Aug 25, 2017 - MULTIPLE VLTELLOGENINS IN XENOPUS LAEVIS GIVE RISE TO MULTIPLE FORMS OF THE YOLK. PROTEINS*. (Received for publication ...
JOURNALOF BIVLOCICAL CHEMISTRY Val. 256, N o 16. Issue of August 25. pp. 8626-8634,1981
THE
P r m f e d i n C.S.A.
The Structure of Vitellogenin MULTIPLE VLTELLOGENINS IN XENOPUS LAEVIS GIVE RISE TO MULTIPLE FORMS OF THE YOLK PROTEINS* (Received for publication, December 22, 1980)
H. Steven WileyS and Robin A. Wallace From the University of Tennessee-Oak Ridge GraduateSchool of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
We describe the purification and characterization of the major vitellogenin-derived yolk polypeptides from the platelets of Xenopus oocytes. Five different types of yolk polypeptides were isolated, three of which (lipovitellin 1,lipovitellin 2, and phosvitin) correspond to previously described yolk proteins. Two newphosphorylated yolk polypeptides, named phosvette 1 and phosvette 2, were also isolated and found to have M, = 19,000 and 13,000- 14,000, respectively. While phosvitin had 7.4% protein-phosphorus, phosvette 1 was found to have 4.8% protein-phosphorus and phosvette 2 contained 10.7%protein-phosphorus. Amino acid analyses of phosvette 1 and phosvette 2 showed that these proteins were not derived from the other yolk proteins. Lipovitellin 1 and lipovitellin 2 could each be resolved into three components by sodium dodecyl sulfate-gel electrophoresis, termed the a, /I, and y forms in order of their increasing electrophoretic mobility. Ferguson plot analysis of lipovitellin l a , p, and y yielded M , = 121,000,116,000, and 111,000, respectively. Lipovitellins 2a, p, and y have M, = 34,000, 31,500, and 30,500, respectively. Phosvitin displayed very anomalous behavior during sodium dodecyl sulfate-gel electrophoresis, but dephosphorylated phosvitin demonstrated no anomalous behavior and resolved as two bands of M, = 37,500 and 39,000. Sedimentation equilibrium and gradient gel electrophoresis of native phosvitin yielded values of M, = 33,000 and 34,000, respectively. An analysis of the stoichiometry among the yolk proteins showed that the ratioamong the lipovitellins, phosvitin, and the phosvettes was 1:0.69:0.25. We propose that the multiple yolk proteins are products of the cleavage of multiple vitellogenin molecules, and that phosvitin and the phosvettes are alternate cleavage products arisingfrom homologous regions of different parent vitellogenin molecules.
vitro (3) or in vivo (4) by estrogens, and it has recently been used as a model for steroid-induced protein ( 5 ) . VTG’ was thoughtto exist in the blood as a dimer containing two identical polypeptide chains with an approximate M, = 200,000 (6). Recently, however, it has been found that the mRNAs coding for VTG in Xenopus do not comprise a homogeneous population but instead are composed of two families, each having at least two distinct sets of sequences (7). Thus, the mRNA population inXenopus liver would seem to have the capacity to produce four different VTG molecules. We have shown previously that there are least at three distinct molecular weight forms ofVTG in the blood of estrogentreated animals (8).However, the very close molecular weights of the VTG monomer peptides (197,000, 188,000,and 182,000), as well as the homogenous behavior of Xenopus VTG on ion exchange columns, make the isolation of the different forms of VTG a difficult task. The existence of several different VTGs is a complicating factor in structuralstudies of the intactprotein. If one wishes to reconstructa molecule from a mixture of its cleavage fragments, one must be able to distinguish between overlapping fragments and fragments derived from different parts of the parentmolecule. If the VTG population contains members with different primary sequences, then cleavage of the population at specific peptide bonds will yield corresponding polypeptides that would be impossible to classify as such. This is further complicated by the fact that even a single M , = 200,000 peptide could yield an enormous number of cleavage fragments. However, in the case of VTG, another approach to an investigation of the structure of the molecule exists. VTG is the precursor from which are derived the smaller, more readily characterized yolk proteins. These proteins exist as an insoluble complex in the oocyte; however, the complex can be separated into its various components by (NH&S04 fractionation (9) and column chromatography (10). To date, three main classes of yolk proteins have been described: LV,, M , 115,000; LV2, M, 31,000; and PV, M , 35,000 (6). In addition,several smaller phosphopeptideshave also been observed (6). Since all of these yolk proteins are derived from VTG (11),we decided to purify and characterize them as an aid to reconstructing the parent molecule. We have characterized several new yolk proteins and have also found that all of the previously described yolk proteins show a heterogeneity in molecular weights which is similar to that seen in the parent VTG population.
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Vitellogenin is the sex-limited yolk precursor protein in oviparous vertebrates (1).The hepatic synthesis of this large ( M , 460,000) phospholipoglycoprotein (2) can be induced in
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* This research was supported by jointly by National Institutes of HealthGrantT32 GM07431, EnvironmentalProtection Agency Agreement 79-D-X0533, and the Office of Health and Environmental Research, United States Department of Energy, under Contract W7405-eng-26 with the Union Carbide Corporation. The costs of publication of this article were defrayed in part by the payment of page “aduertisecharges. This article must therefore be hereby marked ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom all correspondence should be addressed a t Department of Microbiology, University of California, Irvine, CA 92717.
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The abbreviations used are: VTG, vitellogenin; Bicine, N,N‘-bis(Phydroxyethy1)glycine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; LVI, lipovitellin 1; LV2,lipovitellin 2; mono-Tris, 2hydroxyethylaminotris(hydroxymethy1)metbane; PMSF, phenylmethanesulfonyl fluoride; PV, phosvitin; PVTI, phosvette 1; PVT2, phosvette 2; SDS, sodium dodecyl sulfate.
