Expression, Tyrosine Sulfation, and Secretion of Yolk Protein 2 of

0 %I? by The American Society for Biochemistry and Moleruiar Bic-iogy, Inc.

Expression, Tyrosine Sulfation, and Secretion of Yolk Protein 2 of Drosophita melanogaster in Mouse Fibroblasts*

(Received for publication, May 2, 1988)

Evelyne FriederichS, Patrick A. Baeuerle$, Henrik Garoff7, Bernd Hovemann]/ I and Wiefand B. Huttner** From the Cd Siology Pro~rarn~~ Ersroperm ~o~&~~r Biobgy ~rut~~~ Postfuch 102209, and the ~Center for Mokcular 3iu~o~, ~~iuers~~ of Heidelberg, ~-~~ ~e~e~erg, Federal ~ep~~~ af~er~~~

Tyrosine sulfation is a post-translational modification in the trana Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeieh, F., and Huttner, W. B. (1988) J. BioL Chem. 263,14925-14929), we have identified the site af tyrosiue sulfation in an insect secretory protein, yolk protein 2 (YP2) of Dr~s~~hilu mehznogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heteroiogo~s mammalian cell system. Mouse fibroblasts, transfected with tlrosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies, YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was -1 mol of sulfate/m01 of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172, These results show that essential parameters of the tyrosine sulfation reactian are very similar in insects and mammals and thus highly conserved in evolution.

Proteins with sulfated tyrosine residues have been found in all animals species studied. The tyrosine-sulfated proteins identified so far are all synthesized in the rough endoplasmic reticulum, and many of them are secretory (for reviews, see Refs. 1-3). Protein tyrosine sulfation has been shown to be catalyzed by a membr~e-bound enzyme, tyrosylprotei~ sulfotransferase, in the trans Golgi (4,5). The structural require- ments for tyrosine sulfation in substrate proteins have been deduced from a comparison of identified tyrosine sulfation sites which, except for hirudin, are all from vertebrate proteins (1,6). These sites share certain structural features which have been referred to as consensus features.

We have previously reported on the tyrosine sulfation of --

+ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ~‘~ue~~serne~t” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact-

$ Present address: Institut Pasteur, Laborstoire de Biologie des Membranes, 25, rue du Dr. Roux, F-75724 Paris C6dex 15, France.

cj Present address: Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142.

ll Present address: Karolinska Institutet, Center for Biotechnology, Dept. of Molecular Biology KS7, Huddinge University Hospital, S- 14186 Huddinge, Sweden.

**Recipient of Grants Hu 27513-Z and Hu 275/3-3 from the Dsutsche Forschungsgemeinscbaft. To whom correspondents and reprint requests should be addressed.

insect secretory protAns, the yolk proteins of Drosophila melanogaster (7). In the preceding paper (8), the site of sulfation of yolk protein 2 (YP2)l was localized to a single tyrosine residue, tyrosine 172. Comparison of the sequence surrounding this tyrosine showed an apparent similarity with the consensus features proposed for vertebrate sulfation sites. This similarity raises the possibility that the structural features involved in the recognition b&ween substrate proteins and ty~ylprote~n sulfotra~sferase might be conserved between insects and vertebrates. One approach to directly test this possibility is to express an insect tyrosine-sulfated secretory protein in a mammalian cell by gene transfer and to investigate whether it undergoes suIfation by the mammalian tyrosylprotein sulfotransferase.

In the present study, we show that Drosophila YPZ, after expression in mouse fibroblasts, is specifically and stoichio- metrically sulfated on tyrosine 172, followed by efficient secretion from the mammalian cell. These results provide direct iG uiuo evidence for a remarkably high evolutionary conser- vation of the tyrosine sulfation reaction and establish the basis for future functional studies.

EXPERIMENTAL PROCEDURES

Isotopes [3SS]Sulfate (carrier-free) and L-[%]methionine (50 TBq/mmol)

were purchased from Amer.&am Corp. t-f3,5-3H]Tyrosine (2.5 TBq/ mmol) was obtained from Du Pont-New England Nuclear.

