4 Yolk Proteins JOSEPH G. KUNKEL and JOHN H. NORDIN University of Massachusetts, Amherst, Massachusetts, USA 1 The study of yolk proteins 84 1.1 Central issues 84 1.2 Constitution of yolk 85 1.3 Location in the egg and embryo 85 1.4 Function of the yolk 85 1.5 Evolutionary homology of yolk proteins 87 1.6 Structural classes of vitellogenins 89 1.7 Genetic approaches to vitellin structure 90 2 Yolk protein chemistry 90 2.1 Table of yolk protein properties 90 2.2 Vitellin, the major yolk protein 90 2.2.1 Vitellogenin amino acid composition 91 2.2.2 Vitellogenin peptide compositions 94 2.2.3 Vitellogenin oligosaccharide 95 2.2.4 Microheterogeneity of vitellin oligosaccharide 96 2.2.5 Vitellogenin lipids 97 2.2.6 Vitellin phosphate 98 2.3 Lipophorin 98 2.3.1 Lipophorin properties 98 2.3.2 Lipophorin amino acid composition 99 2.3.3 Lipophorin carbohydrate 100 2.3.4 Lipophorin lipid 100 3 Synthesis of yolk protein 101 3.1 Hormonal induction of vitellogenin 101 3.2 Vitellogenin protein synthesis 102 3.3 Vitellogenin carbohydrate biosynthesis 102 3.4 Vitellogenin lipid biosynthesis 103 3.5 Vitellogenin phosphate biosynthesis 103 3.6 Post-translational modification of vitellogenin 103 3.6.1 Carbohydrate modification 104 3.6.2 Protein modification 104 4 Secretion 105 5 Uptake of vitellogenin 105 6 Integration of vitellogenin gene expression 106 References 107 Addendum 110 83 1THE STUDY OF YOLK PROTEINS three classes of perhaps non-homologous Vgs emerging (see section 1.6; Harnish, D. and White, B., 1982). 1.1 Central issues The status and importance of yolk protein was thrown into doubt by the finding that Bombyx mori Knowledge of the chemistry and biology of yolk eggs and embryos can survive and develop to hatch- proteins has expanded explosively in the past ing in a male milieu without the female-specific decade and promises to be equally as volatile in the protein, Vg (Yamashita, O. and Irie, K., 1980). The present one. Some old issues have been partially significance of this finding is somewhat diluted by resolved but new issues are arising with the applica- the fact that this species' Vg, although the most tion of new technology. Reviews in the past decade abundant single protein in the egg, makes up only (Engelmann, F., 1979; Gong, H. and Chai, C.-H., 40% of the soluble proteins in the egg. Other en- 1979; Hagedorn, H. and Kunkel, J., 1979) have con- dogenous ovary-specific (Borovsky, D. and Van- sidered the biology and biochemistry of the prin- Handel, E., 1980) and non-female-specific proteins ciple yolk protein precursor vitellogenin (Vg) as (Irie, K. and Yamashita, L., 1980) may also serve as well as those other serum proteins (Wyatt, G. and yolk protein. Nonetheless, there are perhaps Pan, M., 1978) likely to be the major or at least proteins in yolks of insects in general other than the minor "contaminants" of any yolk extract. The roles female-specific vitellin (Vt) that may have roles as of some of these minor components will be important, or in light of the observations on important issues for research in the coming decade. Bombyx, more important, to play in support of Data on yolk protein chemistry have expanded embryonic development. We will review the debate considerably now that new techniques of DNA on lipophorin (Lp), one of those proteins described analysis are providing cloned cDNA and genomic- in several insect orders which may exemplify a DNA with which to study the relationships between minor but important yolk component. the Vg gene and the final secretion products Another confusing point which has not as yet (Bownes, M., 1979; see Berger, E. and Ireland, R., been entirely resolved is the tissue site of Vt vol. 2). Our understanding of the genes for yolk synthesis. While it is clear that insect Vg is proteins in a few select species may soon transcend synthesized in fat body in widely different insect that encompassing the physiology and biology of groups (Pan, M. et al., 1969), it now appears that the translation products. The primary translation dipteran Vg is also synthesized in a number of product, or previtellogenin, and the post- ovarian cell types (Srdic, Z. et al., 1979; Jowett, T. translational processing of this protein have also and Postlethwait, J., 1980; Brennan, M. et al., been lively issues. Vg is a particularly good model 1982). This again raises the problem of homology of a complex protein in which to study processing between Vgs of different groups. Distinct fat body because of the massive amounts of Vg that are and follicle cell products with separate non-nutritive produced (Tata, J. and Smith, D., 1979) and the functions have been proposed previously extensive changes in subunit structure that occur in (Anderson, L. and Telfer, W., 1969; Bast, R. and its physiological lifespan. The primary translation