8626
Multiple Vitelbgenins Derived and EXPERIMENTALPROCEDURES
The care of animals, together with the injection of hormones and isotopes, was performed as previously described (12).Acrylamide and N,N'-methylene-bisacrylamide were recrystallized 3 times from acetone prior to use. SDS was purchasedfrom Polysciences (electrophoresis grade). Ammediol (2-amino-2-methyl-1,3-propanediol) was recrystallized from ethanol, rinsed with acetone, and dried under vacuum. Mono-Tris was synthesized as described by Lewis (13) and was recrystallized from ethanol 3 times. Isotopes were purchased from Amersham, and all other chemicals and reagents were the highest quality available. Gel Electrophoresis-SDS-gel electrophoresis was performed by a modification (8) of the procedure of Wyckoff et al. (14). Tube gels for the Ferguson plot analysis (15, 16) were 0.6 X 10 cm and contained from 5 to 14% total acrylamide with aconstant 2% of the total acrylamide as bisacrylamide (2% C).Slab gel electrophoresis in straight percentage gels employed gels 13 cm long and 1.5 mm thick. Linear gradient SDS gels were cast as slabs (14 X 8 X 0.1 cm) from 5 to 15% total acrylamide, with the higher percentage acrylamide containing 5% glycerol. All gels were fixed overnight in 45% methanol and 10%acetic acid. Gels were stained 6 hwith 0.05% Coomassie blue R-250 in 20 mM AlC13,25% isopropyl alcohol, 10% acetic acid, and destained with 10%acetic acid, 5% ethanol. The inclusion of the AlCln in the staining solution was necessary for the visualization of the phosphoprotein bands (17). For quantitation of the distribution of in labeled yolk proteins, a discontinuous SDS-gel electrophoresis system was developed that had a running pH of 8.1. This was used to avoid alkaline dephosphorylation of the proteins (18).The system used a mono-Tris/Bicine buffer calculated from the equations of Jovin (19).The gels (10 X 0.6 cm) were cast containing 0.09 M mono-Tris and 0.058 M HCI. Polymerization was achieved using a final potassium persulfate concentration of 200 pg/ml and 0.5 pl/ml of N,N,N',N'-tetramethylethylenediamine as the catalyst for 10% acrylamide gels. The upper reservoir buffer contained 45 mM mono-Tris, 45 mM Bicine, 0.05% SDS, while the lower reservoir buffer was100 mM mono-Tris, pH 7.6.Yolk proteins were denatured at1 mg/ml in 2% SDS, 2 mM EDTA, 10 mM dithiothreitol, 10 mM Tris, pH 8.0, at 100 "C for 10 min. Samples were diluted 1O:l with denaturing buffer lacking SDS but containing 10% glycerol and 0.05% bromophenol blue and loaded on gels having a 1cm stacking gel of 3% T, 20% C acrylamide cast in the gel buffer. Gels were run at 2 mA/gel until the tracking dye reached the bottom of the tube, after which the gels were removed and sliced into 1-mm segments prior to counting in a Beckman Lobetta I1 planchet counter. Molecular weight estimation by gel electrophoresis employed the following standards: @-galactosidase (116,000), phosphorylase b (96,000).bovine serum albumin (68,000),catalase (59,000),ovalbumin (45,000), aldolase (40,000). yeast glyceraldehyde-3-phosphate dehydrogenase (34,000), carbonic anhydrase (30,000), PMSF-inactivated trypsinogen (24,000),soybean trypsin inhibitor (21,500),@-lactoglobulin (18,400),hemoglobin (16,000),a-lactalbumin (14,400),and cytochrome c (12,500). Radioactivity in column fractions was assayed by spotting an aliquot (generally 100 pl) on Whatman No. 3MM discs and processing them toremove trichloroacetic acid-soluble counts andphospholipids (20).They were then eithercounted in a Beckman Lobetta I1 planchet counter or placed in scintillation fluid for counting in a scintillation counter. Protein Purification-Unless otherwise stated, all isolation procedures were performed at 0-4 "C. Xenopus VTG was isolated by Mg":EDTA precipitation (21) in the presence of 2 mM PMSF. ,"Plabeled yolk proteins were isolated from animals that had been injected with 1,OOO IU of human chorionic gonadotropin and 2 mCi of [.'2P]orthophosphate, 72 and 24 h, respectively, prior to killing. The animals were f i s t bled by heart incision (22) and the ovaries were removed and rinsed in solution 0-R2 (23). The ovaries were homogenized in 5 volumes of 0.25 M sucrose, 10 mM Hepes, 1 mM EDTA, 2 mM PMSF, 1%polyvinylpyrrolidone pH 7.2, and the yolk platelets were isolated as described by Wallace (9). The platelets were dissolved in 10 volumes of 1 M NaCI, 10 mM Hepes pH 7.2, and any insoluble material was removed by centrifugation at 10,OOO X g for 20 min. Isolation of the Lipovitellin Subunits-Lipovitellin was separated from the other yolk proteins by the addition of 2 volumes of a saturated (NH,),SO, solution, dropwise over a period of 15 min, to the dissolved yolk platelets (9). The precipitated lipovitellin was removed by centrifugation at 10,OOO X g for 20 min and redissolved by the addition of an equal volume of water. After an overnight dialysis against 1 M NaCl, 10 mM Hepes, pH 7.2, the lipovitellin was
Yolk Proteins
8627
