Characterization of ribosomal protein phosphorylation in maize axes during germination

Abstract

Ribosomal proteins from maize ( Zea mays L.) axes were in vivo 32P-labeled at different germinating periods. These proteins were analyzed by 1- and 2-D gel electrophoresis and autoradiography. Specific 32P-ribosomal protein patterns were found within the 24 h tested period. The main labeled bands corresponded to 37, 31, 16 and 14.5 kDa. The 31 kDa labeled band appeared on the labeled pattern from 8 h incubation onward, while 32P-incorporation on the 16 and 14.5 kDa bands decreased along the germination period. Early phosphorylation of the 31 kDa protein was achieved by stress (heat or anoxia shock) but not by phytohormones. Hydrolysis of the phosphoproteins and paper electrophoretic analysis of the amino acids showed phosphoserine threonine as the labeled residues. Separation of the ribosomal subunits and analysis of the correspondent proteins indicated that the 31 kDa phosphoprotein is located in the small subunit, while the 14.5, 16 and 37 kDa phosphoproteins are part of the large subunit. Furthermore, the 31 kDa protein appeared labeled in the 2-D gel autoradiography and showed cross reaction with antibodies raised vs. rat S6 ribosomal protein by immunoprecipitation. Acidic ribosomal proteins were extracted from the total ribosomal proteins and analyzed by gel electrophoresis and autoradiography. Results indicated that the extracted proteins corresponded to the 37, 16 and 14.5 kDa phosphoproteins. These proteins gave positive reaction in slot blot analysis using antibodies raised vs. the correspondent yeast acidic ribosomal proteins. The possible significance of the phosphorylation of these proteins in regulating protein synthesis during seed germination is discussed.