Phosphorylation of the Saccharomyces cerevisiae La protein does not appear to be required for its functions in tRNA maturation and nascent RNA stabilization.
An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.
Long
K S
KS
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Cedervall
T
T
Walch-Solimena
C
C
Noe
D A
DA
Huddleston
M J
MJ
Annan
R S
RS
Wolin
S L
SL
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000378:metabolism; D001665:Binding Sites; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020871:RNA Stability; D012331:RNA, Fungal / Q000096:biosynthesis*; D012343:RNA, Transfer / Q000096:biosynthesis*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D000097585:SS-B Antigen
D001324:Autoantigens; D005656:Fungal Proteins; D020033:Protein Isoforms; D012313:RNA; D012331:RNA, Fungal; D012343:RNA, Transfer; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000097585:SS-B Antigen; C088396:LHP1 protein, S cerevisiae