Proc. Natl. Acad. Sci. USA Vol. 86, pp. 906-910, February 1989 Cell Biology Specific postendocytic proteolysis of B in oocytes does not abolish receptor recognition (/pepstatin A/endosome) JOHANNES NIMPF, MARKUS RADOSAVLJEVIC, AND WOLFGANG J. SCHNEIDER* Department of Biochemistry and and Research Group, University of Alberta, Edmonton, AB, Canadi T6G 2H7 Communicated by Richard J. Havel, October 11, 1988

ABSTRACT Upon receptor-mediated transfer of plasma family ofglycophospholipoproteins with molecular masses of very low density lipoprotein (VLDL) particles into growing -240 kDa, which are degraded inside the oocyte to lipovi- chicken oocytes, their major apolipoprotein (apo) component, tellin(s) (the amino-terminal portion of ), phos- apoB, is proteolytically cleaved. apoB fragmentation appears vitin(s) (the central, phosphoserine/serine-rich domain), and to be catalyzed by cathepsin D or a similar pepstatin A-sensitive carboxyl-terminal polypeptides termed phosvettes or lipovi- protease and results in the presence of a characteristic set of tellin(s) II. The specific breakdown of has been polypeptides on VLDL particles. The nicks introduced suggested to constitute a regulatory step in the fusion into the apoB backbone during postendocytic processing occur between endocytic compartments in Xenopus oocytes (18). in yolk platelets and appear to prepare internalized VLDL for Lastly, it is known that chicken egg yolk does not contain storage in yolk. Since yolk VLDL binds to chicken receptors intact (apoB), the that directs specific for apoB-containing lipoproteins in identical fashion to VLDL particles to receptors on the oocyte plasma membrane plasma VLDL, the possibility exists that the developing embryo (1, 19-21). utilizes yolk VLDL as a by way of receptor-mediated Besides having a function in maintenance of a high rate of endocytosis. yolk formation, specific proteolysis may play a role in assuring the developing embryo access to macromolecular Specific cell-surface receptors are involved in the endocyto- , such as lipoproteins. Since this access might, at into least in part depend on receptor-mediated endocytosis, we sis of very low density lipoprotein (VLDL) particles have become interested in the binding behavior of VLDL growing oocytes of the laying hen (1). The massive import of particles stored in yolk. Here we report on a possible VLDL is part of the process of yolk deposition that assures mechanism for the processing of plasma VLDL by in- accumulation of sufficient amounts of nutrients in the oocyte traoocytic proteolysis and demonstrate that the proteolyzed for growth of the avian embryo. In addition to VLDL, most, yolk VLDL has identical receptor-binding characteristics as if not all, yolk components are believed to be transported the lipoprotein in the circulation. across the plasma membrane ofthe oocyte by way of specific receptors. Systems of receptor-mediated endocytosis may exist for transferrin (2, 3), various vitamin-binding MATERIALS AND METHODS (4-12), and IgG (13, 14). Though direct biochemical demon- Materials. We obtained pepstatin A, thrombin (human stration ofoocyte receptors for most ofthese proteins has not plasma, no. T-6759), kallikrein (porcine pancreas, no. K- been reported, we have recently characterized the chicken 3627), cathepsin D (bovine spleen, no. C-3138), and bovine oocyte plasma membrane proteins responsible for the trans- serum albumin (no. P-5763) from Sigma; molecular mass port of VLDL (1) and vitellogenin, another major yolk standards from BRL; acrylamide, bisacrylamide, and glycine component (15). from Schwartz/Mann; nitrocellulose paper BA 85 from We are now concerned with the mechanisms by which the Schleicher & Schuell; Nuflow acetate membrane filters N25/ chicken embryo utilizes the nutrient stores of the oocyte 45 from Oxoid (Basingstoke, U.K.); goat anti-rabbit IgG (no. content for growth. Presumably, the lipoproteins VLDL and 0612-3151) from Cooper Biomedical; and Na'25I from Ed- vitellogenin are a major source not only for