Standard procedures as described by Man&is et aE (9) were used to construct pSV%YP2 (see Fig. 1). A 2.2&b DNA fragment, containing the YP2 gene including its 5’-flanking region (referred to as YPaprom), was cut out from pBR322-YP2 (10) by WindHI digestion, purified on a low melting agaiose gel and ligated with T4 DNA ligase (Boehrineer Mannheim) into HindIII-digested, dephosphorylated icalf inte&inal phos~h~~se, Boehringer Mannheim) <SP64 (Xl). The resulting pSP64-YP2prom DNA (see Fig. 1) was digested with SphI which removed an -5OO-bp fragment. This fragment included the 353 bp of the 5’-flanking region of the YP2 gene which contained the YP2 gene promoter (see Fig. 1). The overlapping ends were blunt- ended by treating 6 pg of DNA with 10 units of T4 DNA polymer&se (Boehringer Mannheim) in the presence of the four dNTPs in 100 gl at 11 “C for 2 h. Phospho~lated HindIfI linkers were ligated to the blunt-ended DNA. The linear plasmid DNA was separated from the 500-bp fragment an a low melting agarose gel and recircularized with

’ The abbreviations used are: YP2, yolk protein 2; kb, kilobase; bp, base pair; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BHK celis, baby hamster kidney cells; PBS, phos- phate-buffered saline; Hepes, 4-(2-hydroxyethyl)-l-piperszineeth- anesulfonic acid, NEPHGE, nonequilibrium pH gradient electrophoresis; TPCK, L-l-ch~oro-3-(4-tosylam~do)-4-phenyl~Z-~~~one; HPLC, high pressure liquid chromatography; TLC, thin-layer chromatography.

14930

Tyrosine Sulfation of Drosophila Yolk Protein 2 in Mouse Fibroblasts 14931

T4 DNA ligase in the presence of agarose (12) at a DNA concentration of 8 pg/ml. The resulting pSP64-YP2 (see Fig. 1) was digested with HindIII, and the 5'-deleted, 1.8-kb YP2 gene purified on a low melting agarose gel and ligated into ~ind111-digested, dephosphorylatedpSV2 (13), yielding pSV2-YP2. The orientation of the HindIII insert was determined by ClaI/BamHI digestion (see Fig. 1).

~ r i f i ~ a t i o n of YP2 from D. melanogaster Flies Unlabeled yolk proteins were purified from Drosophila flies as

described in the preceding paper (Ref. 8, fraction D). For the purification of [35SJsu~ate- la~led YP2, fraction C obtained from about 50 flies, labeled for 24 h with 110 MBd0.5 ml [95S]sulfate as described in the preceding paper (8), was subjected to two-dimensional isoelectric focusing/SDS-PAGE (see below). The radiolabeled YP2 contained in the fured, stained, destained, and dried polyac~lamide gel is referred to purified [36S]sulfate-labeled fly YP2.

Yolk Protein Antibodies 2-mg yolk proteins of D. mehogaster purified from flies as de-

scribed (Ref. 8, fraction D) was subjected to preparative SDS-PAGE, and gel pieces enriched in YP2 were excised from the center of the major Coomassie Blue-stained band. A gel piece corresponding to 50- 100 pg of protein was eluted for 18 h at room temperature with 1% SDS in PBS (150 mM NaCl, 20 mM NaHzPOa, pH 7.4), and protein in the eluate precipitated by 80% acetone at -20 "C. The resulting precipitate was dissolved in 0.5 ml of PBS, mixed with 0.5 ml of complete Freund's adjuvant, and injected intradermally into a female white New Zealand rabbit. For booster injections (4-6-week inter- vals), the same antigen material in incomplete Freund's adjuvant was used. Yolk protein antiserum was obtained by bleeding the rabbit after the fourth booster injection.

For affinity purification, an affinity matrix ("yolk protein-Sepha- rose") was prepared by reacting 3 ml of a 50% (packed gel/volume) suspension of cyanogen bromide-activated Sepharose 4B (Pharmacia LKB Biotechnology Inc.) with 1 mg of purified yolk proteins (fraction D, Ref. 8) as described by the manufacturer. The yolk protein antiserum was preincubated for 24 h at 4 "C with total adult male fly protein (final concentration of 0.15 mg/ml) which had been denatured by boiling in Laemmli sample buffer (14) under reducing conditions, precipitated by 80% acetone, and dissolved in PBS. Subsequently, 2 ml of this preincubated yolk protein antiserum was passed at room temperature over the yolk protein-Sepharose a t a flow rate of 0.1 ml/ min, followed by washing for 2 h with 500 mM NaC1, 50 mM Tris-

Sph I blunt ends

Hind Ill linker ligatmn Hind Ill Hind

la I

pSP64-YP2

linker ATG

1 Hind Ill

5' untranrirrbed sequence a YP2gene. untranslated exon sequences I YP2gene translated exon sequences FB C3 YPZgene, Tuntranscribed intron tequenrc sequence (J- €COR I

