Phosphorylation of the Saccharomyces cerevisiae La protein does not appearto be required for its functions in tRNA maturation and nascent RNA stabilization

An abundant nuclear phosphoprotein, the La autoantigen, is the first protei n to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcri pts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal p athway of tRNA maturation. Experiments in which the human protein was expre ssed in S. pombe have suggested that phosphorylation of the La protein regu lates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phos phorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p , in which each of the serines was mutated to alanine, were expressed in ye ast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we dete rmined that we had identified and mutated all major sites of phosphorylatio n in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting rev ealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturat ion or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 protei ns localized to both nucleoplasm and nucleoli, demonstrating that phosphory lation does not affect subcellular location. Thus, although La proteins fro m yeast to humans are phosphoproteins, phosphorylation does not appear to b e required for any of the identified functions of the S. cerevisiae protein .