protein and monton Radiopharmaceutical Center (Edmonton, AB, Can- carbohydrate components but also for essential lipid building ada). Other materials were from previously reported sources blocks and energy. It appears that most of those plasma (1, 21). proteins that are concentrated in the oocyte through receptor- Animals and Diets. White Leghorn layers (8-18 months old) mediated endocytosis undergo specific proteolytic process- were obtained from a local poultry farm and maintained on ing following uptake from the plasma. This has been sug- layer mash fed ad libidum, with a light period of 12 hr. White gested for riboflavin-binding protein (16), which exists in Leghorn roosters (4-8 weeks old) were kindly provided by F. three closely related but distinct forms in the laying hen that Robinson (Department of Animal Sciences, The University can be isolated from plasma, egg white, and yolk. The egg of Alberta, Edmonton, AB, Canada) and maintained on white form is distinguished from the other two forms by its grower mash at the same conditions as the hens. We used different carbohydrate composition; the yolk protein is a adult female New Zealand White rabbits for raising antibod- truncated form of the plasma protein, lacking 11 or 13 amino ies. acid residues at the carboxyl terminus (5, 6). The carboxyl- Preparation and Analysis of Lipoproteins, Filtration Assay. terminal truncation in yolk riboflavin-binding protein has Lipoprotein fractions were isolated as described (21). VLDL been proposed to be elicited by specific intraoocytically from yolk (yVLDL) was isolated as follows: the yolk from localized protease(s) (6, 17). Another example of specific one freshly laid egg was separated carefully from the egg postendocytic processing is provided by the vitellogenins, a white and rinsed with ice-cold saline. The yolk membrane

The publication costs of this article were defrayed in part by page charge Abbreviations: apo, apolipoprotein; (V)LDL, (very) low density payment. This article must therefore be hereby marked "advertisement" lipoprotein; pVLDL, plasma VLDL; yVLDL, yolk VLDL. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 906 Downloaded by guest on September 28, 2021 Cell Biology: Nimpf et al. Proc. Natl. Acad. Sci. USA 86 (1989) 907 was punctured with a needle and the yolk was squeezed out Table 1. Comparison of pVLDL and yVLDL composition and mixed with 5 vol of ice-cold buffer (20 mM Tris HCl/150 Relative % mM NaCI/0.2 mM EDTA/1 mM phenylmethylsulfonyl flu- oride/5 AM leupeptin, pH 8). The suspended yolk was Component pVLDL yVLDL subjected to ultracentrifugation at 150,000 x ga, for 12 hr at Protein 12.3 12.9 40C. The floating waxy yellow material was mixed with 10 vol Cholesterol, free + esterified 4.6 4.3 of buffer as above and subjected to a second centrifugation Phospholipid 16.8 17.0 under the same conditions. The yVLDL was recovered from Triglyceride 66.3 65.8 the top of the tube. Lipoproteins were radiolabeled with 1251 by the iodine monochloride method as described (1). All pVLDL and yVLDL were purified, and their content of protein, lipoproteins were extensively dialyzed against 0.15 M NaCl/ total cholesterol, phospholipid, and triglyceride was determined. 0.2 mM EDTA, pH 7.4, and stored at 40C. Lipoprotein g/ml and compared the composition (Table 1), morphology, concentrations are expressed in terms ofprotein content (22). and protein components (Fig. 1) ofthe particles. As shown in Triglyceride, cholesterol, and phospholipid contents of iso- Table 1, the relative amounts of triglyceride, cholesterol, lated VLDL fractions were determined with commercially phospholipid, and protein were essentially identical in the available assay kits. Oocyte membranes were prepared from two particles. These data suggested that pVLDL particles are small oocytes (3-15 mm in diameter) and extracted with octyl transported into the oocyte without major disruption of their glucoside as described (1). After dilution of the detergent to structural integrity, such as by lipolytic processes. This