Teller, W., 1976; Kelly, T. and Telfer, W., 1979). product's size is currently an important issue Currently it appears that the higher dipteran Vts because it is expected to be a principal conserved from the various tissue sources are all homologous. property by which the degree of homology of the The interesting cell physiological paradox of ap- various egg-bound forms (vitellins) can be deter- parently identical gene products in different tissues mined. The details of post-translational processing being processed differently, for export in one tissue are important because they can be investigated to and for storage in another, is also raised. The elucidate protein function and can be used to deter- resolution of this question may contribute to our mine molecular and physiological homologies be- basic knowledge of how macromolecules evolve tween species. These homologies may be important and how they are directed to their destinations. indicators of conserved functions. The issues of size The question of sexual dimorphism of vitello- and processing are at present being resolved with genic competence in insects is also in a resolution 84 stage. While it was thought that both male and tial yolk to allow development to a larva which can female egg-laying vertebrates respond equally to start drinking a uterine milk, the nourishment for estrogen stimulation by secretion of Vg from the extended growth in the female's uterus (Ingram, M. liver, insect males were thought incapable of Vg et al., 1977). This makes the study of yolk a synthesis (see Hagedorn, H., vol. 8). Neither of the nutritional, as well as biochemical and develop- foregoing assertions is correct. Male insects of at mental, problem. While other stored components of least four major orders have been experimentally the egg (mitochondria, ribosomes, lipid, glycogen, induced to produce Vg (Mundall, E. et al., 1979; uric acid, vitamins, conjugated hormones and Kambysellis, M., 1977; Dhadialla, T. and Wyatt, minerals) are undoubtedly important in the physi- G., 1981; Chalaye, D., 1979) and male livers of ology of the embryo, protein and lipid yolk some vertebrate species, e.g. turtles (Ho, S. and predominate and this chapter concentrates on the Callard, I., 1980), are reluctant to do so following yolk proteins. These proteins are stored primarily in simple estrogen treatment in vitro. Focus of yolk granules, large membrane-bound quasi- research efforts will surely now be on relative rates crystalline storage vesicles which are formed by the of Vg synthesis and hormonal and cellular accretion of smaller pinocytotic vesicles (Anderson, mechanisms that permit the observed sexual E., 1970; Roth, T. et al., 1976). dimorphisms. For example the recent study of Nair, K. et al. (1981) has eliminated the post- 1.3 Location in the egg and embryo metamorphic polyploidization of the fat body as the sexual dimorphic mechanism in Locusta. Yolk granules are relatively evenly distributed in Few of the above issues can be totally resolved the centrolecithal egg of the insect. An outer cortex without a thorough understanding of the chemistry of the egg is relatively free of large yolk granules. of the molecules concerned. In the present chapter As cleavage of the egg proceeds, the central yolky we hope to provide a description of the chemistry of area does not take part in any cleavage process and yolk proteins, and interpret that chemistry to en- comes to lie within a cavity surrounded by the lighten the important issues at hand: sexual dimor- epithelium of the blastoderm (see Sander, K., et al., phism, complex protein synthesis, secretion, post- this volume). The yolk is thus somewhat separate translational processing and macromolecular from the tissues to which it will be providing nutri- evolution. ment, and mechanisms must be established by which the yolk is efficiently accessed and utilized 1.2 Constitution of yolk by the developing embryo. A yolky egg has substantial stored products in its 1.4 Function of the yolk cytoplasm which transcend the needs of the embryo for survival through early cleavage. (Non-yolky By virtue of its central location in the egg the yolk eggs, such as that of the sea urchin or Amphioxus, must be called upon by the peripheral embryo to pass through early embryology and hatch quickly to release its nutriment. This process is not well under- begin feeding and developing at an early embryonic stood. It may require an extra embryonic cell type, age.) It is interesting that the small amount of the vitellophage, to invade the yolky area (Tanaka, reserve material in these "non-yolky eggs" is a A., 1976) and actively digest the yolk materials, this protein yolk. Protein storage is thus a normal cell type serving as an intermediary for the embryo feature of even "non-yolky" eggs.