Abstract
Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.
Highlights
Yolk deposition into rapidly growing oocytes in a variety of oviparous species is achieved by receptormediated endocytosis of circulating yolk protein precursors
The homology of the two yolk precursor molecules extends to their intracellular fates following receptor-mediated endocytosis in oocytes: both proteins are proteolytically processed into a small number of fragments
In order to be used in ligand blotting experiments, lipovitellin was first dialyzed for 12 h at 4 “C against a buffer containing 500 mM
Summary
The homology of the two yolk precursor molecules extends to their intracellular fates following receptor-mediated endocytosis in oocytes: both proteins are proteolytically processed into a small number of fragments These fragments are: the amino-terminal region, termed lipovitellin I, which forms the insoluble fraction of yolk; the phosphoseryl-rich domain (phosvitin) and smaller, less well characterized fragments, most likely derived from the carboxyl-terminal region of VTG that include lipovitellin II and the phosvettes [1]. We have begun to address the issue of specificity of these receptors in regards to the VTG domain they recognize Such studies can be expected to advance our knowledge of molecular mechanisms underlying receptor-mediated endocytosis, because elegant kinetic [19, 20] and cell biological [21,22,23] investigations have shed light on the overall endocytic pathway of vitellogenin in Xenopus oocytes, but the receptor protein itself has resisted characterization to date
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