biosynthesis of mosquito vitellogenin

0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Biosynthesis of Mosquito Vitellogenin”

Vol. 265, No. 17, Issue of June 15, pp. 9924-9933.1990 Printed in U.S.A.

(Received for publication, July 27, 1989)

Tarlochan S. Dhadialla and Alexander S. RaikhelS From the Department of Entomology and Program in Cell and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

Vitellogenin (Vg), the hemolymph precursor to the major yolk protein in mosquitoes, is synthesized in the fat body of blood-fed females. Mosquito Vg consists of two subunits with M, = 200,000 and 66,000. Here, we demonstrate that both the Vg subunits are first synthesized as a single precursor. The identity of this Vg precursor was confirmed by immunoprecipitation with subunit-specific monoclonal antibodies. In cell-free translation of fat body poly(A)+ RNA, the Vg precursor had M, = 224,000 which increased to 240,000 in the presence of canine pancreatic microsomal membranes. A precursor with M, = 250,000 was immunoprecipitated in microsomal fractions isolated from fat bodies. With in vitro pulse labeling, the 250-kDa precursor could be detected in homogenates of fat bodies from blood-fed mosquitoes only during the first few hours after the initiation of Vg synthesis. A much greater accumulation of the Vg precursor was achieved by an in vitro stimulation of Vg synthesis in previtellogenic fat bodies cultured with an insect hormone, 20-hydroxyecdysone. The 250-kDa precursor was glycosylated and to a much lesser degree phosphorylated. Treatment of fat bodies with tunicamycin yielded the precursor with M, = 226,000 which was neither glycosylated nor phosphorylated. The reduction in molecular mass of the 250-kDa Vg precursor and of both mature Vg subunits combined was similar after digestion with endoglycosidase H, indicating that giycosylation is completed prior to cleavage of the Vg precursor. In vitro pulse-chase experiments revealed rapid proteolytic cleavage of the 250-kDa precursor to two polypeptides with M, = 190,000 and 62,000 which trans- formed into mature Vg subunits of 200- and 66-kDa as the last step prior to Vg secretion. This last step in Vg processing was inhibited by an ionophore, monensin, and therefore occurred in the Golgi complex. Sul- fation as an additional, previously unknown, modification of mosquito Vg was revealed by the incorporation of sodium [36S]sulfate into both Vg subunits. Since sulfation of Vg was predominantly blocked by monensin, the final maturation of Vg subunits in the Golgi complex is, at least in part, due to this modification.

Knowledge of co- and post-translational events, which take place during the production of a mature protein molecule, is central to understanding both the structure-activity relation of mature protein products and regulation of their biosyn-

*This research was supported by Grant AI-24716 from the Na- tional Institutes of Health (to A. S. R.) The costs of publication of this article were defrayed in part by the payment of-page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should be addressed. Tel.: 517-353- 7144.

thesis. Considerable progress in this area of protein biochemistry has been achieved recently by analysis of both the nucleic acid sequences of various genes and the deduced amino acid composition of their protein products. As much as information on the nucleic acid sequence of a gene is important, however, it alone cannot reveal the modifications of the amino acids that follow after the translation of the primary gene products (Anderegg et al., 1988; Treston and Mulshine, 1989).

The development of an insect egg is dependent upon the availability of vitellogenin (Vg)’ which is synthesized and secreted by the fat body. The secretion of this major yolk protein and its specific endocytosis by the developing oocytes relies on the synthesis of Vg which is correctly modified and processed in the fat body cell. Therefore, it is important to know about modifications which occur during Vg biosynthesis.

The insect and vertebrate Vgs also provide useful model systems to study the structure and regulation of their genes (Bownes, 1986; Schubiger and Wahli, 1986; Dhadialla et al., 1987; Wahli, 1988) and the biochemical characterization of the mature protein products (Wang and Williams, 1980; Kun- kel and Nordin, 1985). Among vertebrates, the primary translation products of Vg genes were best studied in chicken (Wang and Williams, 1982) and Xenopus lueois (Gottlieb and Wallace, 1981, 1982). In both cases, the precursors to Vg are high molecular weight molecules (about 200,000) that undergo glycosylation followed by phosphorylation without additional cleavage. Most insect Vgs are phospholipoglycoproteins of high molecular weight (Masuda and Oliveira, 1985; Osir et al., 1986; Borovski and Whitney, 1987; Della-Ciopa and Engel- mann, 1987; Raikhel and Bose, 1988; also reviewed by Kunkel and Nordin, 1985). Insect Vgs are first synthesized either as one or two large precursors (about 200-220 kDa) that then undergo processing as well as proteolytic cleavage into two or more subunits of smaller size. These are assembled and secreted together as high molecular weight oligomeric proteins (Chen et al., 1978; Chen, 1980; Wyatt et al., 1984; Wojchowski et al., 1986; Della-Cioppa and Engelmann, 1987). Information on gene structure and the complete sequence of most of the co- and post-translational modifications of insect Vg is only available for Drosophila, whose yolk polypeptides are strik- ingly different in molecular weight from other insect vitello- genins (Bownes and Hames, 1978; Postlethwait and Kas- chnitz, 1978; Warren et al., 1979; Barnett et al., 1980; Brennan et al., 1980; Hung and Wensink, 1981, 1983; Minoo and Postlethwait, 1985; Friedrich et al., 1988).

’ The abbreviations used are: Vg, vitellogenin; EGTA, [ethylene- bis(oxyethylenenitrilo) tetraacetic acid; Endo-H, endo-@-N-acetylglu- cosaminidase; Me&O, dimethyl sulfoxide; mAB, monoclonal anti- body; PBM, post-blood meal; PMSF, phenylmethylsulfonyl fluoride; DOMA)’ RNA, nolvadenvlated RNA: SDS-PAGE, sodium dodecvl _ - sulfate-polyacrylamide gel electrophoresis; TES, N-tris[hydroxy- methyllmethyl-2-aminoethanesulfonic acid; 20-OHE, ZO-hydroxyecdysone.

