Purification of Euglena gracilis chloroplast elongation factor G and comparison with other prokaryotic and eukaryotic translocases.

C.A Breitenberger,L.L Spremulli

doi:10.1016/s0021-9258(18)43465-5

Abstract

Euglena gracilis chloroplast protein synthesis elongation factor G (EF-Gchl) has been purified to about 80% homogeneity by a two-step procedure which removes all traces of the cytoplasmic and mitochondrial translocases (EF-2 and EF-Gmt). The purification scheme generally results in approximately 130-fold purification with 20% recovery of the total EF-G activity present in whole cell extracts. The Euglena EF-Gchl is a monomeric protein with a molecular weight of approximately 85,000. As observed for all translocases to date, the activity of Euglena EF-Gchl is inhibited by treatment with low concentrations of N-ethylmaleimide, suggesting that a free sulfhydryl group is required for catalytic activity. Treatment with 3 microM fusidic acid results in a 50% inhibition of Euglena EF-Gchl activity and of the EF-G activity present in Chlamydomonas reinhardtii. About 10-fold higher concentrations of this antibiotic are required to inhibit the mitochondrial EF-G of Euglena and Escherichia coli EF-G to the same extent. Yeast mitochondrial EF-G is clearly distinguishable from the other organellar translocases tested, requiring 1 mM fusidic acid for 50% inhibition. Fusidic acid also inhibits the cytoplasmic translocases from yeast, wheat germ, and Euglena, although a wide range of sensitivities is observed. When antiserum raised against highly purified Euglena EF-Gchl is used to inhibit enzymatic translocation, a low degree of cross-reaction of the antiserum with Chlamydomonas EF-G and with E. coli EF-G is observed. The EF-G activity present in spinach is very slightly inhibited by the antiserum, whereas that of yeast is not affected. The mitochondrial and cytoplasmic translocases of Euglena are also unaffected by the antiserum against EF-Gchl. The evolutionary implications of these observations are discussed.

Highlights

The apparent similaritybetween prokaryotic and organellar mechanisms of protein synthesis has been cited as evidence that eukaryotic organelles may have evolved from an endosymbiotic prokaryote which eventually lost complete autoninhibition of Euglena EF-Gehk activity and of the EF-G omy and became a eukaryotic organelle
Fusidicacid inhibits mitochondria, unlike other known organellar translocases, the cytoplasmic translocases from yeast, wheatgerm, may not be functionally interchangeable with E. coli EF-G3 and Euglena, a wide range of sensitivities is on E . coli ribosomes [3]
Inhibition of EF-G activity was measured in the following preparam), tions: phototrophically grown Chlamydomonas reinhardtii (O),part i d y purified E. coli EF-G and yeast extract containing mitochondrial EF-G (0)A. blank of -1.5 pmol was subtracted and 100%

Summary

We have been investigating the enzyme responsiblefor the

The mechanism of protein biosynthesis in the cytoplasm of eukaryotes has been the subject of many studies, but much less is known about protein synthesis in eukaryotic organelles’ such as chloroplasts and mitochondria. Organellar ribosomes, with sedimentation coefficients of 55 S to 80 S , are generally translocation step of protein synthesis on the chloroplast ribosomes of Euglena gracilis, a photosynthetic microorganism with three potential sites of protein synthesis (cytoplasm, mitochondria, and chloroplasts). The cytoplasmic translocase (EF-2)catalyzestranslocation only on eukaryotic cytoplasmic ribosomes such as those from wheat germ, whereas the organellar translocases (EF-Gmtand E F - G c ~c)atalyze translocation on prokaryotic ribosomes [2].In this report, a simple twosmaller than those found in the eukaryotic cell cytoplasm

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Euglena B Euglena B

Yield mg

Phenylalanine polymerized

Findings

Theories on the evolution of present day organisms are