again precipitated with (NH,),SO,. The final pellet was dissolved in asmallamount of waterand then exhaustively dialyzed against distilled water containing 2 mM PMSF. The lipovitellin was then delipidated (24), and 140 mg of the resulting powder were dissolved in 20 ml of 10% SDS, 10 mM Tris pH7.5, with 10 mM dithiothreitol at 100 "C. Any insoluble material was removed by centrifugation, and the sample was applied to a column (5 X 100 cm) of Bio-Gel A-1.5m equilibrated with 0.1% SDS, 50 mM NHdHCO:,, 0.02% NaN.3.Elution was carried out at 20 "C at a flow rate of 59 ml/h. The LVL andLV, subunits of lipovitellin were resolved by this procedure (25). Fractions containing either LV, or LV, (as determined by SDS-gel electrophoresis) were pooled and lyophilized. The SDS was then removed by ion pair extraction (26), and the proteins were dried under vacuum. The sodium content of these preparations was approximately 1%as determined by atomic absorption spectroscopy. Yolk Phosphoprotein Isolation-The soluble fraction of the yolk that remainedafter (NH4)2S04precipitation was exhaustively dialyzed against distilled water containing 2 mM PMSF, using Spectropor 6 membrane tubing ( M , cutoff -2000) and lyophilized. This was then dissolved in 0.5 M NaC1, 50 mM Hepes, 5 mM EDTA, pH 7.0, at a maximum concentration of 10 mg/ml. The sample was then applied to a column (1.6 X 95 cm) of Sephadex G-75 (superfine) equilibrated with the same buffer at 20 "C and eluted at a flow rate of 13 ml/h. Fractions containing the desired protein peaks were collected and exhaustively dialyzed against distilled water prior to lyophilization. Selected protein samples were further purified by DEAE-cellulose chromatography by dissolving in 5 ml of 50 mM Tris, pH 7.5, and applying them to columns of DEAE-cellulose (Selectacel type 40, Schleicher and Schuell, Lot D1391, 0.94 meq/g). The prepared DEAE-cellulose (10) was autoclaved in0.2 M sodium citrate, 0.26 Triton X-100 to destroy anycontaminating proteases, and then packed andequilibrated with 50 mM Tris, pH 7.5, as previously described (10). Proteinswere then eluted at a constant flow rate with a 400- to 1OOO-ml linear gradient from 0.0to 0.35 M in KC1 or NaCl in the equilibration buffer. The protein load on these columns did not exceed 50 mg/100-ml bed volume. Appropriate fractions were pooled and dialyzed against distilled water prior to lyophilization. Dephosphorylation of PV-PV was dephosphorylated by the use of calf intestine alkaline phosphatase (Sigma type VII). ["2P]PVwas incubated at concentration a of 1 mg/ml in 50 mM 2-(Nmorpho1ino)ethanesulfonic acid, pH 5.5, a t 20 "C with constant stirring. Twenty micrograms of alkaline phosphatase were added, and at several time intervals aliquotsof the reaction mixture were removed and assayed for the presence of acid-insoluble radioactivity (20). Dephosphorylation was completed (>95%) by approximately 2 h, at which time the reaction was terminated by adding SDS to a final concentration of 1%and incubating the sample at 100 "C for 2 min. Analytical Procedures-Amino acid analyses on samples were performed after acid hydrolysis in evacuated ( 9) for extended periods of time. Such exposure could result in alkaline-catalyzed p elimination reactions in the phosphoproteins and a consequent loss of protein phosphorus (18). To retainany possible small yolk peptides, small pore dialysis tubing (cutoff M , 2000) was used routinely. In addition, since some yolk phosphopeptides do not absorb light at 280 nm (41), initial purification attempts employed yolk platelets derived from animals injected with [32P]orthophosphateprior to killing. Yolk platelets obtained by low speed centrifugation of homogenized ovaries were dissolved in 1M NaC1. This initial salt step also serves to dissociate the phosvitins from the lipovitellins. The lipovitellins were then separated from the yolk phosphoproteins by several rounds of (NH4)&304precipitation. The lipovitellins thus obtained were totally dissociated by treatment with SDS at 100 "C and then fractionated by gel filtration in the presence of SDS. As seen inthe Fig. 1,two main peaks of UV-absorbing material were obtained. The first
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0.15-
LV1 r"----.I
L"2 c " ,
SDS
010
l r ,
n
FIG. 1. Gel filtration of Xenopus lipovitellin. Delipidated ["2P]lipovitellin (120 mg) was dissolved in 20mlof 10% SDS and applied to a column (5 X 100 cm) of Bio-Gel A-1.5m equilibrated with 0.1%SDS, 50 mM NH4HCO:1,0.02% NaN3. Flow rate was 59 ml/h and 12-min fractions were collected. Aliquots (100 pl) of each fraction were counted for the presence of 32P.Indicated fractions were pooled and lyophilized for further analysis. The peak designated SDS yielded a large amount of material on lyophilization that was completely dissolved in acetone.
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D
015
l 005
F R A C T I O N NO
FIG. 2. Gel filtration of (NH4)2S04-soluble["P]phosphoproteins from Xenopus yolk. The material remaining after the (NH&SO, precipitation of yolk lipovitellin was dialyzed against distilled water and lyophilized. This material (40 mg) was dissolved in 4.5 ml of 0.5 M NaCI, 50 mM Hepes, 5 mM EDTA, pH 7.0, and loaded on a column (1.6 X 95 cm) of Sephadex G-75 (superfine) equilibrated with the same buffer. The column was run at 20 "C at a flow rate of 12.7 ml/h and 6-min fractions were collected and assayed for the presence of "P. Designated fractions were pooled for further analysis.
