Acidic Phosphoprotein Complex of the 60S Ribosomal Subunit of Maize Seedling Roots (Components and Changes in Response to Flooding)

Abstract

We determined that ribosomes of seedling roots of maize (Zea mays 1.) contain the acidic phosphoproteins (P-proteins) known to form a flexible lateral stalk structure of the 60s subunit of eukaryotic ribosomes. l h e P-protein stalk, composed of PO, P1, and P2, interacts with elongation factors, mRNA, and tRNA during translation. Acidic proteins of 13 to 15.5 k D were released as a complex from ribosomes with 0.4 M NH,CI/SO% ethanol. Protein and cDNA sequence analysis confirmed that maize ribosomes contain one type of P1, two types of P2, and a fourth and novel Pl/PZ-type protein.This novel P-protein, designated P3, has the conserved C terminus of P1 and P2. P1, P2, and P3 are similar in deduced m a s (1 1.4-1 2.2 kD) and isoelectric point (4.1-4.3). A 35.5-t o 36-kD acidic protein was released at low levels from ribosomes with 1 .O M NH,C1/50% ethanol and identified as PO. Labeling of roots with [32P]inorganic phosphate confirmed the in vivo phosphorylation of the P-proteins.Flooding caused dynamic changes in the P-protein complex, which affected the potential of ribosome-associated kinases and casein kinase II t o phosphorylate the P-proteins. We discuss possible alterations of the ribosomal P-protein complex and consider that these changes may be involved in the selective translation of mRNA in flooded roots.Ribosomes are a two-subunit organelle, and are the site of mRNA translation into protein in a11 organisms. The large ribosomal subunit is a complex macromolecule that is composed of rRNAs, a large number of basic (high-pI) proteins, and a small number of acidic (low-pI) proteins. Across evolutionary kingdoms and phyla the large ribosoma1 subunit is variable in size, but possesses a number of morphological features that are universally conserved. For example, a universal feature of the peptidyl transferase region of the large subunit is a complex of acidic proteins that form the body and stalk of a lateral protuberance (Moller, 1990; Liljas, 1991).In bacteria the acidic protein stalk of the 50s ribosomal subunit is composed of ribosomal protein, L10, and two dimers of L7 and L12 in a (L7/L12),-LlO pentameric complex (Moller, 1990; Liljas, 1991). L10 is a 17-kD acidic pro- tein that interacts with the 23s rRNA scaffold of the large subunit within the GTPase domain of the rRNA. L7 and L12 are 12-kD acidic proteins that are encoded by a single gene, but differ in that the N-terminal Ser of L7 is posttranslationally aminoacetylated. The N-terminal domain of L7 and L12 forms an a-helical structure that is responsible for dimer formation and binding to M O . A central region of acidic residues forms a flexible hinge that allows the dimers to assume an elongated conformation that forms the stalk or a closed conformation in which the C-and N-terminal domains are in close proximity on the body of the 50s ribosomal subunit (Oleinikov et al., 1993;Traut et al., 1993). The C-terminal regions of L7 and L12 are required for binding of elongation factor G, and the subsequent hydrolysis of GTP that occurs in the transloca...

Regulated Heterogeneity in 12-kDa P-protein Phosphorylation and Composition of Ribosomes in Maize (Zea mays L.)

“…P1 antisera recognized polypeptides with apparent molecular masses of ϳ14.5 and 50 kDa in coleoptile ribosomes ( Fig Maize P2a is encoded by a gene family of approximately four members, has a predicted molecular mass of 11.5 kDa, and five or six putative phosphorylation sites (26,27). The P2a antiserum detected a group of ϳ12-kDa polypeptides in coleoptile ribosomes ( Fig.…”

“…Maize P3 is encoded by one to two genes, has a predicted molecular mass of 12.2 kDa, and three putative phosphorylation sites (26,27). The P3 antiserum detected a 15-kDa polypeptide in coleoptile ribosomes ( Fig.…”

“…Maize P1 is encoded by a single-copy gene, has a predicted molecular mass of 11.0 kDa and three putative phosphorylation sites (26,27). P1 antisera recognized polypeptides with apparent molecular masses of ϳ14.5 and 50 kDa in coleoptile ribosomes ( Fig Maize P2a is encoded by a gene family of approximately four members, has a predicted molecular mass of 11.5 kDa, and five or six putative phosphorylation sites (26,27).…”

“…Acidic ribosomal proteins were purified from coleoptile ribosomes as previously described by Bailey-Serres et al (26) in the absence of sodium molybdate.…”

“…We reported that maize (Zea mays L.) possesses one form of P1, two forms of P2 (P2a and P2b), and a third, plant-specific P1/P2-type protein designated P3 (26,27). Here we examined whether maize ribosomes vary with respect to the composition and phosphorylation of these proteins.…”