Biochemistry 1995,34, 7941 -7948
794 1
Eukaryotic Acidic Phosphoproteins Interact with the Ribosome through Their Amino-Terminal Domain? M. Payo Jose,$ Humberto Santana-Roman,# Miguel Remacha,' J. P. G. Ballesta,' and Samuel Zinker*,§ Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Canto Blanco, 28049 Madrid, and Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, 07000 Mexico Received November 15, 1994; Revised Manuscript Received March 6, 1995@
Variable-size fragments of the four yeast acidic ribosomal protein genes rpYPla, rpYPlP, rpYP2a and rpYP2P were fused to the LacZ gene in the vector series YEp356-358. The constructs were used to transform wild-type Saccharomyces cerevisiae and several gene-disrupted strains lacking different acidic ribosomal protein genes. The distribution of the chimeric proteins between the cytoplasm and the ribosomes, tested as P-galactosidase activity, was estimated. Hybrid proteins containing around a minimum of 65-75 amino acids from their amino-terminal domain are able to bind to the ribosomes in the presence of the complete native proteins. Hybrid proteins containing no more than 36 amino terminal amino acids bind to the ribosomes in the absence of a competing native protein. The fused YPl-P-galactosidase proteins are also able to form a complex with the native YP2 type proteins, promoting their binding to the ribosome. The stability of the hybrid polypeptides seems to be inversely proportional to the size of their P protein fragment. These results indicate that only the amino-terminal domain of the eukaryotic P proteins is needed for the Pl-P2 complex formation required for interaction with the ribosome. The highly conserved P protein carboxyl end is not implicated in the binding to the particles and is exposed to the medium. ABSTRACT:
The large ribosomal subunits of cytoplasmic ribosomes from all organisms have a set of very acidic proteins (pl
