Polyadenylate-Binding Protein from Pea (Pisum sativum)'

A polyadenylate-binding protein (PABP) was purified from cellfree extracts prepared from pea seedlings (Pisum sativum) by ammonium sulfate precipitation and Affi-Gel Blue and polyadenylate-Sepharose 4B affinity chromatography. The final preparation from polyadenylate-Sepharose 4B columns contained a single 70-kilodalton polypeptide with high polyadenylate-binding activity. The purified protein was active over a broad range of ionic strengths and showed temperature and pH optima of 370C and pH 6.5, respectively. Specificity studies indicated that the pea PABP was most active with polyadenylic acids, showed some activity with polyguanylic acid, and did not bind to polycytidylic acid. Moreover, longer polyadenylate molecules were bound more effectively than shorter ones. Because these properties are similar to PABPs isolated from other sources, we conclude that we have identified, purified, and characterized a plant PABP analogous to those described in yeast and animal systems. PABPs2 have been isolated from several eukaryotic organisms (8, 10, 16, 19). Usually, there are two types of PABPs within the same cell, a cytoplasmic protein of 70 kD and a nuclear protein of 55 kD. In yeast, both proteins are products of the same gene (16). Comparison of PABP genes from different organisms revealed that all have four conserved RNA-binding domains, including a highly conserved octapeptide Arg/Lys-Gly-Phe/Tyr-Gly/Ala-Phe/Tyr-Val-X-Phe/Tyr, within their N-terminal regions (10, 12, 16, 26). In general, PABPs have higher affinities for poly(A) than for poly(G) or poly(U) and do not bind with poly(C) (14, 23). The yeast PABP requires polyadenylate tracts of 12 or more nts for optimal binding; oligoadenylates of 12 to 25 nt were bound with KD values< 3 x 10-8 M, whereas shorter molecules were bound with KD values of 2 x 10-7 M or greater (18). With longer poly(A) molecules, the PABPs bind in a cooperative manner so as to form "beads on string" complexes with a periodicity of 25 to 27 nts (2, 3). 'This work was supported in part by U.S. Department of Agriculture-University of Kentucky Cooperative Agreement No. 58-43YK7-0025. The investigation reported in this paper (No. 91-3-162) is in connection with a project ofthe Kentucky Agricultural Experiment Station and is published with approval of the Director. 2Abbreviations: PABP, polyadenylate-binding protein; poly(A), polyadenylic acid; poly(G), polyguanylic acid; poly(C), polycytidylic acid; poly(U), polyuridylic acid; VRC, vanadyl ribonucleoside complex; nt, nucleotide. In yeast, PABPs are essential because mutants that lack a functional PABP gene are not viable (17, 18). The requirement for PABP is related to its role in initiation of mRNA translation; PABPs are apparently involved in recruitment of 60S subunits during translation initiation. In yeast, PABPs are also involved in mRNA turnover, because they are required for poly(A) tail shortening of cytoplasmic RNAs (17). Others have suggested that PABPs also play other roles in regulating mRNA stability (4) and mRNA translocation from the nucleus to the cytoplasm (15). Posttranscriptional events are being increasingly recognized as important for regulation of gene expression in plants (see, for example, refs. 9, 21, 22, 24). Because PABPs are needed for two crucial posttranscriptional aspects of gene expression in yeast, an understanding of plant PABPs should lend insight into posttranscriptional processes in plants. At present, there is no reported information about PABPs from plant cells. Here, we report the purification of a plant PABP from cellfree extracts of pea (Pisum sativum) seedlings by affinity chromatography and a study of the binding properties of the purified protein. MATERIALS AND METHODS