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Mosquito Vitellogenin Biosynthesis 9925

For mosquitoes, there are conflicting reports as to the molecular weight of the mature Vg and its subunit composition (Hagedorn and Judson, 1972; Atlas et al., 1978; Harnish and White, 1982; Hagedorn, 1985; Ma et al., 1984; Borovsky and Whitney, 1987). However, by several radiolabeling and immunoprecipitation techniques utilizing mABs specific to Vg we have conclusively demonstrated that the Aedes aegypti Vg is composed of only two subunits, M, = 200,000 and 65,000 (Raikhel and Bose, 1988). Both subunits were glycosylated and phosphorylated (Raikhel and Bose, 1988). In a preliminary study, by cell-free translation of poly(A)+ RNA from vitellogenic female fat body of the mosquito, A. aegypti, it was shown that the two Vg subunits originate from a common high molecular weight precursor (Bose and Raikhel, 1988). In this report, we present data on characterization of the high molecular weight Vg precursor and its co- and post-translational processing in the mosquito fat body.

EXPERIMENTAL PROCEDURES’

RESULTS

Identification of the Vg Precursor by Cell-free Translation- A polypeptide with an apparent molecular weight of 224,000 was the predominant and specific cell-free translation product of poly(A)+ RNA from fat bodies of vitellogenic females. Immunoprecipitation analysis of the translation products demonstrated that the 224-kDa polypeptide was the primary product of translation of Vg mRNA. Monoclonal antibodies to different Vg subunits were effective in precipitating this polypeptide from the pool of cell-free translation products (Fig. 1). When fat body poly(A)+ RNA from vitellogenic mosquitoes was translated in vitro in the presence of canine pancreatic microsomal membranes and the translation products immunoprecipitated with mABs, the size of the Vg precursor increased to 240 kDa (Fig. 1).

Identification of the Vg Precursor in the Fat Body Cells- Next, we attempted to identify the Vg precursor forms in fat bodies at various times after initiation of Vg synthesis by a blood meal and by an in vitro hormone induction without a blood meal. Analysis of radiolabeled proteins from such fat bodies by immunoprecipitation and SDS-PAGE revealed differences in protein patterns (Fig. 2). Protein bands with M, = 190,000-200,000 and 62,000-66,000 corresponding to the large and small Vg subunits at various stages of their processing (see below) were present at all times analyzed. In contrast, a polypeptide with M, = 250,000 was evident only in fat bodies either induced with 20-OHE or dissected from mosquitoes at 3 h PBM. The relative intensity of the 250-kDa band was the greatest in 20-OHE-induced fat bodies (Fig. 2). Additional immunoprecipitation analysis confirmed that the 250-kDa polypeptide was the high molecular weight Vg precursor. As can be seen in Fig. 3, the 250-kDa polypeptide was immunoprecipitated by mABs specific to different Vg subunits or a mixture of these mABs. Thus, the 250-kDa polypeptide rep- resents a cellular form of the Vg precursor distinct from the 224-kDa precursor detected by the cell-free translation assay in the absence of canine pancreatic microsomal membranes (Fig. 1).

Identification of the Vg Precursor in Microsomal Fraction of Fat Bodies-Due to our inability to detect a high molecular weight Vg precursor in pulse labeled fat bodies 18-22 h PBM,

’ Portions of this paper (including “Experimental Procedures” and Figs. 14-16) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

69 - 46

FIG. 1. Immunoprecipitation analysis of cell-free translation products of poly(A)+ RNA from the vitellogenic mosquito fat body. Poly(A)’ RNA from fat bodies of vitellogenic female mosquitoes was isolated and translated in cell-free translation system in the presence (+) or absence (-) of canine pancreatic microsomal membranes (CMM) as described under “Experimental Procedures” (Miniprint). Products of translation were either immunoprecipitated with individual mAB (AlD12 specific to the large Vg subunit or BllD12 specific to the small Vg subunit) or precipitated with trichlo- roacetic acid (7’CA) and analyzed by SDS-PAGE on 5-10% gradient gels under reducing conditions. The gels were fluorographed to visu- alize the radiolabeled polypeptides. The molecular weights of translation products recognized by Vg mABs are indicated on the left and the molecular weight standards on the right. The standards in order of decreasing M, were myosin, phosphorylase b, bovine serum albumin, ovalbumin, and carbonic anhydrase (Amersham Carp).

EC 3 6 1220 2430

(kDa)

250k proVg- - -’ VgL- rrllnwmllll -200

-116 -97.4

vgs - ---m-i- -66.2

-42.7

FIG. 2. In vitro pulse labeling of mosquito fat bodies of various physiological ages. Fat bodies dissected from previtello- genie female mosquitoes were incubated for 6 h in culture medium containing 10-j M 20-OHE (EC) as described under “Experimental Procedures.” Fat bodies from female mosquitoes were dissected at the indicated hours after the initiation of Vg synthesis by blood feeding. lmmunoprecipitates of extracts of fat bodies pulse labeled with [“S] methionine for 2 h were analyzed by SDS-PAGE on 5-10% gradient gels under reducing conditions. Equal amounts of radioactivity was loaded in each lane. Labeled proteins were visualized by fluorography as described under “Experimental Procedures.” 250k pro-Vg, Vg precursor; VgL, large Vg subunit; VgS, small Vg subunit. The standards in order of decreasing M, are myosin, fl-galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin (Bio-Rad).

we sought for its existence and enrichment by isolating microsomal fractions. We followed this approach on the as- sumption that the Vg precursor is processed into its cleavage products at a much greater rate in fat bodies at peak vitellogenesis (20-26 h PBM) than soon after initiation of Vg synthesis (3 h PBM). Under such circumstances during peak


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9926 Mosquito Vitellogenin Biosynthesis

