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The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725–736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781–786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.

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.

The polypeptide, lipid, and carbohydrate components of the lipovitellin complex from anostracan crustacean, Artemia salina, have been investigated. The lipovitellin isolated from the yolk granules is a carotenoid.lipoglycoprotein complex, containing 3.3% carbohydrate and 8.6% lipid. About half of the carbohydrate fraction is mannose and the rest consists of galactosamine, galactose, and glucosamine. The lipid fraction contained 67% phospholipid and 33% neutral lipid. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine are three major phospholipid components and their relative ratio changes by developmental stages. The neutral lipid fraction is composed of mainly triacylglycerol and cholesterol. Two polypeptide chains are identified as apoprotein components of the lipovitellin complex. Their molecular weights are estimated by sodium dodecyl sulfate gel electrophoresis to be 190,000 and 68,000, respectively. The third polypeptide with Mr = 85,000 is a very minor component and exists in nonstoichiometric levels in the lipovitellin complex. On the basis of an apparent molecular weight of the lipovitellin complex determined by gel filtration chromatography, the dimeric lipovitellin form is suggested, each monomer of which contains one set of Mr = 190,000 and one set of Mr = 68,000 apoproteins. Each dimeric lipovitellin complex contains approximately 70 associated lipid molecules, of which about 4 are attributed to canthaxantin, and 100 bound carbohydrate molecules.

We have investigated the effects of ligand concentration on the intracellular behavior of the yolk protein precursor vitellogenin in Xenopus oocytes. After internalization by receptor-mediated endocytosis, vitellogenin is delivered to a final storage compartment in mature, high density (1.23 g/ml sucrose) yolk platelets by a biphasic transport pathway. The first phase consists of a relatively rapid increase in the density of ligand-containing organelles to 1.21 g/ml, a region of the sucrose gradient containing a subpopulation of light yolk platelets and lysosomal enzyme activity. The second and rate-limiting phase in vitellogenin transport is the appearance of ligand in mature yolk platelets. The first phase of transport is accompanied by cleavage of vitellogenin into the mature yolk proteins. The majority of the light yolk platelets are not converted into heavy platelets when vitellogenin is absent from the medium for 24 h, indicating that they are a relatively stable organelle which does not mature into heavy platelets during this time period. The progress of vitellogenin through both phases of transport exhibits a concentration dependence, with much slower rates of density increase and lack of appearance in heavy yolk platelets at subsaturating ligand levels. The majority of vitellogenin entering oocytes at low ligand concentrations remains for at least 60 min in a low density endocytic compartment which is accessible to a second pulse of ligand. Vitellogenin-colloidal gold tracer studies indicate that this low density compartment is composed primarily of multivesicular bodies. Low concentrations of vitellogenin residing in this compartment for 4 h are no longer accessible to newly internalized ligand, suggesting that movement into an organelle incapable of fusing with endosomes has occurred.

We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.