PHOSPHORYLATION OF SRP1P, THE YEAST NUCLEAR-LOCALIZATION SIGNAL RECEPTOR, IN-VITRO AND IN-VIVO

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisi ae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extr acts that phosphorylates Srp1p complexed with NLS peptides/proteins. F rom partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophos phorylation of the 36 kDa subunit of the yeast enzyme was stimulated b y the substrate, GST-Srp1p. However, with the use of a more refined sy stem, no stimulation of autophosphorylation of the 36 kDa subunit of y east CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It wa s shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-termi nal end causes great stimulation of phosphorylation without NLS peptid es/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region , probably between amino acids 403 and 516. Binding of NLS peptides/pr oteins most likely causes a change in protein conformation, exposing t he CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell gr owth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ab ility of Srp1p purified from E coli and treated with calf intestinal p hosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significan t difference was observed. It appears that NLS binding does not requir e any phosphorylation of Srp1p, either by CKII or by some other protei n kinase.