250k pro’Jg- ..- -

VgL- -200

FIG. 3. Immunoprecipitation analysis of the high molecular weight Vg precursor in mosquito fat bodies. Previtellogenic fat bodies were first incubated in the culture medium containing lo-” M 20-OHE for 6 h and then labeled with [““Slmethionine for 2 h. Proteins were immunoprecipitated from fat body homogenates as described in the legend to Fig. 16 under “Experimental Procedures” (Miniprint) and analyzed by SDS-PAGE as described in the legend to Fig. 2. Abbreviations and molecular weight of standards are as listed in Fig. 2.

bodies stimulated either in uitro by 20-OHE for 6 h or in uiuo by blood feeding of mosquitoes and dissected 3 h PBM. Processing of the 250-kDa polypeptide was similar in fat bodies under either conditions of stimulation of Vg synthesis. In both cases, while the 250-kDa polypeptide continued to decrease over 2.5 h of chase from the fat body cells, it was not secreted into the culture medium (Fig. 5A). The maturation of the Vg subunits into higher molecular weight forms was not as evident as seen in fat bodies taken at mid-vitellogenesis (see below, Fig. 5B).

20h 3h

(kDa)

250k proVg- VgL-I - -200

Therefore, in order to follow processing of Vg subunits, we used fat bodies of mosquitoes 18-22 h PBM. When extracts of fat bodies pulse labeled for as little as 5 min were immunoprecipitated, only two polypeptides with M, = 190,000 and 62,000 were detected. The protein profile obtained after a 30- min pulse was no different (Fig. 5B). A transition in the maturation of these two polypeptides became evident when fat bodies pulsed for 5 min or longer were chased for more than 15 min. While the small Vg subunit precursor appeared as a doublet with M, = 62,000 and 66,000, the large subunit, represented by a wide band, appeared to increase in its apparent molecular weight to 200,000 (Fig. 5B). The mature secreted Vg consists of only two subunits with molecular weights of 200,000 and 66,000.

-116 -97.4

vgs- II, - -66.2

Glycosylation of Vitellogenin-Pulse labeling of 20-OHE stimulated fat bodies with D-[2-3H]mannose and [3H]iV-acetylglucosamine revealed that both the 250-kDa Vg precursor and Vg subunits were glycosylated (Fig. 6). The oligosaccharide moiety on the 250-kDa Vg precursor was sensitive to Endo-H resulting in the reduction of its size to 227 kDa (Fig. 7).

-42.7 Additional pulse labeling of fat bodies 18-22 h PBM cul-

FIG. 4. Identification of the Vg precursor in microsomes isolated from vitellogenic mosquito fat bodies. Fat bodies, 20 and 3 h after the initiation of Vg synthesis by a blood meal, were pulse labeled with [““Slmethionine for 2 h. Microsomal fraction was prepared as described under “Experimental Procedures” and immunoprecipitates analyzed by SDS-PAGE. Abbreviations and the rest of the details are as described in the legend to Fig. 2. ,-42.7

Vg synthesis, the precursor is never present in quantities as easily detectable as during early stages of Vg synthesis (Fig. 2). Microsomal membranes were prepared from vitellogenic fat bodies 20 h PBM (peak Vg synthesis). As an internal control, microsomal membranes were also isolated from fat bodies 3 h PBM, a stage when we observed the 250-kDa precursor in immunoprecipitates of fat body extracts after pulse labeling. Immunoprecipitation and SDS-PAGE anal- yses of both the preparations from fat bodies 3 and 20 h PBM revealed a high molecular weight polypeptide with an apparent molecular weight of 250,000 (Fig. 4). The predominant polypeptides with M, = 190,000 and 62,000 corresponding to incompletely processed large and small Vg subunits, respectively, were also present (Fig. 4). These results indicate that the 250-kDa Vg precursor is a discrete intermediate during Vg biosynthesis at all stages of the vitellogenic cycle.

-1 16 -97.4

BB”\ e2k,- - - - - - - -66.2

-42.7

In Vitro Puke-Chase Labeling of Vitellogenin-Precursor- product relations during Vg biosynthesis were also investigated by in vitro pulse-chase experiments. Preliminary experiments showed identical patterns of immunoprecipitated polypeptides after labeling with [““Slmethionine, [35S]cysteine, or a “‘C-labeled amino acid mixture, and subsequent experiments were done using [3”S]methionine.

For pulse-chase analysis of the Vg precursor, we used fat

FIG. 5. Time course of [%]methionine labeling of the Vg precursor (A) and Vg subunits (B) in mosquito fat bodies analyzed by SDS-PAGE. A, previtellogenic fat bodies, stimulated with 20-OHE as described in the legend to Fig. 2, were pulse labeled with [%]methionine for 30 min and chased for the indicated times in complete culture medium as described under “Experimental Pro- cedures.” B, fat bodies from vitellogenic mosquitoes (18-22 h post- blood feeding) were pulse labeled with [“%]methionine for 30 min and chased for the indicated times as in A. Fat body extracts were prepared at the indicated times and the extracts immunoprecipitated with a mixture of mAB to Vg. The immunoprecipitates were resolved by SDS-PAGE and analyzed as described in the legend to Fig. 2. Proteins secreted into the 120-min chase medium (M) were also analyzed. The molecular weights of the Vg polypeptides are indicated on the left and the standards on the right. The standards and abbreviations are as described in the legend to Fig. 2.


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20-OHE or by a blood meal, were incubated in vitro in the presence of [“‘Plorthophosphate for 1 or 2 h, and the immunoprecipitates of the fat body extracts were analyzed by SDS- PAGE. The results demonstrated that the 250-kDa Vg precursor and both Vg subunits were phosphorylated (Fig. 6). The intensity of the phosphorylated 250-kDa band relative to Vg subunits, as observed on autoradiograms, was lower than that after labeling with [%]methionine or radioactive sugars, seen on respective fluorograms. This observation was confirmed quantitatively using scanning densitometry of auto- radiographs or fluorographs of different gels on which polypeptides labeled with [“‘Plorthophosphate, tritiated sugar precursors, and [%I methionine had been electrophoresed (Fig. 6). The relative peak area ratios of the 250-kDa precursor to the large and small Vg subunits combined labeled with [““S] methionine, [‘“Plorthophosphate, and tritiated sugar precursors were 0.53, 0.11, and 0.80, respectively.