I SV40 early promoter ..v

FIG. 1. Insertion of the Llrosophila YP2 gene, after deletion of its 5'4anking region, into the expression vector pSV2. Plasmid pSP64-YP2prom (top left), which contains the YP2 gene (2.2-kb HindIII-fragment, Refs. 10 and 34; see description on bottom left) inserted into the HindIII site, was used for deletion of the 5'- flanking region of the YP2 gene (approximately 350 bp). pSP64- YP2prom was digested with SphI, which cuts into pSP64 and into the YP2 gene at the capping site 48 bp upstream of the initiation codon. After blunt-ending and addition of HindIII iinker, the large fragment was purified and recircularized, yielding pSP64-YP2 (top rfght). The 5'-deleted YP2 gene, containing only 48 bp upstream from the ATG, was excised from pSP64-YP2 with HindIII and inserted into the HindIII site of the expression vector pSV2 (bottom right), yielding pSV2-YP2.

HCl, pH 7.5, at a flow rate of 0.4 ml/min. Bound antibodies were eluted with 4 M MgClz and concentrated and dialyzed by vacuum dialysis against 0.15 M NaCl, 50 mM Tris-HC1, pH 7.5. The purity of the affinity-puri~ed yolk protein antibodies was determined by reducing SDS-PAGE and their concentration by the Bio-Rad protein mieroassay and by ODw.

Cell Culture Unless indicated otherwise, all cells were grown in Dulbecco's

minimum essential medium supplemented with 10% fetal calf serum at 37 "C in an atmosphere containing 10% COZ.

DNA Transfer into Mammalian Cells BHK cells were microinjected as described by Timm et at. (12)

with 1 pg/pl pSV2-YP2 DNA or with pSV2 DNA c o n ~ a i ~ n g the 5'- deleted, 1.8-kb YP2 gene in the anti-sense orientation. After microinjection, cells were incubated for 17 h and then analyzed for YP2 expression by indirect immunofluorescence as described below.

For transient expression of YP2 in Ltk- cells, two 10-em dishes of cells were grown to subconfluency. DNA transfer was performed as described by Sussman and Milman (15). One dish received 4 ml of culture medium containing 500 pg/ml DEAE-dextran (Sigma) and 2 pg of pSV2-YP2 DNA, whereas the other dish received the same medium lacking DNA (mock transfection). After 36 hours, cells were labeled with [s6S]sulfate and analyzed for YP2 expression as described below.

L cells stably expressing YP2 were obtained as follows. 10-em dishes containing 105-106 Ltk- cells were co-transfected with 2 pg of pSV2-YP2 DNA and 100 ng of pFG5 DNA containing the Herpes simplex thymidine kinase gene (16), using a modification (17) of the method described by Graham and van der Eb (18). Transfected cells were first grown in the presence of 0.4 p M aminopterin, 100 pM hypoxanthine, and 16 p~ thymidine (Boehringer Mannheim). 50 aminopterin-resistant colonies were picked, and each colony was transferred to a 3.5-cm dish, whereas the remaining aminopterin- resistant colonies on each 10-cm dish were mixed. The mixed clones were passaged several times before the cells were used for labeling experiments. The individual clones on the 3.5-cm dishes were grown for 2 months in selection medium and then for 2 weeks in medium containing only hypoxanthine and thymidine and thereafter in nor- mal medium before being used for the labeling experiments shown.

Indirect Immunoflmrescence Microinjected BHK cells were fixed, permeabilized, and analyzed

by indirect immunofluorescence as described by Timm et al. (12), using affinity-purified yolk protein antibodies (0.5 pg/ml) followed by staining with rhodamine-conjugated anti-rabbit IgG secondary antibody (Biosys).

M e t a ~ l i c Labeling of L Cells Confluent 3.5-, 6-, or 10-cm dishes of L cells were used for labeling.

Before addition of radiolabel, L cells were washed twice with Dulbec- co's PBS and preincubated for 2 h in Dulbecco's minimum essential medium lacking methionine, sulfate, and/or tyrosine (depending on the radiolabel used later) and supplemented with 1% fetal calf serum which had previously been dialyzed against 10 mM Hepes, pH 7.3, and 150 mM NaC1. Cells then received the same medium again (1,2, and 4 ml for 3.5-, 6-, and 10-cm dishes, respectively), this time containing radioactive methionine (14.8 MBq,/ml), sulfate (11-37 MBq/ml), and/or tyrosine (7-30 MBq/ml) as indicated in the figure legends, followed by incubation for 18-24 h. In some experiments with mixed clones, the medium in addition contained the above concentrations of ~ i n o p t e ~ n , hypoxanthine, and thymidine. The medium was collected, the cells were washed with cold Dulbecco's PBS, and medium and cells used for immunoprecipitation and SDS- PAGE as described below.