below its critical micellar concentration, the receptor protein notion was supported by morphological analysis of the was recovered by centrifugation and the pellet was used for isolated particles (Fig. 1 A and B)-namely, VLDL particles a solid-phase filtration assay as described (1, 23). of both plasma and yolk were spherical and uniform in size, Electron Microscopy. VLDL samples were negatively with a diameter of 38 + 0.8 nm (mean ± SEM, determined on stained with 2% sodium phosphotungstate (pH 7) and pho- samples of 400 particles from each source). These findings tographed in a Philips EM420 operated at 100 kV. The agree well with those of Burley et al. (20), who determined instrumental magnification was x60,000 and the low-dose mean particle diameters of 35 and 36 nm for pVLDL and unit was employed to minimize specimen damage. "yolk lipoprotein," respectively. Together with the obser- Electrophoretic and Blotting Procedures. One-dimensional vations ofGilbert and collaborators (27, 28), who showed that sodium dodecyl sulfate (SDS) gel electrophoresis was per- VLDL particles traverse the interstitial space of the granu- formed according to Laemmli (24) using a mini-gel system losa cell layer as well as the basal lamina unchanged, our data (mini Protean II slab cell) or a regular gel system (Protean II strongly suggest that intact pVLDL particles are transported slab cell) from Bio-Rad. Electrophoresis was conducted on into the oocyte. The accumulated evidence does not support gradient gels (4.5-18% polyacrylamide) at 200 V at room a previous suggestion (29) that lipoprotein lipase produced by temperature for 40-50 min for mini gels and at 35 mA per gel the granulosa cells might act on pVLDL in order to mediate at 10°C for 6-7 hr for regular gels (16 x 12 x 0.15 cm). the transport of VLDL-derived triglyceride fatty acids into Lipoprotein samples were delipidated by successive extrac- the oocyte. If that were the case, we would expect, unlike tion with 20 vol of CHCl3/CH30H, 2:1 (vol/vol), and presently observed, altered morphology (e.g., smaller size) diethylether/ethanol, 3:1 (vol/vol). Electrophoretic transfer and loss of triglyceride in yVLDL compared to pVLDL. of proteins to nitrocellulose and blotting procedures were In contrast, a comparative analysis by SDS/polyacryl- performed as described (21). amide gel electrophoresis of the apolipoprotein composition Preparation of Yolk Fractions. Yolk was extruded from of VLDL isolated from plasma and yolk (Fig. 1 C and D) small oocytes (3-15 mm diameter), diluted with 4 vol ofbuffer demonstrated that their protein components were strikingly containing 5 mM Hepes, pH 7.4/0.25 M sucrose/1 mM different. In particular, the highest band in pVLDL, repre- EGTA/0.5 mM MgCl2 (buffer A), and subjected to centrif- senting apoB (-500 kDa) (30), was completely absent from ugation at 5000 x ga, for 10 min. The resulting supernatant was saved, and the pellet was gently suspended in 2 vol of A. pVLDL C. D. buffer A and resedimented at 5000 x ga, for 5 min. For digestion studies, the pellet, designated yolk platelets, was kDa in 1 vol of buffer A, subjected to one cycle of 200 resuspended _ freeze-thawing, and sonicated for 20 sec (Sonifier model W 185, Heat Systems/Ultrasonics) with a microprobe at setting 6, followed by two cycles offreeze-thawing. The supernatant - 97 from the first centrifugation step was used immediately. Digestion of 1251I-labeled VLDL from plasma (1251I-pVLDL) - 68 was performed as described in the legend to Fig. 3. B. vVLDL Preparation of Antibodies. Polyclonal antibodies against - 43 chicken apoB were raised in adult female New Zealand rabbits as described (21). apoB was isolated by electroelution - 29 (25) from a 4.5-18% gradient SDS/polyacrylamide -gel on which pVLDL apoproteins had been separated. The was prepared for injection by mixing equal volumes of the -amoomw .