Biosynthesis of the vitellogenins. Identification and characterization of nonphosphorylated precursors to avian vitellogenin I and vitellogenin II.

Avian vitellogenin consists of two major species designated VTG I and VTG II. Rooster hepatocytes were employed to identify intracellular forms of the vitellogenins and to characterize biosynthetic intermediates of VTG I and VTG II. After labeling with [3H]serine, intracellular vitellogenin radioactivity was seen in mature VTG I and VTG II but was primarily found in two species, pVTG I and pVTG II, which showed greater mobilities in sodium dodecyl sulfate-polyacrylamide gels. The pVTG species were identified as vitellogenins by reaction with antibodies against plasma VTG II and against the mixture of VTG I and VTG II. Immunological and peptide mapping procedures were used to relate pVTG I and pVTG II to secreted VTG I and VTG II, respectively. Pulse-labeling and pulse-chase experiments showed that the pVTG species are precursors to the secreted vitellogenins and are thus discrete intermediates in the biosynthesis of the vitellogenins. Additional labeling experiments showed that the pVTG species are glycosylated but not phosphorylated. The stages of vitellogenin biosynthesis may be ordered as follows: polypeptide synthesis leads to glycosylation leads to phosphorylation leads to secretion. The presence of only small quantities of the phosphorylated vitellogenins intracellularly indicates that when phosphorylation is completed, the vitellogenins are rapidly secreted from the hepatocyte. The differences in the electrophoretic mobilities of the pVTG and VTG species suggested that sodium dodecyl sulfate-polyacrylamide gel electrophoresis does not accurately estimate the molecular weights of the heavily phosphorylated vitellogenins. This was confirmed directly by showing that the mobility of plasma vitellogenin increased upon dephosphorylation. An independent estimate of vitellogenin molecular weight was made by gel chromatography in 7 M guanidine-HCl. With this method, the molecular weights of the pVTG and VTG species were indistinguishable and in agreement with the molecular weight of the pVTG species as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These analyses indicate that the vitellogenin polypeptide has Mr approximately equal to 180,000. This value is 60,000-70,000 less than commonly estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The implications of this lower molecular weight are discussed in relation to vitellogenin structure and the egg yolk polypeptides which may derive from each vitellogenin.