FIG. 6. Analysis of the ratio of the mosquito Vg precursor to its subunits, labeled with different radioactive precursors, by scanning densitometry. Fat bodies. stimulated with 20-OHE as described inthe legend to Fig. 2, were pulse labeled with (a) a mixture of n-[2-“Hjmannose and [‘H]N-acetylglucosamine, (b) [,r2P]orthophosphate, or (c) [““Slmethionine, and proteins were immunoprecipitated and subjected to SDS-PAGE. Radiograms were scanned at 600 nm using a Gilson spectrophotometer. pro- Vg, 250 kDa Vg precursor; VgL, large Vg subunit; V&G’, small Vg subunit.

Phosphorylation of Vg subunits was further studied using pulse-chase analysis of fat bodies 18-22 h PBM. When immunoprecipitates of extracts of fat bodies pulsed with [“‘PI orthophosphate for l-15 min in vitro were analyzed by SDS- PAGE, the two polypeptides with apparent molecular weights of 190,000 and 62,000 were detected. Analysis of phosphorylated Vg in pulsed fat bodies chased for 15-120 min revealed an increase in the size of both phosphorylated Vg subunits to M, = 200,000 and 66,000 (not shown). The changes in mobility of phosphorylated Vg subunits are also seen after the inhibition of processing by monensin (Fig. 12B).

32 35 P s

Endo HI+-

(kDa)

-116 -97.4

66k- m -66.2

53k-- I

-42.7

Digestion of the secreted [“‘Plorthophosphate-labeled fat body proteins (predominantly Vg) with calf alkaline phosphatase resulted in the complete removal of phosphorous moiety on the Vg molecule (Fig. 8). Analysis of the fluorographs of [““S]methionine-labeled Vg subjected to alkaline phosphatase showed a reduction of about I and 4 kDa in the size of the large and small Vg subunits, respectively (Fig. 8). Additional bands in between the two dephosphorylated [%]methionine- labeled Vg subunits after digestion with calf alkaline phosphatase presumably resulted from endogenous proteolytic activity in the enzyme. Purified Vg incubated under similar conditions without the enzyme did not result in any degra- dation. Similar reduction of Vg subunits was identified after total secreted fat body proteins were digested with alkaline phosphatase for various times, O-3 h (not shown).

FK 7. Effect of Endo-H on the mosquito Vg precursor and Vg subunits. Immunoprecipitates were prepared from proteins secreted into culture medium from fat bodies pulse labeled with [,“P] orthophosphate and homogenates of fat bodies stimulated with 20- OHE and pulse labeled with [“‘Slmethionine. In both cases, the immunoprecipitates were incubated with (+) or without (-) Endo-H for 16 h at 37 “C. After the digestion period, the products were analyzed by SDS-PAGE as described in the legend to Fig. 2. 25Oh, processed Vg precursor; 227h, Vg precursor reduced by Endo-H; 200k and 66k, the fully processed large and small Vg subunits, respectively; 1.90k and 53k, the two subunits after Endo-H digestion. Molecular weights of the standards shown on the right are the same as described in the legend to Fig. 2.

Immunoprecipitated “‘P-labeled Vg was treated with Endo- H to determine if phosphorylation on the Vg molecule was primarily on the protein backbone or the carbohydrate moiety.

32 35 P s

CAP==

(kDa)

200k--, - 193lc-

c -200

-116 -97.4

66k--- 62k- , I -66.2

-42.7 tured in vitro in the presence of D-[2-“Hlmannose also showed that the 190- and 62-kDa polypeptides were already glycosylated and that these could be chased into mature glycosylated Vg subunits with M, = 200,000 and 66,000 (not shown). When we tested the mature secreted Vg, the oligosaccharide moiety of both the subunits, 200 and 66 kDa, remained sensitive to Endo-H. The Endo-H reduction of the apparent molecular weight of the mature Vg was 23,000 for both subunits (Fig. 7).

Phosphoylation of Vitellogenin-Fat bodies, stimulated by

FIG. 8. Calf alkaline phosphatase (CAP) digestion of 32P and %3-labeled Vg. Secreted proteins, from vitellogenic fat bodies, labeled with [‘“Plorthophosphoric acid or [“Slmethionine-labeled purified Vg were incubated without (-) or with (+) CAP at 37 “C. After 3 h, the proteins were mixed with an equal volume of 2 X SDS sample buffer and resolved by SDS-PAGE as described under “Ex- perimental Procedures.” The molecular weights of standards on the right are the same as in Fig. 2.


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After digestion with Endo-H, both the “‘P-labeled Vg subunits remained phosphorylated and their mobility on SDS gels increased similar to that of [““Slmethionine-labeled Vg subunits (Fig. 7). Quantitatively, treatment with Endo-H resulted in a 10% loss of the “‘P radiolabel.

B3h 20-OHE TMI+-I I+

Sulfation of Vitellogenin-Fat body proteins labeled in vitro with sodium [““Slsulfate for 2 or 3 h and analyzed by immunoprecipitation followed by SDS-PAGE and fluorography demonstrated that both the Vg subunits were sulfated (Fig. 9). Although the relative degree of sulfation of the two Vg subunits was not quantified, fluorographs of the gels revealed that the larger Vg subunit is more sulfated than the smaller subunit. However, on molar ratio, there might not be any difference in the degree of sulfation of the two subunits. In order to determine that the observed labeling of Vg subunits is due to sulfation of tyrosine residues rather than metabolic utilization of ‘% into methionine or cysteine, we subjected SDS gels on which immunoprecipitates of sodium [““S]sulfate- and [““Slmethionine-labeled Vg had been separated to HCl hydrolysis. Fig. 9 demonstrates, that while the HCl treatment of gels completely released the radiolabel from the sodium [““Slsulfate-labeled polypeptides, the [““Slmethionine-labeled Vg subunits were not affected.

