Synthesis and processing of mammalian protamines and transition proteins.
Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms.
Green
G R
GR
Department of Biology, Amherst College, MA 01002.
Balhorn
R
R
Poccia
D L
DL
Hecht
N B
NB
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis* / Q000378:metabolism; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D010766:Phosphorylation; D011479:Protamines / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D012671:Seminiferous Tubules / Q000378:metabolism*; D013087:Spermatids / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D011479:Protamines; C053886:spermatid transition proteins; D010450:Endopeptidases