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Abstract. The 3' terminal nucleotide of turnip yellow mosaic virus (TYMV) RNA (23-25 S) may be esterified with valine in the presence of ATP and an enzyme preparation from Escherichia coli. The nucleotide composition near the valine-binding site is different for TYMV RNA and tRNAVaL from cabbage, as shown by comparison of the valine adducts of nucleotides labeled with radioactive valine in T1 RNase digests. Consequently, host tRNAVal is not involved in the observed charging of TYMV RNA with valine. The TYMV RNA appears to have a tRNA-like structure, at or near its 3' end, that is recognized by three different enzymes which specifically catalyze reactions involving tRNA.We have recently shown that RNA extracted from Turnip Yellow Mosaic Virus (TYMV) binds valine when incubated with ATP and cell-free extracts from Escherichia coli that are devoid of nucleic acids. The amino acid is bound by an ester linkage to the 3' terminal nucleotide of the RNA molecule, in a manner analogous to that encountered in aminoacyl-tRNAs.I Purified E. coli valyltRNA synthetase catalyses this reaction, but only after the RNA isolated from TYMV has had an AMP residue linked to its 3' end, from an ATP donor, by tRNA nucleotidyltransferase (EC 2.7.7.25; manuscript in preparation).In our previous paper,' we argued against the possibility that valine is bound to host tRNAVal entrapped within the virus particle. We now present experiments that further confirm our conclusion that valine is indeed bound to TYMV RNA. Our results also exclude the existence of a covalent linkage between host tRNAVal, or part of its structure and the 3' end of TYMV RNA. All of our data suggest that the viral RNA bears a tRNAVal-like structure in the vicinity of its 3' terminus.Methods and Materials. Extraction of RNA from TYMV: TYMV, isolated from infected cabbage (Brassica chinensis) leaves, was kindly supplied to us by Dr.L. Bosch (Leiden), Dr. J. M. Bov6 (Versailles), and Dr. L. Hirth (Strasbourg). RNA was extracted from the virus particles by phenol treatments in the presence of bentonite,3 and stored at -30'C in 0.01 M sodium cacodylate, pH 6.Extraction of tRNA from Chinese cabbage: RNA was extracted from uninfected cabbage leaves by the phenol method, in the presence of 0.01 M Tris (pH 7.4)-0.125 mM EDTA-1% sodium dodecylsulfate, and precipitated by ethanol. The RNA that is soluble in 1.5 M NaCl was isolated, incubated with DNase, and chromato-1345

The complete amino acid sequence phospholipid-transfer protein isolated from spinach leaves has been determined. The primary structure of the spinach protein was elucidated by analyses by HPLC of cyanogen bromide fragments and peptides obtained by tryptic digestions. The single polypeptide chain of the spinach protein consists of 91 amino acid residues. The protein contains six cysteines whereas phenylalanine and glutamine are absent. The present data, which are the first to be obtained with a phospholipid-transfer protein from a photosynthetic tissue, are compared to the amino acid sequences determined with plant and animal proteins involved in the intracellular transport of hydrophobic compounds.Several proteins involved in the intracellular transport or binding of hydrophobic compounds: phospholipids, fatty acids, glycolipids, cholesterol, vitamin A, have been discovered (for reviews, see [l -111. In particular, proteins able to bind or transfer phospholipids, called phospholipid-transfer proteins, have been purified to homogeneity from animals [l -5, 10, 111, higher plants [3,4, 8, 121, yeasts [I31 or bacteria [14]. These proteins have in common the property of facilitating an intermembrane transfer of phospholipids. However, they differ in specificity. Some of these proteins are specific for phosphatidylcholine, like the protein specific to bovine liver [l, 2, 151, or for phosphatidylinositol and phosphatidylcholine, like the bovine brain protein [lo], whereas other proteins are non-specific. Non-specific proteins have been isolated from animal sources (bovine liver [2,4]) or plant tissues (spinach leaf [16], maize seed [17] or castor bean seed [12]); these proteins are for the major part basic (PI around 9 -10). A better knowledge of the structures of these proteins can provide information about their mode of action. The elucidation of the primary structure has only been obtained with two animal phospholipid-transfer proteins: the specific (18, 191 and the non-specific [20, 211 proteins from bovine liver. The recent obtention of high amounts of purified protein from spinach leaf [I61 has allowed a study of its structure; the NH,-terminal end has been determined in previous experiments [22]. This paper reports the complete amino acid sequence of this protein, which is the first phospholipidtransfer protein to be analyzed from a photosynthetic tissue. This structure was compared to that recently obtained with a similar protein purified from castor bean seeds [23]. MATERIALS AND METHODS Purification of phospholipid-transfer proteinPhospholipid transfer protein was isolated from spinach leaves as previously described [16]. The protein was desalted by gel filtration on a column of Sephadex G-25, equilibrated and eluted with 3.5 M acetic acid. Amino acid analysisAmino acid analyses were performed as in [16] on a Technicon TSMl amino acid analyzer. Oxidized protein samples (4 x 1 nmol) were hydrolyzed in 6 M HCl for 24, 48, 72 and 120 h. The values for serine and threonine were determined after 24 h hydrolysis...

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