In vivo thiophosphorylation of chromosomal proteins. Recovery and analysis of HeLa histones and derivative phosphopeptides.

HeLa cells are shown to incorporate [35S]thiophosphate (added to the medium as sodium thiophosphate) into the gamma-positions of ATP and GTP, and into other nucleotides. Under these conditions there is a transfer of radioactive thiophosphoryl groups to histones H1, H2A, H3, and H4. The newly thiophosphorylated chromosomal proteins can be recovered selectively by affinity chromatography on organomercurial-Sepharose columns. The thiophosphorylated histone H1 and its NH2-terminal and COOH-terminal fragments were subjected to tryptic digestion and the sulfur-derivatized phosphopeptides were isolated by Hg-affinity chromatography prior to electrophoretic separation of different sites of modification. Thiophosphate was found to be incorporated into both serine and threonine residues of histone H1 in HeLa cells during logarithmic growth. When [3H]threonine and nonradioactive thiophosphate were employed simultaneously as precursors, the thiophosphorylated H1 molecules retained on the mercury column also showed the presence of the [3H]threonine label. It follows that newly synthesized H1 molecules are subject to thiophosphorylation in the growing cell cultures.