- 18 electroeluate and Freund's adjuvant as described (21). Anti- - 14 sera were tested by Western blotting, and IgG fractions were purified by affinity chromatography on protein A-Sepharose -50 nm p y (26). FIG. 1. Comparison of VLDL from plasma (p) and yolk (y). Pure VLDL fractions were prepared and stained with 2% sodium phos- RESULTS AND DISCUSSION photungstate and processed for electron microscopy (A and B) or delipidated and 20 ,ug of protein each was subjected to SDS/ Characterization of VLDL from Plasma Versus Yolk. We polyacrylamide gradient gel electrophoresis (4.5-18%), followed by isolated lipoprotein fractions from both plasma oflaying hens staining with Coomassie blue (C and D). Size standards are indicated and egg yolk by ultracentrifugal flotation at a density of 1.006 in kDa. Downloaded by guest on September 28, 2021 908 Cell Biology: Nimpf et al. Proc. Natl. Acad. Sci. USA 86 (1989) yVLDL, which contained six major and three minor bands, exception oftwo or three minor bands, indistinguishable from all of lower molecular mass than apoB. The two bands in that of yVLDL. In lane E of Fig. 2, pVLDL was incubated common to both particles (migrating to positions of 16 and 9.5 with cathepsin D but pepstatin A (200 ,g/ml) was present kDa, respectively) represent the dimeric and monomeric throughout. Proteolysis was totally blocked by the inhibitor, forms of apo-VLDL-II, respectively (21, 31, 32). demonstrating that the breakdown observed with the com- Are the higher molecular mass polypeptides in yVLDL mercial preparation of cathepsin D was not due to a contam- derived from plasma apoB? Extensive postendocytic prote- inating, pepstatin A-resistant protease. These results sug- olysis of apoB has been previously suggested by studies gested strongly that cathepsin D or a similar pepstatin tracing the fate of 3H-labeled VLDL proteins following A-inhibitable protease is the physiologically active agent in intravenous injection (19). However, a direct comparison of the production of yolk apoB. 3H-labeled apoB-derived yolk products with unlabeled pro- Second, we performed immunoblotting analysis ofyVLDL teins isolated from yolk lipoproteins was not presented in proteins and cathepsin D-generated fragments with polyclo- these studies. In an earlier report the same group (20) had nal IgG raised against intact chicken apoB. Almost all of the concluded that not all high molecular mass apoproteins ofthe yVLDL proteins were cross-reactive with the polyclonal yolk lipoprotein were derived from apoB. We wanted to anti-apo-B IgG and, more importantly, the immunoreactive elucidate the of the polypeptides on yVLDL particles fragments generated by cathepsin D were very similar to by a different approach: first, we investigated the effect of those in yolk apoB (not shown). various proteolytic enzymes on the protein components of Finally, we attempted to directly demonstrate the pepstatin intact pVLDL. The goal ofthese experiments was possib'ly to A-inhibitable proteolytic activity within the oocyte. Since identify enzyme(s) that would produce polypeptides from yolk platelets were the most likely source for the enzyme(s) apoB that were identical in mass to the bands seen in the for reasons outlined below, we performed the experiment apolipoprotein moiety of yVLDL. The rationale for the shown in Fig. 3. Yolk was obtained from small oocytes (3- selection of proteases was based on two previous reports 15 mm diameter) and separated into platelets and supernatant dealing with the proteolysis ofyolk proteins. First, Evans and as described in Materials and Methods. When 125I-pVLDL Burley (19) attempted, but failed, to reproduce oocytic was incubated with disrupted platelets, proteolysis of apoB breakdown of chicken apoB in vitro with the enzymes was observed; the resulting radiolabeled fragments were kallikrein, thrombin, chymotrypsin, and trypsin. Thus, these identical to those in 125I-yVLDL (lanes A and B). The proteases could'almost certainly be excluded as candidates. proteolytic activity responsible for this degradation was Second, Opresko and Karpf (18) have shown that an early completely inhibited by pepstatin A (lane C) and absent from step in the