts (3400 units/mg) observed in crude E. coli extracts (15). Fig. 2 shows the effect of light on the EF-Ts activity in Euglena extracts. In these experiments, dark-grown E, gracilis cells were placed in a phototrophic medium and incubated for up to 96 h in the dark or in light (see "Materials and Methods"). As seen in Fig. 2, exposure of the cells to light resulted in a large increase in the EF-Ts specific activity over the time course of the experiment. Cells kept in the dark showed no increase in EF-TS activity with time. Since it is known that the synthesis of chloroplast proteins can be regulated by light (whether coded by nuclear or chloroplast DNA) the above results suggest that the observed EF-Ts activity is due to chloroplast EF-TS (EF-TScd.

Effect of Light on EF-Ts Formation-
Evidence That EF-Ts Is Nuclear-coded-To determine whether the EF-Tschl gene is chloroplast-or nuclear-coded, a mutant of E. gracilis (W3BUL) that is lacking chloroplast E. gracilis

EF-Ts activity in wild type and W:,BUL Euglena cells
The cells were grown in the dark and then suspended in phototrophic medium as described in the text. The cells were divided and then incubated for 48 h under the conditions shown in the table. The preparation of the cell-free extracts and the assay for EF-Ts are described under "Materials and Methods." Cycloheximide and chloramphenicol concentrations were 10 and 100 pg/ml, respectively. Methods"). Cells were removed at the times indicated and the extracts were prepared as described under "Materials and Methods." EF-Ts activity was measured as described under "Materials and Methods." DNA was examined. As seen in Table I, extracts of these cells also had high EF-Ts activity. In addition, the activity was higher in light-grown cells, a result similar to that observed in the wild type cells.
Further evidence suggesting that the EF-Ts,hr gene is located in the nucleus was obtained by the use of antibiotics. If cytoplasmic ribosomes are the site of EF-Ts'hl synthesis, cycloheximide would inhibit its synthesis, whereas chloramphenicol should inhibit EF-TsChl synthesis, if it occurs on chloroplast ribosomes. As also shown in Table I, cycloheximide resulted in an 80% inhibition of EF-T%.hl synthesis, while chloramphenicol had no effect.
Although EF-TGhl and EF-Gchl have been purified from spinach chloroplasts (6,16) and EF-Gchl from E. gracilis extracts (7), EF-Tschl activity has not been previously identified. Tiboni et al. (6) did report that some of the EF-TuChl in spinach extracts was present as an EF. T complex (presumed to be an EF-Tu. EF-Ts complex), but no evidence for EF-Ts activity was presented.
Although the spinach factors EF-Tuchl and EF-Grhl appear to be coded by chloroplast genes (9), E. gracilis EF-G<hl is nuclear-coded (8). The present study indicates that EF-Ts'hI in this organism is also nuclear-coded and that the synthesis of this factor, similar to that observed with EF-G from Euglena (8), is stimulated by light.
Preliminary studies indicate that the bulk of the EF-Ts,hl in E. gracilis extracts is complexed to EF-Tu. Thus, it was observed that the two activities co-migrate on both DEAE-Sephadex and Ultrogel chromatography columns (data r?ot shown). In E. coli, much of the EF-Ts present in crude extracts is also complexed with EF-Tu (17) and an EF-Tu-EF-Ts complex can readily be formed in vitro (18). Since the EF-Tschl from E. gracilis has not been purified to homogeneity, it is not yet possible to compare its properties with E . coli EF-Ts. However; EF-Ts preparations from both sources contain one similar property, i.e. the ability to interact with E. coli EF-Tu.