main peak eluting off the column was designated LV,, as suggested by Bergink and Wallace (6). The smaller peak eluting ahead of the LV, peak (fraction 130-140) was found to comigrate with LVI when subjected to SDS-gel electrophoresis, and probably represents an aggregate of LV,. The second major peak eluting off the column (Fig. 1) at fraction 195 contained almost all of the protein phosphorus in the lipovitellins and was designated LV2 (6). SDS-gel analysis of the column fractions revealed that all of the protein loaded on the column eluted with either the LVI or LV2 peak. The material left in solution after the (NH&S04 precipitation of the lipovitellins contains the highly phosphorylated yolk polypeptide(s).Fractionation of this material over a column of Sephadex G-75 gave the profile presented in Fig. 2. Surprisingly, we observed two main peaks of phosphoproteins instead of the expected single peak of phosvitin. We later found that the use of standard dialysis tubing (cutoff M , 10,000) during the preparation of the yolk phosphoproteins resulted in the loss of most of the material in peak 11. The asymmetry between the 32P and UV absorbance profile of peak I1 (Fig. 2 ) was seen inevery run of the yolk phosphoproteins. The fractions comprising the first major peak were pooled (Fig. 2 ) , and the material was further subjected to DEAE-cellulose chromatography. Only a single main peak of "P-labeled and UV-absorbing material was obtained (Fig. 3 ) . SDS-gel electrophoresis in single percentage gels revealed that this material migrated as a major band and a much fainter, minor band, with the major band corresponding to the migration position of Xenopus PV (6).The second major peak obtained bygel fitration (Fig. 2 ) was also fractionated by DEAE-cellulose chromatography and gave the result seen in Fig. 4. Two "P-containing components were obtained, one corresponding to a UV-absorbing peak and the otherlacking significant UV absorbance. These two components are designated PVT, and PVT2 inorder of their elution from the column of DEAE-cellulose. The "phosvette" designation was chosen because gel filtration revealed that they were smaller than what has previously been designated as Xenopus PV (Fig. 2 ) . We also collected and lyophilized the peaks of the smaller peptides obtained by gel filtration (fractions 95-115 and fractions 130-150, Fig. 2 ) but were unable to obtain significant quantities of protein material. In order to determine if there was a significant cross-con-
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Multiple Vitellogenins Derived and
Yolk Proteins
8629
(Fig. 6 4 . LV1 migrated as a broad band with an apparentM , between 115,000 and 105,000. LV, migrated as three closely spaced bands of M , = 33,000,31,500, and 30,000, as previously observed by Ohlendorf et al. (25). PV appeared as a major M , = 34,000 and a minor band of band with an apparent 28,000, while PVTl and PVTpwere composed of major bands of 19,000 and 13,000-14,000, respectively(Fig. 6B). Minor bands were also seen in all the phosvette preparations. PV and PVTzwere only visualized in these gels by the inclusion of 20 mM Al"+(17). However, PVTl stains faintly with Coomassie bluealone. Unfortunately, the staining intensity of PV and the phosvettes was never sufficient to demonstrate their presence in the total yolk platelet fraction by staining alone. We isolatedthe (NH4)2SOs-soluble phosphopeptides from yolk platelets labeled in vivo with ["P]orthophosphate, elecFIG. 3. DEAE-cellulose chromatography of the first phostrophoresed them in a constant percentage (10% T) gel, and phoprotein peak eluted by gel filtration of (NH&S04-soluble then subjected the driedgel to film autoradiography to visu[32P]phosphoproteins (Fig. 2). An approximately20-mgsample was applied to a column (1.9 X 32 cm) of DEAE-cellulose in 50 mM alize the labeled proteins. The result of this experiment is Tris, pH 7.5, and eluted with a 1000-ml gradient from 0.0 to 0.5 M seen in Fig. 6C. Indicated next to the autoradiograph is the NaCl in the column elution buffer. The flow rate was 150 mI/h and 6- migration position of the purified yolk phosphoproteins visumin fractions were collected and assayed forthe presence of J2P.The alized by staining in adjacent lanes. It can be seen that the indicated fractionwas pooled for further analysis. phosvettes are indeed normal constituentsof the yolk platelets. Both the major and minor PV bands areinseen the total yolk phosphoproteins. PVTl again migrated essentially as a single-labeled band, while the PVTz region was observed to be composed of several closely spaced bands with a higher molecular weight minor band in these gels (also seen in Fig. 5 5). It should be noted that on electrophoresis in gradient gels, *1 the minor PVT2 band was always observed to have a lower 0 4; molecular weight than PVT, (see Fig. 6B,lanes 1 and 2). z Molecular Weights of the Yolk Proteins-In order to reconstruct the VTGmolecule from an analysis of the yolk 3 i proteins, it is essential to accurately determine the molecular 2 weights of the proteins. This task is somewhat complicated by the fact that highly phosphorylated proteins behave anom1 alously during SDS-gel electrophoresis (25).Ferguson plot analysis canidentify anomalously migrating proteins but canI3 not provide an accurate molecular weight for those proteins notdemonstrating "normal" behavior. We performed this FIG. 4. DEAE-cellulose chromatography of the second phos- analysis on theyolk proteins and found (as did Ohlendorf et phoprotein peak eluted by gel filtration of (N&)2SO4-soluble al. (25)) that while LV1 and LV2 behaved essentially as the [32P]phosphoproteins(Fig. 2). The "P-labeled material was loaded standard proteins on these plots, PV,as well as the phosvettes, on a column (1.5 X 18 cm) of DEAE-cellulose in 50 mM Tris, pH 7.5, did not. LVp resolved as three closely spaced bands in the and eluted with a 400-ml linear KC1 gradient from 0.0 to 0.35 M KC1 single percentage gels with M , = 34,000, 31,500 and 30,500, in in column equilibration buffer at a flow rate of 112 ml/h. The 3-min The fractions werecollected and assayedfor the presence of indicated fractionswere pooled for further analysis. tamination of the differentisolated yolk phosphoproteins, samples of each purified 32P-labeledprotein were electrophoresed in 12% T, 2% C gels in the presence of SDS. The gels were sliced and evaluated for the presence of 32P.The results are seen inFig. 5. On these gels, PV and PVTI both migrated as essentiallysingle "'P-containing peaks, while PVT, migrated as a major and a minor peak. The slower mobility of PV on these gels indicated that it has a higher molecular weight than either of the two phosvettes. When the protein samples were treated withcyanogen bromide prior to electrophoresis (34), the PV and PVT, peaks were reduced in size and smaller fragments appeared, while PVT2 was not affected 2i bycyanogen bromide treatment (results not shown). This "-7%' result indicates that both PV and PVTI contain methionine 0 10 20 30 40 50 while PVT2 does not. SLICE NO. Purified samples of each major yolk protein were obtained FIG. 5. SDS-gel electrophoresis of the major (NH&3O4-so1and subjected to SDS-gradient gel electrophoresis after reuble yolk phosphoproteins. Purified samples of each "'P-containduction and alkylation (35). The results seen in Fig. 6 were ing protein were electrophoresed in gels (9.6 X 5 cm) in 12% T, 2% C obtained. Purified LVI and LV, migrate closely with their acrylamide (14). The gels were slicedinto 1-mm segments whichwere counterparts seen during electrophoresis of total yolk platelets counted for the presence of "'P.