proteolytic modification of endocytosed vitello- the post-platelet supernatant (lane D). Incubation of 125[_ genin in Xenopus oocytes can be inhibited by peptstatin A, a pVLDL with the incubation buffer alone did not result in specific inhibitor of cathepsin D, which is localized to the significant degradation of apoB (lane E). Furthermore, un- endosomal/lysosomal fraction of a wide variety of cells (33). disrupted yolk platelets failed to degrade apoB (not shown). Since VLDL and vitellogenin presumably follow a common These findings suggest strongly that yolk platelets contain the intracellular route, at least early in their pathways, we pepstatin A-sensitive protease(s) that effects the posten- reasoned from the available data that cathepsin D, or a docytic degradation of pVLDL-derived apoB in oocytes. cathepsin D-like, pepstatin A-sensitive protease might be Receptor Binding of yVLDL. Our results support the responsible for apoB degradation inside the chicken oocyte. following scenario: pVLDL particles are imported into the Thus, we incubated isolated pVLDL with various proteases oocyte by way of receptor-mediated endocytosis; upon and compared the pattern of proteolytic fragments generated entering endosomal compartments, the apoB moiety is acted with that present in isolated yVLDL (Fig. 2). Thrombin on by cathepsin D or a similar protease; the fragmented apoB treatment resulted in two major and a minor breakdown remains associated with the VLDL particles, which are product (lane F), none of which was identical to any of the stored as part of the yolk without further degradation. Next, yVLDL proteins (lane C); tissue kallikrein (lane G) also we wanted to test whether the apoB-specific avian lipopro- generated polypeptides that were different from those found tein receptor (1, 21) was capable ofbinding yVLDL particles. in yVLDL; however, the pattern obtained by treatment of As outlined in the Introduction, such capacity might suggest pVLDL with cathepsin D at pH 5 (lane D) was, with the A B C D E A B C D E F G 89Wr,- ]-___ _lp_'- kDa 200- 4II

uI,. ' 97-

68- 1_

43-

FIG. 3. Digestion of apoB by yolk fractions. Aliquots of 125I-pVLDL (3 jig of protein, 1 x 105 cpm) were incubated at pH 5 FIG. 2. Enzymatic digestion of pVLDL. Aliquots (50 ug of with yolk platelets (60 ,ug ofprotein; the platelets had been disrupted protein) of pVLDL were digested with 2 jig of cathepsin D at pH 5.0 as described in the text) in the absence (lane B) or presence (lane C) in the absence (lane D) or presence (lane E) of 10 ,g of pepstatin A; of pepstatin A at 200 Mtg/ml or with post-platelet supernatant (60 ,ug with 2 Mg of thrombin at pH 7.0 (lane F); or with 2 ,g of kallikrein of protein, lane D), or in incubation buffer alone (lane E) for 16 hr at at pH 7.0 (lane G) at 37°C for 16 hr in a volume of 50 Ml. Lane B 37°C. Lane A shows 1251-labeled yVLDL (1251-yVLDL) for compar- contains 20 Mg of pVLDL, and lane C contains 75 ug of yVLDL ison. SDS/polyacrylamide gradient gel electrophoresis (4.5-18%) on . SPS/polyacrylamide gradient gel electrophoresis minigels and autoradiography were performed as described in the (4.5-18%) was performed on minigels; proteins were stained with text. Exposure to Kodak XR-5 film was for 10 hr. Size standards are Coomassie blue. Size standards in lane A are the same as in Fig. 1. indicated in kDa. Downloaded by guest on September 28, 2021 Cell Biology: Nimpf et al. Proc. Natl. Acad. Sci. USA 86 (1989) 909 a function ofreceptor-mediated endocytosis in the delivery of 1 2 3 4 5 6 7 8 yVLDL particles to the developing embryo. As shown in Fig. 4, isolated radioiodinated yVLDL indeed bound to the receptor with high affinity and in saturating fashion. In the -- 200 kDa presence of excess unlabeled yVLDL, only a linear binding component representing nonspecific binding was observed. When binding parameters for yVLDL and pVLDL were compared, Kd values of 17.8 and 16.4 pug/ml, and Bma. values . i 97 of 48 and 46 Ag/ml for VLDL from yolk and plasma, respectively, were obtained. These