Heterologous expression of the highly conserved acidic ribosomal phosphoproteins from Dictyostelium (changed from Dictyosteliumm) discoideum in Saccharomyces cerevisiae.
The genes encoding the acidic ribosomal phosphoproteins DdP1 and DdP2 from Dictyostelium discoideum have been cloned into yeast plasmid vectors under the control of the inducible GAL1 promoter. These constructions have been used to transform S. cerevisiae strains D45 and D67 lacking the equivalent ribosomal components. The D. discoideum genes are properly transcribed when cells are grown in the presence of the inducer galactose and the mRNAs incorporated into polysomes. However, the heterologous ribosomal proteins are not able to rescue the growth deficiency in S. cerevisiae caused by the absence of their own ribosomal proteins. When the heterologous proteins are analyzed using specific antibodies, only protein DdP1 is found in the ribosomes of the transformed S. cerevisiae D67 strain. No other heterologous protein is found in any other transformed strain, suggesting that the heterologous acidic ribosomal components are rapidly degraded when they are not bound to the ribosomes. The results indicate that D. discoideum DdP1 protein is able to interact with the yeast ribosome, though the interaction is functionally inefficient. Protein DdP2, in spite of having a higher sequence similarity to its yeast counterparts, is completely inactive in S. cerevisiae. Since the P proteins from both organisms have extensive amino acid sequence similarity ranging from 60% to 70%, these results warns about establishing a direct relationship between the extent of amino acid sequence similarity and the capacity of heterologous proteins to be functional in host species. Moreover, our data suggest that evolution affected the interaction of the acidic proteins with the ribosome rather than the structural features responsible for their primary functions.
Bermejo
B
B
Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Madrid, Spain.
Prieto
J
J
Remacha
M
M
Coloma
A
A
Ballesta
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D004023:Dictyostelium / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010750:Phosphoproteins; D012269:Ribosomal Proteins