\.
Yolk
Derived Yolk Proteins
Multiple Vitellogenins and
8630 A VTG Yolk LV, LV,
B PVT2 PVT, PV
PV
C
Mr ( k )
FIG.6. SDS-gel electrophoresis of the yolk proteins. A, stained gradient
-21
1
-14
2 3 4 5 6
1
2
gel (5 to 157) of the lipovitellins. Each lane contains 3 pg of the purified proteins except lanes 1 and 3, which contain 20 pg and 6 pg, respectively, of the total yolk proteins. Note that LV, resolves as three bands in this gel system (lane 1 ) . B, stained gradient gel (7.5 to 20%) of the purified yolk phosphoproteins. Approximately 10 pg of each protein were electrophoresed. C, autoradiogram of a straight percentage (10%) gelofyolk phosphoproteins labeled in Litlo with [”?]orthophosphate and isolated by (NH1)2S0.1 fractionation.
3
excellent agreement with the results of the gradientSDS gels, 08 and also in agreement with the values of 35,500, 32,000, and 31,600 reported by Ohlendorf et al. (25). We refer to these forms of LV2 as LVn,, LV2,{, and LV2, in order of increasing electrophoretic mobility.Wewerealso able to resolve the larger LVI protein into three bands as seen in Fig. 7. These 0.E three bands had apparentM , = 121,000,116,000, and 111,000, and are designated LV,,,, LVI/{,and LVI,, respectively. The three molecular weight forms of LVI are in contrast to the findings of Ohlendorf et al. (25), who only identified a single E 0 band with a M , = 105,500. We ascribe ourfinding of the three % 04 LV, bands to thelow amount of protein electrophoresed ( e 1 0 a pg), since we can visualize three LVI bands using the exact electrophoretic conditions specified by Ohlendorf et al. (25). High loads of LVI, asused in previous investigations,give rise to a single broad LVI band. Gradient gel electrophoresis (Fig. 02 6 A ) also does not seem capable of resolving the three LV, bands. The three LVI bands also are seen in total yolk platelet samplesprepared in thepresence of PMSF, so theyare unlikely to be an artifact of the isolation procedure. Ferguson plot analysis of Xenopus PV led Ohlendorf et al. 0 (25) to propose that the molecular weight of PV was only 6 5 4 3 2 1 0 about 17,000 instead of the valueof 35,000 previously reported DISTANCE FROM FRONT (an) (6). However, there is no consistentevidence that a Ferguson FIG. 7. Absorbance scan of an SDS-tube gel containing LV1. plot is capableof predicting themolecular weight of a protein A 5%gel containing 10 pg of LVI was run as described under “Experthat displays anomalous behavior (15,36).In order toresolve imental Procedures” and stained with Coomassie blue K-250. The gel the question of the “true” molecular weight of phosvitin, we was then scanned at 560 nm. The three LVI peaks are designated performed Ferguson plot analysis of native PV and PV de- LVI,. LVlp, and LVI, in the order of their increasing electrophoretic phosphorylated by alkaline phosphatase. Since the anomalousmobility. Only the bottom half of the IO-cm gel was scanned. behavior of PV on SDS gels is presumably due to the presence of the highly charged phosphate groups on the protein, we In order to further verify that PV has an average M , > reasoned that their removal would result in a “normal” protein 30,000, we performed sedimentation equilibrium analysis of with respect to its mobility on SDS gels. The result of this purified PV. The result of this experiment is seen in Fig. 9. The plot of log C versus r’ is linear and shows no sign of experiment is seen in Fig. 8. The plot of the initialPV preparation intercepted the Y axis a t a point considerably aggregation. The molecular weight of the PV sodium salt (32) below that of the standard proteins. The slope of this plot derived by this method is34,900. Subtracting the contribution gave a molecular weightvalue of 16,000 for PV. However,95% of the sodiumions to themolecular weight of P V gives a value dephosphorylated PV behaved normally with respect to itsY for native PV of approximately 33,000, slightly lower than the intercept and resolved as two closely spaced bands of 37,500 Ferguson plot-derived value of dephosphorylated PV (Fig. 8). and 39,000 (Fig. 8).Thus the slopeof the Ferguson plot does However, the sedimentationequilibrium value for the molecnot seem to be a reliable estimate of the molecular weight of ular weight of PV is in excellent agreement with that derived by gradient gel electrophoresis (34,000; see Fig. 6). Gradient highly conjugated proteins.