data demonstrate that pVLDL and yVLDL display very similar binding character- - 68 istics for the receptor(s) present in oocyte membrane ex- tracts. To identify the receptor protein(s) to which yVLDL and pVLDL bound, we performed direct and competitive -43 ligand blotting (Fig. 5). Previously, we had demonstrated that plasma LDL and VLDL were specifically recognized by a 95-kDa protein in oocyte membrane extracts (1, 21). Fig. 5 - 29 shows that yVLDL indeed bound to the same receptor as pVLDL, in that both lipoproteins reacted with a 95-kDa protein in crude extracts (lanes 3 and 8) and their binding was mutually exclusive (lanes 4-7). As an additional control, we performed immunoblotting with rabbit IgG directed against the bovine LDL receptor, which recognizes the chicken 1. y P I Y P oocyte receptor as we have previously shown (Fig. 5, lanes IgGC.- 1-yVLDL 15 - pVLDL 1 and 2; ref. 1). In conclusion, both pVLDL and yVLDL bind in identical fashion to the previously identified chicken FIG. 5. Electrophoretic transfer (Western) and ligand blotting of receptor for apoB-containing lipoproteins (1). chicken oocyte receptors. To each lane of a 4.5-18% gradient gel, 35 ,ug of protein of chicken oocyte membrane extract was applied and, Several aspects of our findings are ofgeneral interest with after electrophoretic separation, was transferred to nitrocellulose respect to receptor-mediated lipoprotein metabolism. First, sheets. Individual strips were incubated with the following: rabbit in contrast to human apoB-100, which appears, at least in anti-bovine LDL receptor IgG and nonimmune IgG at 10 ug/ml some subjects, to be cleaved by plasma or tissue kallikrein (lanes 1 and 2, respectively), followed by 1251-labeled goat anti-rabbit (34-36) into two major fragments termed apoB-74 and apoB- IgG (5 ,ug/ml; specific activity = 350 cpm/ng of protein); 125I- 26 (37) while in the circulation as resident of lipoprotein yVLDL (5 Ag/ml; 183 cpm/ng of protein) in the absence (-) (lane 3) particles, the proteolysis of chicken apoB in the oocyte or presence of unlabeled yVLDL (lane 4) or pVLDL (lane 5) at 1 provides an example of intracellular and cell-specific limited mg/ml; and 1251-pVLDL (5 ug/ml; 202 cpm/ng of protein) in the degradation of this large apolipoprotein. apoB-74 and apoB- absence (-) (lane 8) or presence of unlabeled yVLDL (lane 6) or 26, as well as the apoB fragments identified in the present pVLDL (lane 7) at 1 mg/ml. Exposure to Kodak XR-5 film was for study, remain particle bound; this should allow for direct 24 hr. Size standards are indicated in kDa. testing of such particles' capacity to interact with apoB(E) absent from yVLDL, which enabled us to determine its receptors (38, 39). Binding studies with apoB-74 and apoB-26 receptor-binding characteristics in a meaningful way. Fur- particles have not been reported to date, presumably because thermore, inasmuch as the fragmented yolk apoB interacts a subfraction ofLDL in which apoB-100 has been completely with the apoB-specific chicken receptor (21) in identical proteolyzed cannot be isolated. However, intact apoB is fashion to plasma apoB, it might be possible to identify the 60 fragment(s) of chicken apoB that contain the receptor rec- ognition site(s). Kirchgessner et al. (30) recently isolated a 1491-base-pair cDNA clone containing an open reading frame for 433 amino acid residues at the carboxyl terminus of chicken apoB; however, this cDNA clone did not reveal any sequences similar to those implicated in binding of human E apoB to the mammalian LDL receptor (40, 41). Identification C) of active apoB fragment(s) from yolk might facilitate eluci- dation of receptor-binding region(s) on chicken apoB. ~0 Second, the specific processing of plasma apoB within 20 0 5010-5 chicken oocytes may also play regulatory roles in endocyto- sis, ligand traffic, and sustenance ofstructural integrity inside these giant single cells-namely, ofall the cell types express- 0 ing apoB receptors studied to date, only the oocyte appears to generate