Effect of Tunicamycin on Vg Processing-In order to look for the effect of tunicamycin on the Vg precursor, we compared its effect on processing of the Vg precursor in fat bodies stimulated either with 20-OHE or removed 3 h PBM. The effect of tunicamycin was similar in both cases; the size of the Vg precursor was reduced to 226 kDa (Fig. 10). The fact that the 226-kDa precursor was not glycosylated was also evident when fat bodies 3 h PBM were pulse-labeled with a mixture of D-[2-“H]mannose and [“H]N-acetylglucosamine in the presence of tunicamycin. Incorporation of the radioactive sugar precursors was inhibited by 87% and the 226-kDa precursor produced as a result of tunicamycin inhibition was not detected (not shown). Interestingly, the ratio of the Vg precursor to its subunits was effected by tunicamycin treatment only in the case of 20-OHE-stimulated fat bodies in which the Vg subunits were just barely detectable (Fig. 10).

The effect of tunicamycin on the processing of Vg subunits was investigated further using fat bodies at the peak of Vg synthesis, when pro-Vg was not detectable. As shown in Fig.

-HCI +HCI SM”SM’

1 (kDa)

mr, 0 -200

-1 16 -97.4

m _. -66.2

-42.7

FIG. 9. Sulfation of mosquito Vg. Vitellogenic fat bodies were preincubated in sulfation medium (“Experimental Procedures”) for 30 min before pulse labeling with sodium [““S]sulfate for 2 h in the same medium. Proteins secreted into the medium were used for analysis. Secreted proteins labeled with [%]methionine were obtained as described in the legend to Fig. 2. Labeled Vg was immunoprecipitated from both the samples and analyzed in duplicate by SDS- PAGE as described in the legend to Fig. 2. After staining the gels for proteins, half of the gel was subjected to HCl treatment to confirm sulfation of Vg. Gel portions containing sulfated or methionine- labeled Vg are indicated by S and M, respectively. The molecular weights on the right are of the same standards as mentioned in the legend to Fig. 2.

- 226k- -- --. -250k proVg

I -vgL

FIG. 10. Effect of tunicamycin on processing of the mosquito Vg precursor. Fat bodies, stimulated either by a blood meal in uiuo (R3h) or by 20-OHE in uitro, were incubated in the presence (+) or absence (-) of tunicamycin for 1 h as described under “Experimental Procedures” and then labeled with [ “Slmethionine. Proteins were analyzed by immunoprecipitation and SDS-PAGE as described in the legend to Fig. 2. 226k is the unprocessed Vg precursor produced in the presence of tunicamycin. Other abbreviations are as listed in the legend to Fig. 2.

356 3+ 3H

TM(+ - +- C-1

FIG. 11. Tunicamycin inhibition of Vg processing. Experi- mental details are the same as in Table I. Proteins extracted from 18-20 h PBM fat bodies pulse labeled with [“%]methionine, [“‘PI orthophosphoric acid, or D-[2-‘H]mannose were immunoprecipitated for Vs. Tunicamvcin (TM) inhibition of both alvcosvlation and phosplhorylation of Vg was significant but not complete (Table I). Since the same amounts of radioactivity were loaded in lanes from samples labeled in the presence (+) or absence (-) of tunicamycin, fully processed glycosylated and phosphorylated Vg subunits were evident in tunicamycin+ samples. Note that the 185k and 49k bands cannot be detected after labeling with either [“‘Plorthophosphate or D-[2-RH]mannose in the presence of tunicamycin. The molecular weights of standards shown on the right are the same as in Fig. 2.

11, when these fat bodies were incubated in the presence of tunicamycin, the large and small Vg subunits had apparent molecular weights of 185,000 and 49,000, indicating that glycosylation and processing but not the proteolytic cleavage of both the subunits was blocked. However, since glycosylation and subsequent processing of Vg was not completely inhibited, mature Vg subunits appeared as faint bands on the gel (Fig. 11). When extracts of fat bodies incubated in the presence or absence of tunicamycin were passed through a concanavalin A-Sepharose column and the unbound fractions analyzed by SDS-PAGE, the 185 and 49-kDa peptides were detected in extracts of tunicamycin-treated fat bodies only (data not shown). The same was also evident by the relative incorporation of D-[2-“Hlmannose into Vg in fat bodies cultured in the presence or absence of tunicamycin (Table I). Moreover, the 185- and 49-kDa Vg precursors were not detected in fat bodies pulse labeled with D- [ 2-3H]mannose in the presence of tunicamycin (Fig. ll), in contrast to the [“‘Slmethionine- labeled Vg from tunicamycin exposed fat bodies. These results indicate the absence of glycosylation of the 185- and 49-kDa Vg polypeptides.

Next, we asked whether phosphorylation of Vg would be effected by tunicamycin inhibition of glycosylation. Pulse labeling of fat bodies 3 h PBM with [32P]orthophosphoric acid in the presence of tunicamycin resulted in 70% inhibition of phosphorylation of the Vg polypeptides. Moreover, the Vg precursor was not detected with SDS-PAGE analysis of ‘*P-


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TABLE I

Effect of tunicamycin on synthesis and secretion of uitellogenin in the mosquito fat body Vitt.elogenic fat bodies (18-22 h post-blood feeding) were preincubated in mosquito Ringer’s solution buffered

with 25 mM TES, pH 7.5, for 1 h. The fat bodies were pulse labeled for 1 h in incomplete media with [““S] methionine, n-[2-“Hlmannose, or [‘“Plorthophosphoric acid as described under “Experimental Procedures.” An- other set of pulse-labeled fat bodies were chased in complete media for another hour. Preincubation and pulse- chase labeling was done in the continued presence or absence of tunicamycin. Vg was immunoprecipitated from fat body extracts or chase media and radioactivity incorporated into Vg was determined. Each value is the mean c S.E. from three replicates of 3 fat bodies each.

