The structural relationship between vitellogenin and the yolk proteins in Xenopus laevis

The crystalline inclusions in amphibiam oocytes known as yolk platelets are formed by the incorporation and proteolytic processing of the soluble serum protein vitellogenin. The nature of this transformation was studied by isolating the yolk proteins and then reconstructing the parent molecule by the use of chemical and biological cleavage techniques. Several new methodologies had to be developed to achieve this goal.

Vitellogenin was isolated from the plasma of estrogen-treated animals by selective precipitation with Mg ²⁺ in the presece of EDTA. This technique apparently relies on the specific complexing of the phosphate moieties of the protein by a Mg²⁺:EDTA chelate. The vitellogenin thus obtained is greater than 99% pure and is relatively undegraded. This technique can also be used for determining the vitellogenin titer in the blood of estrogen-treated animals. Vitellogenin was also isolated by DEAE-cellulose chromatography. In combination, these two isolation procedures yield vitellogenin preparations of very high purity.

In order to study the processing of vitellogenin by the oocytes, a method was developed for labeling vitellogenin in vitro to a very high specific activity by using [γ^{32<.sup>p]ATP and a phosphoprotein kinase isolated from Xenopus liver. The phosphoprotein kinase was purified 21,00o-fold from liver cytosol by employing ammonium sulfate fractionation, DEAE-cellulose chromatography and phosphocellulose chromatography. It is specific for phosphoproteins such as casein and phosvitin and its K_m for ATP (2.2 uM) is one of the lowest yet reported for a protein kinase. Using the phosphoprotein kinase, specific activities of over 5x108 cpm/mg have been obtained fr milligram quantities of vitellogenin. The labeled vitellogenin was incorporated into oocytes at a high rate (approximately 300 ng/oocyte/hr) and was processed normally into the yolk proteins.}

Vitellogenin was found to be a heterogeneous population of molecules that could be resolved into at least 3 different molecular weight forms by SDS gel electrophoresis. Native gel electrophoresis of vitellogenin resolves the population into 4 bands, three of which seem to differ in molecular weight and one of which seems to differ in net charge. Isoelectric focusing of vitellogenin confirmed that the protein population was heterogeneous with respect to charge. Chemical cleavage of vitellogenin at the cysteine residues with nitrothiocyanobenzoic acid demonstrated that there were two forms of molecules whose cysteine residues were at different positions with respect to the phosphorylated region of vitellogenin. Therefore, Xenopus vitellogenin seems to be the product of multiple genes.

The vitellogenin-derived yolk proteins were isolated from oocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography and gel filtration. In addition to three previously described yolk proteins (lipovitellin 1, lipovitellin 2 and phosvitin), I found two additional populations of phosphoproteins: phosvette I, which contains 4.8% phosphorus and has a molecular weight of 19,000, and phosvette II, which contains 10.7% phosphorus and has a molecular weight ranging from 9,500 to 14,500. Amino acid analysis of the yolk proteins

vii demonstrated that phosvette I and phosvette II are not derived from the other yolk proteins. A comparison of the amino acid composition of vitellogenin and the yolk proteins revealed that all of the yolk proteins are not present in equimolar amounts in the platelets. The ratio between lipovitellin 2, phosvitin and the phosvettes was determined by protein phosphorus analysis and yielded a lipovitellin 2:phosvitin: phosvettes ratio of 1.00:0.70:0.27. The time course of the cleavage of vitellogenin into the yolk proteins was studied by incubating oocytes with [32p]vitellogenin and examining the labeled cleavage products by SDS gel electrophoresis. This study showed that the phosvettes were indeed derived from vitellogenin and that the time course of their appearance was different from that of phosvitin. It thus appears that there are two classes of vitellogenin molecules that give rise to alternate cleavage products.

The placement of the yolk proteins in the vitellogenin molecules was investigated by N-terminal analysis and cyanogen bromide cleavage of vitellogenin and the yolk proteins. This study showed that the N-terminal of vitellogenin contains the future lipovitellin l molecule while the C-terminal region of vitellogenin gives rise to the lipovitellin 2 molecule. Phosvitin and the phosvettes seem to be derived from an internal region of vitellogenin. Since both the lipovitellin l and lipovitellin 2 region of vitellogenin are in tight association, and since phosvitin was found to be on the outside of the molecule, it is proposed that the cleavage of vitellogenin into the yolk proteins is a mechanism by which the phosvitin region of vitellogenin is removed, thus exposing the insoluble lipovitellin core proteins.