Multiple Proteins Vitellogenins Derived Yolkand
8631
I 0
2
4
6
o,oT
8
10
12
,1
I
0 0
14
25
50 M,
75
100
the yolk in an equimolar ratio. There are several noteworthy features of this analysis. Our analysis of LV, and LV2 agrees quite well with the values previously reported by Bergink and Wallace (6) and Ohlendorf et al. (25). However, our PV preparations have a much higher methionine level than previously reported. Sinceour PV samples lack three amino acids previously reported to exist in Xenopus PV (threonine, valine, and isoleucine (6,25)),our preparations seem to be quite pure with respect to previous preparations. The high methionine level can best be ascribed to improved methodology in amino acid analysis. Xenopus PV also has a considerable number of unesterified serine residues, a situation in contrast to that observed for chicken PV (39).The high number of unesterified serine residues is probably the reason that Xenopus PV has a significantly lower protein-phosphorus content than most other vertebrate phosvitins (33). The amino acid analysis of PVTzis highly unusual since six
125
10-3
FIG. 8. Apparent molecular weight of native phosvitin versus dephosphorylated phosvitin. A , Ferguson plot analysis of PV (0) and 95% dephosphorylated PV (a).Dephosphorylated PV was observed to form two closely spaced bands in these gels while PV resolved as a single band. The Y intercepte of the standard proteins used in this study are indicated as a series of solid points on the Y axis. B, the K, values of the standard proteins derived from the Ferguson plot analysis plotted against their molecular weights. The straight line through the points was derived by a least squares fit. The K,, values of PV and dephosphorylated PV are indicated.
gel electrophoresis in the presence of SDS has previously been reported to yield accurate molecular weight values of conjugated proteins (37, 38). To verify that the gradient gel technique also yielded an accuratemolecular weight value for the phosvettes, PVT2was subjected to sedimentation equilibrium analysis. This analysis gave a nonlinear plot of log C uersus r2 and yielded a M , range between 9,500 and 14,500, in fairly FIG. 9. Sedimentation equilibrium analysis of Xenopus PV. good agreement with the gradient gel value of 13,000-14,000. PV was equilibrated with 0.5 M NaCI, 50 mM Hepes, 5 mM EDTA, pH It thus seems that SDS-gradient gel electrophoresis is the 7.0, by gel filtration and diluted to an initial concentration of 0.2 mg/ best electrophoretic technique for estimating the molecular ml. Equilibrium was achieved a t 27,690 rpm and 20 "C. weight value of highly phosphorylated proteins. TABLE I Analysis of the Yolk Proteins-The Xenopus yolk phosAnalysis of yolk phosphoproteins phoproteins were subjected to elemental analysis and the Yolk phosphoproteins were isolated as described in the text and all results arepresented in Table I,together with their observed molecular weights and calculated phosphoserine content. It is determinations were corrected for the water and sodium content of apparent that theproteins are quite different with respect to the samples. No. of their phosphorus levels. The phosphorus level in Xenopus PV phosphoProtein M , C H N P ' / P o ms res,is somewhat lower than thevalue found for Rana pipiens PV dues/rnolh (7.4 uersus 9.3%)(33), but theN/P ratio (a check on phospho% % % % protein purity) is lower than that of Rana PV. PVT, has a PV 79 29.6 5.4 11.5 7.4 3.44 33,000' considerably lower protein phosphoruscontent thandoes PV, 32 5.3 13.1 4.8 6.04 29 PVTI 19,Wd while PVT, has a somewhat higher level and also has a very 47 PVT2 13,000-14,000d 24.7 5.7 10.3 10.7 2.13 low N/P ratio. LV2 has a much lower protein-phosphorus LV2 30,500-34,000' 1.2 12-13 level than do the other yolk phosphoproteins. a The atomic ratio of nitrogen to phosphorus. Amino acid analyses of the purified yolk proteins were Calculated from the molecular weights and phosphorus content performed together with an analysis of several homogeneous of the proteins. VTG preparations. The results are seen in Table 11. Also ' Determined by sedimentation equilibrium. presented in Table I1 is the total of the amino acid contents Determined by gradient gel electrophoresis. of the different yolk proteins, assuming that all are present in Determined by Ferguson plot analysis.
Multkle Vitellogenins and Derived Yolk Proteins
8632
TABLEI1 Amino acid composition of VTG and the yolk proteins
Proteins were isolated as described in the text and analyzed for aminoacids as describedunder"ExperimentalProcedures." The reportedvalues are inmoles/mol of protein using the following molecular weights: LVI, 117,000, LV,, 32,500, PV, 33,000; PVT,, 19,ooO; PVT2,13,500, VTG, 190,ooO. Amino acid
LV,
LVn
PV
PVT,
Sum of Yolk
PVTn
VTG
pro-
teim
18 11 5.6 Aspartic acid or as-24 78 137 148 paragine Threonine 55 15 0 0 87 4.0 74 66 14 14" 16 Serine 11.0 79 121 3 I3 79 29 Phosphoserine* 47 92 174 140 36 38 33 Glutamic acid or 11.0 258 229 glutamine 44 16 Proline 6.6 4.1 2.0 84 73 50 15 5.5 Glycine 6.8 2.4 84 80 99 24 Alanine 2.8 1.5 1.5 129 135 61 16 0 Valine 1.8 0.33 79 102 28 8.2 4.3 0 Methionine 2.4 42 43 59 12.0 0 Isolucine 73 81 1.3 0.6 99 18 0 3.2 I41 1.0 Leucine 121 30 12 0 2.3 0.5 Tyrosine 45 50 45 11 0 2.1 Phenylalanine 1.5 60 62 10 1.7 30 7.2 14 Histidine 60 63 74 22 28 7.0 1.8 Lysine 133 133 7.7 17 11 21 55 112 87 Arginine 0 0 0 9 2 11 10 Cvsteine' Determined by the sulfite addition technique(27). Calculated from the phosphorus content of each protein. Determined by nitrothiocyanobenzoic acid titration (28).