Radioactivity incorporated into immunoprecipitated Vg Tunicamycin

(0.25 mM) [““S]Methionine D-[2-“H1Mannos.e [‘“PlOrthophosphoric acid

Fat body Secretion Fat Body Secretion Fat body Secretion

CPm cpl7l CPm Present 5.8 x lo” f 7.1 x 10’ 4.2 x lo” + 1.6 x 10’ 7.4 x 10” + 2.3 x lo:’ 2.7 X 10’ f 1.3 X 10“ 350 + 130 200 f 120 Absent 3.8 x lo” + 2.0 x lo” 4.5 x 10” + 1.2 x 10” 1.0 x 10” + 1.4 x lo” 1.4 x 10” + 9.5 x 10” 720 f 160 1,500 + 220 Inhibition of control 0 91% 93% 98% 52%” 87%

” In different experiments, the degree of phosphorylation of fat bodies pulse labeled with [‘“Plorthophosphoric acid in the presence of tunicamycin varied from 50 to 70% of the control values. The above data represent results of a single experiment.

FIG. 12. Processing and secretion of [““S]methionine (A) and [“‘Plorthophosphoric acid (B) labeled Vg in fat bodies cultured in the presence or absence of 1Om6 M monensin. Details of the experiment are as in Table II. Pulse-chase labeled proteins in the fat bodies and media were immunoprecipitated and analyzed by SDS- PAGE as described in the legend to Fig. 2. p, c, and m represent Vg from pulsed, chased fat bodies, and the secreted Vg in media, respectively. Molecular weights shown on the right are of the same standards as listed in the legend to Fig. 2.

labeled proteins from tunicamycin exposed fat bodies (not shown). Pulse labeling of fat bodies 24 h PBM with [‘“PI orthophosphoric acid in the presence of tunicamycin resulted in 50-70% inhibition of phosphorylation of Vg (Table I). Analysis of “2P-labeled proteins from tunicamycin exposed fat bodies showed that the nonglycosylated 185- and 49-kDa Vg precursors were not phosphorylated, but as a result of incomplete inhibition of glycosylation and subsequent processing with tunicamycin the processed Vg subunits (200,000 and 66,000) were (Fig. 11).

Effect of Monensin on Vg Processing-To determine whether any of the Vg processing events are localized in the Golgi complex, we pulse labeled fat bodies with [““Slmethio- nine in the presence or absence of monensin. Analysis of immunoprecipitates of extracts of labeled fat bodies and secreted proteins confirmed that monensin totally inhibited the processing of the 190,000 and 62,000 molecular weight Vg subunit precursors to the mature subunits and hence secretion of Vg (Fig. 12A).

Next, we used inhibition with monensin to elucidate the nature of the change in 190- and 62-kDa polypeptides to mature 200- and 66-kDa subunits. Since, we demonstrated that glycosylation of Vg is completed before the 250..kDa precursor is cleaved into the 190- and 62-kDa Vg subunits, we focused our attention to phosphorylation and sulfation. While phosphorylation of Vg was not effected in fat bodies pulse labeled with [“‘Plorthophosphoric acid in the presence of monensin, processing of the 190- and 62-kDa phosphorylated subunits into the secreted mature subunits was (Table II and Fig. 12B). To determine if this block was due to lack of sulfation, we pulse-chased fat bodies with [““S]methionine or sodium [““Slsulfate, separately, in the presence or absence of monensin. As shown in Table II secretion of Vg was less than 1% with either of the two radiolabeled compounds in fat bodies incubated in the presence of monensin. As would be expected, similar amounts of [““S]methionine was incorporated into Vg in fat bodies incubated in the presence or absence of monensin. However, fat bodies pulsed with sodium [““Slsulfate in the presence of monensin incorporated only about 35% of the label into Vg as compared to those pulsed in the absence of monensin. These results indicate that ap- proximately 65% of Vg sulfation takes place in the trans- portion of the Golgi complex.

DISCUSSION

In this report we have conclusively demonstrated that in the mosquito, A. aegypti, the two Vg subunits originate from a common high molecular weight precursor and are, therefore, the products of the same gene. The common origin of Vg subunits from the same high molecular weight precursor was shown by cell-free translation of poly(A)+ RNA from vitello- genie mosquito females and analysis of the translation products by mABs specific to each of the Vg subunits. Peptide map analysis of the high molecular weight translation product of Vg mRNA and the Vg subunits also revealed similarities between them (Bose and Raikhel, 1988). The size of the primary translation product of Vg mRNA (pre-pro-Vg) detected in our studies closely correlates with the size of the polypeptide that can be derived from the mosquito Vg mRNA of 6.5 kilobases reported previously (Gemmill et al., 1986). In Locusta migratoria, Vg mRNA is 6.3 kilobases in length and its primary product of translation has a molecular mass of 185 kDa (Wyatt, 1988).

In vitro pulse labeling and analysis of microsomal fractions from fat bodies at various times of the vitellogenic cycle


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TABLE II Effect of monensin on phosphorylation and sulfution of mosquito uitellogenin

After preincubation in the presence or absence of monensin for 30 min, fat bodies were pulse labeled for 1 h with [““Slmethionine or sodium [s”S]sulfate and the label chased for 2 h in the continued presence or absence of monensin. In the case of [3ZP]orthophosphoric acid labeling, fat bodies were preincubated and pulse labeled with the radiolabel for 30 min each in the presence or absence of monensin. The radiolabel was not chased in this case. After the pulse-chase or pulse periods, proteins in the fat body and the media were immunoprecipitated to determine the amount of radioactivity incorporated. Each value is a mean of two ([%3]) or three ([32P]) determinations. The numbers in parentheses represent the number of fat bodies used for each determination.

