The posttranslational modification and secretion of vitellogenin in xenopus laevis

A procedure was developed for the preparation of rough and smooth microsomes from small quantities of liver obtained from estrogen stimulated Xenopus laevis females. The separation involves the centrifugation of a postmitochondrial supernatant over a CsCl-containing, discontinuous sucrose gradient. Morphological, biochemical, and enzymatic characterization of these fractions indicates that excellent separation of rough microsomes from smooth microsomes is achieved. In addition, pulse-chase experiments in which (5 min) pulses with [³H]leucine are used to demonstrate that rough and smooth microsomes each exhibit the predictable patterns of incorporation characteristic of secretory protein synthesis and intracellular translocation.

This procedure was combined with suitable incubations for pulse chase experiments which demonstrate the subcellular sites of vitellogenin phosphorylation. The results of these experiments indicate that approximately 70% of the phosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of iv the total incorporated phosphate. This is further supported by the 3 32 analysis of newly synthesized and assembled [³H,³²P]vitellogenin on sodium dodecylsulfate-polyacrylamide gels and measurements of protein kinase activity in microsomal subtractions.

The presence of a divalent-cation requiring protein kinase (E.G. 2.7.1,37, ATP;protein phosphotransferase) is demonstrated in the microsomes derived from the liver of estrogenized female Xenopus laevis. Using Xenopus phosvitin, a proteolytic derivative of vitellogenin, as substrate, we have characterized various requirements and kinetic properties of this enzyme activity. In addition we present data showing that this protein kinase is responsible for phosphorylating hepatic precursors to serum vitellogenin in vivo.

Pulse-chase experiments measuring the rates of incorporation of radiolabeled glucosamine and galactose into intracellular vitellogenin show that glycosylation of this multicomponent protein occurs in a Golgi-enriched fraction isolated from homogenized liver slices. No apparent role for the rough endoplasmic reticulum was demonstrable. Kinestics of the intracellular translocation of glycosylated intermediates of vitellogenin indicate that the galactosylated form is secreted more rapidly than the glucosamine-labeled precursor. This was corroborated by measuring the rates of accumulation of various pulse-labeled forms of vitellogenin in the chase medium. In addition, a negligible amount of mannose was incorporated into intracellular or secreted vitellogenin.

The antibiotic tunicamycin was shown to inhibit [³H]glucosamine incorporation into microsomal vitellogenin by 70%, without any significant effects on the synthesis of the protein backbone. In addition, nonglycosylated vitellogenin showed normal secretion kinetics. After suitable pretreatment with the antibiotic followed by a labeling period in tunicamycin-free medium, mannose was still not incorporated into vitellogenin whereas glucosamine behaved in a typical manner. In contrast to this finding, gas-liquid chromatography of the alditol acetate derivatives of the neutral hexoses of vitellogenin showed that mannose was indeed a major component of the vitellogenin oligosaccharide side chain.

These results indicate that the oligosaccharide component of vitellogenin in Xenopus is a "complex" type of carbohydrate unit which is linked via an N-glycosidic bond between an asparagine residue and N-acetylglucosamine. With respect to subcellular localization of glycoprotein assembly in Xenopus liver, there is a significant departure from currently accepted models of glycoprotein synthesis.