-.-...,*, 0
20
40
60
'
DISTANCE FROM TOP OFGEL ( m m )
.,
.."...
, . . . . 80
2-
100
When the amino acid content of each of the yolk proteins is added together, it does not add up to the amount in VTG. Since all yolk phosphoproteins are derived from VTG (ll), this indicates that they are not present in the same molar amounts in the yolk platelets. The greatest discrepancy in the combined amino acid content of the yolk proteins and VTG is in the serine and phosphoserine content; thus would it seem that PV and the phosvettesin particular are present in fractional amounts in the yolk. Stoichiometry o f the Yolk Proteins-Because of the previous observations, we decided to establish the stoichiometry of the yolk proteins by an analysis of their phosphorus contents. Since the stoichiometry of LV, and LVz have already been determined to be 1:1 by direct protein analysis (6), our analysis was directed a t t h e stoichiometry between LV, and the remainder of the yolk phosphoproteins. For this purpose, an analysiswas made of the relative contribution made by the phosphoserine in each yolk protein to the total yolk phosphorus content. The latter parameter was determined by SDSgel electrophoresis of total yolk platelets labeled in uiuo with [3zP]orthophosphate (12). The SDS gels were run using a discontinuous buffer system running at pH 8.1 in order to avoid alkaline dephosphorylation of the proteins duringelectrophoresis (18). The gels were sliced and the percentage of the total32Pcomigrating with the individual yolk proteins was determined as seen in Fig. 10. PV accounted for 61% of the protein-phosphorus in the platelets,while 14 and 21% of the phosphorus are associated with ZVZ and the phosvettes, respectively. Only -4% of the phosphorus is associated with LVI. These values agree very well with those of Bergink and Wallace ( 6 ) ,who found the ratio of :'2P in the yolk proteins was 1:0.22:0.35 for PV:LV,:phosphopeptides. This result also c o n f i i s t h a t t h e phosvettes are essentially the same small phosphopeptides described by Bergink and Wallace (6). When this information wasused to calculate the molecular ratio of LVz to PV,a ratio of 1:0.69 was obtained.' Thus there is less than one PVmolecule for each LVz molecule. However, the ratioof LV2to the phosvettes was determined tobe L0.25, assuming a 1:l ratio between PVTl and PVT,. Within the error associated with this typeof analysis, the ratio of LVZto PV plus the phosvettes is approximately 1:l. It would thus seem that approximately 70% of the VTG population gives rise to PV during its cleavage into the yolk proteins, while approximately 25 to 30%gives rise to the phosvettes. DISCUSSION
The evidence presented indicates that the multiple molecFIG. 10. Ratio of protein-phosphorus in the different yolk proteins. Yolk platelets labeled in vivo with 32Pwere directly dis- ular weight forms of VTG give rise to multiple yolk proteins solved in 2% SDS, 10 mM Tris, 2 mM EDTA, 10 mM dithiothreitol at in the oocyte. We have identified three main classes of yolk a concentration of 1 mg/ml. The proteins were electrophoresed using proteins: LVI (LV1,, LVIo, LVI,), LVz(LVZ,,LV'p, LVZ,),and the mono-Tris/Bicine gel system (pH 8.1). The gels were sliced and the highly phosphorylatedphosphoproteins(PV, PVT1, counted for the presence of radioactivity. Indicated are the migration positions of the purified proteins on duplicate gels.Note that the PVTz).Each of these classes can be divided into at least three members. Although we cannot rule out the possibility that phosvettes were not resolved in this gel system. the threedifferent members of LV, and LVI are derived from the same parent molecule by differential intra-oocyte cleavamino acids are absent, and also because of the very high age, we feel that this is unlikely for the following reasons. serine and phosphoserinelevels present. Serine and phosphoSince there are three molecular weight forms of VTG, differserine comprise almost 60% of the amino acid residues of of each of these could give rise to more than ential cleavage PVT2. The fractional amounts of isoleucine and valine observed in PVTz were consistently seen at the samelevels in three membersof each lipovitellin class.The difference in the molecular weights of the largest and smallest members of the all PVTz preparations analyzed, and are probably duePVT' to being a mixture of at least two proteins. The presence of As an example, the ratio between LVs and P V was calculated by isoleucine in PVTZ indicates that this protein class is not a the following equation: simple derivativeof PV, while the high phosphoserine content LVa 410 total protein phosphorus in LV, X precludes its derivation from the other yolk proteins. PVTI " PV phosphoserine residues in LV2 also cannot be derived fromPV because it contains threonine, phosphoserine residues in PV valine, and isoleucine, and cannot havebeen derived from the % total protein phosphorus in PV other yolk proteins due to itshigh phosphoserine level.