Radioactivity incorporated into immunoprecipitated Vg

Monensin (lo-” M) [35S]Methionine” [32P]Orthophosphoric acid (“5S]Sulfate

Fat body Secretion Fat body Secretion Fat body Secretion

CPm CPm CPm Present 2.9 x lo6 (5) 1.6 x lo4 202 (5) 24 676 (15) 20 Absent 2.7 x lo6 (5) 2.3 x lo6 216 (5) 786 1884 (15) 2242 Inhibition of control 0 99% 6.5% 97% 65% 99%

’ In an experiment where fat bodies were labeled with [“‘Slmethionine in the presence or absence of monensin under exactly the same conditions as for [“‘Plorthophosphoric acid labeling, percent inhibition of control values in the fat body and secretion was 5.6 and 98.8, respectively.

demonstrated the existence of a high molecular weight Vg precursor of 250 kDa in the rough endoplasmic reticulum of mosquito fat body cells. The identity of this polypeptide as a Vg precursor was confirmed by immunoprecipitation analysis with mABs specific to mosquito Vg subunits. Additional proof that the 250-kDa polypeptide was the intracellular precursor to Vg subunits was obtained by pulse-chase labeling of fat bodies stimulated in vitro by ZO-OHE or in viva by a blood meal. In either case, the 250-kDa polypeptide could be chased from the fat body cells but was not secreted into the culture medium. The high molecular weight precursor of 250 kDa, therefore, is a discrete intermediate in the biosynthesis of mosquito Vg. The size of the Vg precursor found in the cells (MI = 250,000) was much higher than that of the primary translation product of the Vg mRNA (pre-pro-Vg) obtained in the cell-free translation experiments. This suggests that the 250-kDa Vg precursor resulted from processing of the 224-kDa pre-pro-Vg. Using radioactive sugars and [32P]orthophosphate, the 250-kDa Vg precursor in fat body cells was shown to be glycosylated and phosphorylated. The 250-kDa Vg precursor will now be referred to as pro-Vg.

Several lines of evidence indicate that glycosylation of pre- pro-Vg occurs co-translationally and, furthermore, it is completed at this step of processing. First, it was suggested by an increase in molecular weight of pre-pro-Vg in a cell-free translation system in the presence of canine microsomal membranes. An increase in the size of translated products under these conditions is known to be due to co-translational glycosylation (Walter and Blobel, 1983). Second, a polypeptide with M, = 226,000 and similar in size to the 224-kDa primary product of translation of Vg mRNA, was detected only in fat, bodies treated with tunicamycin, an inhibitor of co-translational glycosylation (Takatsuki and Tamura, 1971) and not in untreated fat bodies. Finally, enzymatic digestion of pro-Vg with Endo-H reduced its size to 227-kDa. The reduction in molecular weight of pro-Vg by Endo-H digestion was the same as that observed with enzymatic digestion of the mature Vg subunits. In the latter case, Endo-H reduced the size of the large subunit by 10 kDa and the small one by 13 kDa. Lack of additional sugar modification of Vg beyond the high mannose-type of oligosaccharide was also indicated by the continued sensitivity of mature Vg subunits to Endo- H and is supported by earlier observations that all high mannose chains on proteins become resistant to Endo-H after being processed into complex-type chains (Tarentino et al., 1978).

Scanning densitometry of the 250-kDa pro-Vg and Vg subunits, which were pulse labeled with [32P]orthophosphate, tritiated sugars, and [35S]methionine under identical conditions, showed that the relative degree of phosphorylation of the 250-kDa pro-Vg was much less than its glycosylation. Furthermore, the differences in sizes of pro-Vg precursor reduced by treatment with Endo-H, which removes only high mannose moieties, and that reduced by tunicamycin, which blocks both glycosylation and phosphorylation, were insignif- icant. This analysis suggests that Vg undergoes only limited phosphorylation as a 250-kDa pro-Vg and that additional phosphorylation likely occurs soon after its cleavage into the two Vg subunits. Phosphorylation of a high molecular weight Vg precursor has been documented for the cockroach, Leuco- phaea. In this insect, phosphorylation occurs only post-translationally, since no 32P-labeled Vg precursor was detected in polysomes (Della-Cioppa and Engelmann, 1987). Pulse-chase experiments utilizing [3’P]orthophosphoric acid labeling combined with monensin confirmed that the 190- and 62-kDa Vg polypeptides were fully phosphorylated, most likely in the endoplasmic reticulum soon after cleavage of the 250-kDa pro-Vg and prior to the last modification of the Vg subunits in the Golgi complex. Digestion of 32P-labeled Vg with Endo- H revealed that the resulting 190,000 and 53,000 M, peptides remained phosphorylated. These results demonstrate that most of the phosphorylation of the mosquito Vg is on the protein backbone. Small loss of the radiolabel (10%) observed could be due to some contaminating proteolytic activity in Endo-H or phorphorylation of the oligosacharide moiety. The presence of phosphorylated sugars on Vg molecule was found only for Rhodnius (Masuda and Oliveira, 1985).

Tunicamycin not only inhibited glycosylation of Vg but also blocked phosphorylation of both the pro-Vg and the cleaved 185- and 53-kDa polypeptides. The importance of glycosylation of the mosquito pre-pro-Vg for further processing into mature Vg was suggested by experiments where tunicamycin- induced polypeptides could not be chased into mature Vg subunits after removal of the tunicamycin block.3 These results indicate that during the biosynthesis of the mosquito Vg, the primary product of translation of Vg mRNA is first glycosylated before being phosphorylated. Similar sequence of post-translational modifications was demonstrated for the avian Vgs (Wang and Williams, 1982). Glycosylation as the first modification of pre-pro-Vg may be important for the molecule to assume a stable conformation necessary for phos-

“T. S. Dhadialla and A. S. Raikhel, unpublished observations.


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1. Translation

Vg mRNA 6.5 kb T I

I 224 kDa I pre pro “g

2. Co-translational glycosylatlon

3 Post-translational phosphorylation I

250

4. Cleavage /

/

62

I

i E”

s 6-a .-

P nf “. 6

mat”re vg large subumt small subunit

FIG. 13. Proposed biosynthetic pathway for vitellogenin in the mosquito fat body.

