Identification and mutation of phosphorylation sites in a linker histone -Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena

Linker histone phosphorylation has been suggested to play roles in both chr omosome condensation and transcriptional regulation, In the ciliated protoz oan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that re quirements for phosphorylation of macronuclear H1 may be limited to transcr iptional regulation. Here we report the major sites of phosphorylation of m acronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, pres ent in a single cluster, were identified by sequencing P-32-labeled peptide s isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kina se consensus sequences. Phosphoserine was detected at two non-proline-direc ted sites that do not resemble known kinase consensus sequences. Phosphoryl ation at the two noncanonical sites appears to be hierarchical because it w as observed only when a nearby p34cdc2 Site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phospho rylation was abolished. These data suggest that the five sites identified c omprise the entire collection of sites utilized by Tetrahymena and demonstr ate that phosphorylation of macronuclear H1, like the protein itself, is no t essential for viability in Tetrahymena.