Immunoprecipitation of YP2 Unless otherwise indicated, all steps were performed at 4 "C.

dishes) or 2 ml (IO-cm dishes) of buffer I (0.5 M NaCl, 2% SDS, 1 Preparation of Cell Lysates-Cells were lysed in 0.5 ml (6-cm

mM phenylmethanesulfonyl fluoride). The lysate was boiled for 5 min, centrifuged for 1 h at 10,000 X g, and 0.5-0.7 ml of the supernatant was diluted to 14 ml with buffer I1 (20 mM Tris-HC1, pH 7.6; 0.5 M NaCI; 1% (w/v) Nonidet P-40; 5 D M EDTA, pH 7.0; 1 mM phenylmethanesulfonyl fluoride; 23 ~ g / m l aprotinin) prior to immu-

14932 Tyrosine Sulfation of Drosophila Yolk Protein 2 in Mouse Fibroblasts noprecipitation. In some experiments, an aliquot of the supernatant prior to dilution with buffer I1 was mixed with Laemmli sample buffer (14) and directly subjected to SDS-PAGE ("total cell lysate").

Processing of Media-The medium was cleared by centrifugation for 5 min at 100 X g. For the analysis of total medium, an aliquot of the cleared medium was precipitated by 80% acetone, and the protein pellet was disolved in Laemmli sample buffer (14) and directly subjected to SDS-PAGE ("total medium"). For immunoprecipitation, the cleared medium (1-8 ml) was diluted to 14 ml and adjusted to contain the ingredients of buffer I1 plus 0.1% SDS. In some experiments, cleared medium from 6- and 10-cm dishes (2-8 ml) was subjected to either of the following procedures prior to immunoprecipitation. 1) After addition of 5 volumes of cold acetone, samples were kept for 2 h at -20 "C, centrifuged for 15 min a t 15,000 X g, the pellet was washed once with 85% cold acetone, centrifuged, dissolved in 0.5 ml of buffer I, boiled for 5 min, and diluted to 14 ml buffer 11. 2) The cleared medium was lyophilized, and the residue dissolved in 0.5 ml buffer I, boiled, and diluted to 14 ml with buffer 11.

Immunoprecipitation-Each 14-ml sample (cell lysate or cleared medium) received 20 pl of yolk protein antiserum and was incubated for 2 h. After addition of 400 pl of a 50% (packed gel/volume) suspension of protein A-Sepharose CL-4B (Pharmacia), pre-equili- brated in buffer I1 containing 5% of bovine serum albumin, samples were incubated for 2 h with rotation and centrifuged for 5 min a t 200 x g. Pellets were washed three times in buffer 11, resuspended in Laemmli sample buffer containing 2-mercaptoethanol, and boiled for 5 min. For SDS-PAGE, samples were loaded as suspensions. For two- dimensional PAGE, samples were filtered through glass wool, precipitated with acetone and dissolved in O'Farrell lysis buffer (19) containing appropriate ampholytes as described below.

SDS-PAGE and Two-dimensional PAGE SDS-PAGE under reducing conditions was performed according to

Laemmli (14). For two-dimensional PAGE either NEPHGE (20), using 1% (w/v) ampholytes pH 3.5-9.5 (LKB), or isoelectric focusing (191, using 1% (w/v) pH 3.5-9.5,0.5% (w/v) pH 5-7, and 0.5% (w/v) pH 7-9 ampholytes, was employed as the first dimension. Gels were fixed, stained, destained, treated with sodium salicylate, and fluoro- graphed as described previously (21).

Immunoblotting [35S]Sulfate-labeled female flies (7) were homogenized in O'Farrell

lysis buffer (19) and aliquots of the homogenate (75 pg of protein) subjected to NEPHGE. After electrophoresis, proteins were transferred onto nitrocellulose filters (Schleicher/Schidl BA 83), autoradiographed a t room temperature to detect [35S]sulfate-labeled proteins, immunolabeled using affinity-purified anti-yolk protein antibodies (0.2 pg/ml) followed by lZ5I-protein A, and autoradiographed a t -70 "C with an intensifying screen to detect the additional lZ5I- protein A label bound over the yolk proteins, as described (7).