Multiple Vitellogenins and Derived Yolk Proteins
8633
LV, class (LV,, (121,000) and LV,, (111,000)) is also greater yolk platelets in which a single class of VTG molecules give than the difference in the molecular weights of the members rise to L v I , PV, and two LV2 molecules. However, that model of 17,000for themolecular weight of the otherclasses of yolk proteins. Thusdifferential cleavage was based on their estimate was a single protein with of a common precursorwould have to resultin the production of pV and the assumption that LVI of additional small peptides in significant quantities. No sig- a molecular weight of 105,000 (25). This study contradicts the nificant quantities of small peptides were observed in our findings of Ohlendorf et al. (25), and instead indicates that preparations. We feel that the three molecular weight forms their observation of multiple forms ofLV2 was due to its of LV, and LV2 are most easily explained by their derivation derivation from multiple VTG molecules. Their observation from the threemolecular weight forms of VTG. Likewise, PV that the amino acid compositions of the three forms ofLV2 and the phosvettes are probably derived from different VTG are nearly identical (25) supports our hypothesis that the molecules. Whilethe multiband structure of PVT, on constant multiple LV2 molecules are derived from homologous regions percentage gels (Fig. 6C) could result from its derivation fromof different VTG precursors. Published models for the strucPV as well as other proteins, we feel that this is unlikely. ture of thecrystalline yolk platelets (40) will haveto be observed PVT, lacks at least six different amino acids (TableII), three revised toaccount for theactualstoichiometry among the different yolk proteins. of which are present in PV. PVT, also contains fractional It has been shown that there are atfour least different genes amounts of valine and isoleucine, which PV lacks.These data are not consistent with PVT, being acollection of minor for VTG in Xenopus that give rise to four distinct mRNAs into twofamilies that differ in peptides derived from several partsof the VTG molecule but (7). The VTGgenesfall are, instead, indicativeof its derivation from a unique region sequence by approximately 20%,while there is less than a 5% of the VTG precursor molecule. The same can be said for difference between the sequences within a family. It is not PVT,.SincePVT,containsreasonableamounts of three surprising that we have found differences between members amino acids absent from PV (threonine, valine, and isoleucine; of each class of yolk proteins. However, it is somewhat surprising that all of the multiple yolk proteins form part of a Table 11), it clearly cannothave been derivedfromthis protein. However, the combined molecularweight and num- highly organized crystalline platelet. It is obvious that the in order to be correctly ber of phosphoserine residuesof the two phosvettes(32,000to constraints that VTG must meet beenex33,000 and 76, respectively) are very close to thatof PV (33,000 processed into the crystalline plateletshavenot and 79, respectively), suggesting that the two phosvettes are ceeded. One aspect of the evolution of the VTG genes that is derived from a region of VTG homologous to PV. The stoi- unusual is the presence of a domain that gives rise to the highly unusual protein PV. Approximately 40% of the amino chiometry of the yolk phosphoproteins also supportsthis acid residues of PV are present as serine, an amino acid that hypothesis, since the combined stoichiometry of the phosvettes andPV is approximately1:l with respect toLVI or LV2. has a 6-fold redundancy with respect to itsgenetic code. Thus the PV region of VTG could accommodate considerable base Themultipleproteinsseen in thephosvettepreparations could either be dueto asignificant heterogeneity in the changeswithout asignificant alteration in amino acid secleavage sites giving rise to the phosvettes, or to the phosquence. However, our data on the differences inthe molecular VTG genesmust vettes being derived from more thana single VTG precursor weights of the yolk proteins indicate that the molecule. have undergone insertions or deletions of genomic elements PV behaves more or less as a single component in most of as well. On the other hand, the sites of the molecule that been the fractionation systems described in this paper. However, specify the specific cleavages in the oocyte seem to have in at least one typeof VTG molecule there is evidence that PV is not a single unique protein with conserved. It seems that regardtoitssequence.DephosphorylatedPV wasalways an additionalcleavage site has been incorporated into theP V observed to runas two bands during SDS-gel electrophoresis region, giving rise to thetwo phosvettes insteadof PV. Neveralso sometimes theless, none of the genetic changes in the VTGs seem to have (Fig. 8).We have also observed that native PV resolves as two bands during electrophoresis(Fig. 6, B and C). interfered with their ability to be specifically sequestered by Recently, we have observed that the lower molecular weight oocytes and processed into the crystalline yolk platelets. It band of PV is readilycleaved by cyanogen bromide, while the would thusseemthatananalysis of the exact sequence higher molecular weightform is resistantcyanogen to bromide differences between the yolk proteins in each class would treatment." Thisevidence suggeststhat Xenopus PV may also provide insight into the constraints on the evolution of the be derived from more thana single VTG precursor molecule. VTG genes. The above described yolk proteins seem to comprise most, if not all, of the VTG molecule. The combinedmolecular Acknowledgment-We are grateful to Dr. A. P. Pfuderer for asweight of the largest member of each of the three classes of sistance with the sedimentation equilibrium analyses. the yolk proteins gives a combined value of 188,000 and a combined number of phosphoserine residues of 95, which is REFERENCES very close to themolecular weight and phosphoserine residues 1. Wallace, R. A. (1978) in The Vertebrate Ouary: Comparatiue of the largest of the three observed VTG molecules (197,000 Biology and Evolution (Jones, R. E., ed) pp. 469-502, Plenum and 89-95, respectively) (2, 8). The previously determined Press, New York molecular weight values for XenopusVTG may also be some2. Wallace, R. A. (1970) Biochim. Biophys. Acta 215, 176-183 3. Wangh, L. J., and Knowland, J . (1975) Proc. Natl. Acad. Sci. U. what high since VTG displays anomalous behavior on SDS S A . 72,3172-3175 gels.3 Indeed, VTG isolated from the liver of estrogen-treated 4. Wallace, R. A., and Jared, D. W. (1968) Science 160, 91-92 animals prior to its phosphorylation displays a significantly 5. Tata, J.R., and Smith, D. F. (1979) Recent Prog. Horm. Res. 35, lower molecular weight than does its phosphorylated counter47-95 part.4 Thus it seems that VTG is only large enough to give 6. Bergink, E. W., and Wallace, R. A. (1974) J. Biol. Chem. 249, rise to a single Lv1 molecule, a single LV, molecule, and either 2897-2903 7. Wahli, W., Dawid, I. B., Wyler, T., Jaggi, R. B., Weber, R., and a PV molecule or the phosvettes. Ryffel, G. U. (1979) Cell 16, 535-549 Ohlendorf et al. (25,40) proposed a model of the crystalline 8. Wiley, H. S., and Wallace, R. A. (1978) Biochem. Biophys. Res.
' H. Steven Wiley, unpublished observations. T. Gottlieb, personal communication.
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8634
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and Derived Yolk Proteins
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