defined polypeptides of apoB fit for storage. The usual fate ofapoB in the classical LDL receptor pathway (42) is lysosomal degradation to acid-soluble oligopeptides and 1251-yVLDL, Ag/ml free amino acids. Though we cannot eliminate that such degradative processes take place in oocytes as well, our FIG.4014. Binding of 1251-yVLDL to oocyte receptors. Oocyte current observations and the 3H-labeled apoB tracer studies membrane octyl glucoside extracts were precipitated (18 Ag of Evans make it that protein per assay tube) by dilution and incubated with 1251-yVLDL of and Burley (19) unlikely lysosomal (183 cpm/ng of protein) at the concentrations indicated in the hydrolysis is a major fate of endocytosed pVLDL particles. absence (o) or presence (*) of 4 mg of unlabeled yVLDL per ml. *, Rather, we speculate that the specific proteolysis of apoB Specific binding values, obtained by subtracting the values for that occurs within endosomes-i.e., yolk platelets-is the nonspecific binding (*) from total binding (o). Each point represents major step en route to subsequent storage. In this context, the average of duplicate incubations. certain vesicular structures in Xenopus oocytes, termed light Downloaded by guest on September 28, 2021 910 Cell Biology: Nimpf et al. Proc. Natl. Acad. Sci. USA 86 (1989) yolk platelets, harbor active proteolytic enzymes and appar- 17. White, H. B., III, Armstrong, J. & Whitehead, C. C. (1986) ently are precursors to heavy yolk platelets, a storage Biochem. J. 238, 671-675. 18. Opresko, L. K. & Karpf, R. A. (1987) Cell 51, 557-568. compartment for proteolyzed vitellogenin (43, 44). Inasmuch 19. Evans, A. J. & Burley, R. W. (1987) J. Biol. Chem. 262, 501- as vitellogenin degradation in Xenopus oocytes is inhibited by 504. pepstatin A (18), and the current data support a role of 20. Burley, R. W., Sleigh, R. W. & Shenstone, F. S. (1984) Eur. J. cathepsin D (or a protease with similar specificity) in pro- Biochem. 142, 171-176. duction of yolk apoB in chicken oocytes, we believe that 21. Nimpf, J., George, R. & Schneider, W. J. (1986) J. Lipid Res. endosomal processing is an important part of routing yolk 29, 657-667. of 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. proteins to their final destiny. However, since processing (1951) J. Biol. Chem. 193, 265-275. apoB does not abolish receptor binding, we feel that in the 23. Schneider, W. J., Goldstein, J. L. & Brown, M. S. (1980) J. case of apoB, proteolysis is not involved in receptor-ligand Biol. Chem. 255, 11442-11447. dissociation and receptor retrieval, as has been proposed for 24. Laemmli, U. K. (1970) Nature (London) 227, 680-685. the vitellogenin system (18). Current studies into the role of 25. Hunkapiller, M. W., Lujan, E., Ostrander, F. & Hood, L. E. specific proteolysis in yolk formation and embryonal nutri- (1983) Methods Enzymol. 91, 227-236. and 26. Daniel, T. O., Schneider, W. J., Goldstein, J. L. & Brown, tion are necessary along two lines: attempts to (i) identify M. S. (1983) J. Biol. Chem. 258, 4606-4611. isolate the responsible protease(s) and (ii) obtain evidence for 27. Perry, M. M. & Gilbert, A. B. (1979) J. Cell Sci. 39, 257-272. yVLDL binding to receptors in embryonic tissue. 28. Perry, M. M., Griffin, H. D. & Gilbert, A. B. (1984) Exp. Cell Res. 151, 433-446. We thank Calla Shank for excellent technical assistance and 29. Bensadoun, A. & Kompiang, I. P. (1979) Fed. Proc. Fed. Am. Beverly Bellamy for expert secretarial help in preparing this manu- Soc. Exp. Biol. 38, 2622-2626. script. We appreciate the help of Dr. Doug Scraba in our department 30. Kirchgessner, T. G., Heinzmann, C., Svenson, K. L., Gordon, with electron microscopy. These studies were supported by the D. A., Nicosia, M., Lebherz, H. G., Lusis, A. J. & Williams, Alberta Heritage Foundation for Medical Research and a grant from D. L. (1987) Gene 59, 241-251. the Medical Research Council of Canada (MA-9083). J.N. is sup- 31. Kudzma, D. J., Swaney, J. B. & Ellis, E. N. 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