5. Phosphorylatvx /

190 kDa

6 Sulfation I 7. Secretion 1 200 kDa

I

4 kDa I Pro vg

phorylation and further processing to occur normally. Pulse-chase experiments with fat bodies revealed that the

250-kDa pro-Vg is rapidly cleaved into two polypeptides with M, = 190,000 and 62,000. These polypeptides are then processed into mature secreted Vg. The importance of cleavage of the glycosylated and partially phosphorylated pro-Vg for continued processing of Vg was realized in experiments utilizing tunicamycin. Tunicamycin has been shown to inhibit cleavage of the Vg precursors in the insects, Locusta and Blatella (Wyatt et al., 1984; Wojchowski et al., 1986), as well as of several vertebrate (Roos et al., 1980) and viral glycoproteins (Ng et al., 1982). In contrast to these reports, the mosquito Vg precursor underwent cleavage resulting into two aglyco- sylated and nonphosphorylated polypeptides with reduced sizes (M* = 185,000 and 49,000) in the presence of tunicamy- tin. However, there appear to be differences in the effect of tunicamycin on cleavage of pro-Vg in fat bodies stimulated for Vg synthesis in vivo by a blood meal or in vitro by 20- hydroxyecdysone (20-OHE). Cleavage of the pre-pro-Vg was at least partially inhibited by tunicamycin in fat bodies stimulated by 20-OHE. In separate experiments, cleavage of the mosquito Vg precursor was also not blocked with a protease inhibitor, leupeptin.4 Leupeptin has been shown to block proteolytic cleavage during processing of a number of proteins (Ascoli, 1979; Berg et al., 1985). The possible intracellular site for cleavage of pro-Vg was suggested by results of experiments with the ionophore, monensin. The ineffectiveness of monensin to block cleavage of the 250-kDa pro-Vg along with the presence of the 190- and 62-kDa Vg polypeptides in fat body microsomal preparations indicated that cleavage occurred in the endoplasmic reticulum and not the Golgi complex.

During the search for cellular forms of the Vg precursor, we discovered a unique feature of the physiology of the mosquito fat body. Analysis of microsomal fractions demonstrated that the high molecular weight precursor (pro-Vg) exists as an intermediate in the biosynthesis of Vg. However, this precursor could be detected in whole fat body extracts pulse labeled with radioactive precursors only during the first few hours after the initiation of Vg synthesis by normal blood feeding. Much greater accumulation of pro-Vg was found in fat bodies in which Vg synthesis was stimulated by 20-OHE in vitro. It is clear that 20-OHE effects predominantly the translation of Vg mRNA which resulted in greater accumulation of pro-Vg than its subunits. In normal blood-fed mos-

4 A. S. Raikhel and T. S. Dhadialla, unpublished observations.

quito females, however, other unknown factor(s) must be involved in the accelerated rate of post-translational cleavage of pro-Vg, which results in the accumulation of larger quantities of mature forms of Vg subunits. Thus, Vg processing in the mosquito fat body presents an interesting system for studying the regulation of protein biosynthesis.

The final step of processing of the 190- and 62-kDa Vg polypeptides into the 200- and 66-kDa Vg subunits, respectively, appears to take place in the trans-Golgi as indicated by its block with monensin. Of the many post-translational modifications of proteins, one that has often been found in secretory proteins and that occurs late in the secretory pathway is tyrosine sulfation (Huttner, 1984). When we pulse- labeled vitellogenic fat bodies with sodium [YS]sulfate, the radiolabel was incorporated into both the Vg subunits, appar- ently more so in the subunit with higher molecular weight. Sulfation of the Vg subunits was on the tyrosine residues as evidenced by the sensitivity of the [35S]sulfate-labeled Vg to HCl. Prior to this report, the only other insect yolk proteins known to be sulfated are of Drosophila melanogaster (Baeuerle and Huttner, 1985). Sulfation of Drosophila yolk proteins has been shown to occur in the trans-Golgi and necessary for their transport to the cell surface (Friedrich et al., 1988). Sulfation of other secretory proteins was also found to be monensin sensitive (Baeuerle and Huttner, 1987).

In this report, the main biosynthetic steps for processing of the primary product of translation of mosquito Vg mRNA into mature secreted Vg are identified and summarized in Fig. 13. The primary product of translation identified by cell-free translation of mosquito Vg mRNA is a 224-kDa polypeptide. Processing of this 224-kDa pre-pro-Vg begins with its co- translational glycosylation followed by partial phosphorylation leading to the formation of 250-kDa pro-Vg identified as a distinct intermediate in the biosynthesis of Vg. While glycosylation of Vg is completed during processing of pre-pro-Vg to pro-Vg, its phosphorylation is not completed until after cleavage of the 250-kDa pro-Vg. The phosphorylation occurs before the cleavage products transit through the trans-Golgi. The rate at which the 250-kDa pro-Vg undergoes proteolytic cleavage varies during the vitellogenic cycle, being slow soon after initiation of vitellogenesis and very rapid during mid- to peak vitellogenesis. The 190- and 62-kDa intermediates are then processed into mature Vg subunits with M, = 200,000 and 66,000. During this step, the 190- and 62-kDa Vg polypeptides undergo sulfation. The transition in molecular weight of Vg peptides into mature subunits is monensin- sensitive, indicating that it occurs in the trans-Golgi, and is at least in part due to sulfation.

Acknowledgments-We thank Drs. C. Noah Koller and John Ka- wooya for critical reading of the manuscript, and Michael Carlson for preparing gels for electrophoresis.

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Supplementary Material to

Biosynthesis of Mosquito Vitcllogenin

Tarlochan S. Dhadialla and Alexander S. Raikhel


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Mosquito Vitellogenin Biosynthesis

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biosynthesis of mosquito vitellogenin

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