Quantitative Analyses of p5S]Sulfate- and fH]Tyrosine-labeled YP2 For the determination of the ratio of [35S]sulfate to [3H]tyrosine

incorporation into YP2, dried gel pieces containing YP2 double- labeled with [35S]sulfate and [3H]tyrosine were incubated overnight in 1 ml of 30% Hz02 a t 55 "C, and radioactivity was determined by liquid scintillation counting. 3H values were corrected for spillover of "S into the 3H window.

For the analysis of tyrosine [35S]sulfate in YP2, [35S]sulfate-labeled YP2 contained in dried gel pieces was eluted from the gel by Pronase digestion, the eluate was subjected to alkaline hydrolysis using barium hydroxide followed by neutralization with sulfuric acid, and tyrosine sulfate present in the neutral supernatant was determined after thin- layer electrophoresis at pH 3.5 as described (22).

For the determination of the stoichiometry of tyrosine sulfation of YP2, [3H]tyrosine-labeled YP2 contained in dried gel pieces was eluted by Pronase digestion, and the eluate was subjected to alkaline hydrolysis followed by separation of [3H]tyrosine sulfate and unmod- ified [3H]tyrosine by two-dimensional thin-layer electrophoresis/ascending chromatography as described previously (7), using metha- no1:pyridine:water = 80:4:20 in the chromatography.

Sulfopeptide Mapping Dried gel pieces containing [35S]sulfate-labeled YP2 were incubated

for 30 min in 10% acetic acid, 30% methanol, washed in distilled water for 1 h, lyophilized, and incubated for 48 h in 1 ml of 50 mM

NH4HCOa buffer to which either 10 pg of TPCK-trypsin (Boehringer Mannheim) or 10 pg of trypsin (Boehringer Mannheim) plus 10 pg of chymotrypsin A, (Boehringer Mannheim) were added at the begin- ning of the incubation and again after 24 h. The eluates were lyophilized and sulfopeptides separated either by two-dimensional electrophoresis/ascending chromotography on thin-layer cellulose sheets followed by autoradiography or by HPLC. For two-dimensional electrophoresis/ascending chromatography, the buffer systems described in Ref. 23 were used. Electrophoresis was performed for 4 h a t 500 V, a condition in which sulfopeptides were found to be separated from each other without having migrated off the cellulose thin-layer sheet. For HPLC analysis, aliquots of the digests were adjusted to 5% formic acid in a total volume of 100 p1 and subjected to HPLC on an LKB system equiped with a Vydac reverse phase C I S (5 pm) column using a flow rate of 1 ml/min. Trifluoroacetic acid (0.1%) was used as the mobile phase, and gradients were developed with acetonitrile containing 0.07% (v/v) trifluoroacetic acid as indicated in Fig. 7. Fractions

NEPHGE - prote in staining

3 5 ~ 0 ~ +

"

-

yp3 YP2 YPl \? "

[ 1 2 5 ~ 1 pro te in A

basic acidic FIG. 2. Specificity of the anti-yolk protein antibodies. Total

protein (75 pg) of [35S]sulfate-labeled female flies of D. melanogaster was subjected to two-dimensional NEPHGE/SDS-PAGE, transferred onto a nitrocellulose filter, and the filter was autoradiographed for 7 h a t room temperature (middle panel). Proteins on the filter were then immunolabeled with affinity-purified anti-yolk protein antibodies (0.2 pglml) followed by incubation with lZ5I-protein A. The filter was then autoradiographed again for 7 h, this time a t -70 "C using an intensifying screen to selectively enhance the lZ5I radiation (bottom panel). For comparison, the Amido Black staining of a nitrocellulose filter containing total female fly protein (150 pg) is shown in the top panel. The positions of YP1, YP2, and YP3 are indicated by white numbers (top panel) or arrows (middle and bottom panels).


FIG. 3. Immunofluorescence analysis of BHK cells expressing Drosophila YP2 after microinjection of pSV2-YP2 DNA. BHK cells were microinjected with 1 pg/pl DNA of either pSV2-YP2 ( A and B ) or pSV2 containing the 5"deleted YP2 gene in the opposite orientation (C and D). Cells were fixed 17 h after microinjection and analyzed for expression of YP2 by indirect immunofluorescence using affinity-purified anti-yolk protein antibodies ( B and D). A and C show the corresponding areas of B and D photographed using Nomarski optics. Bars = 10 pm.

(1 ml) were collected and [35S]sulfate-labeled peptides detected by liquid scintillation counting.

RESULTS

Construction ofpSV2- YP2 for Expression of Drosophila YP2 Protein in Mammalian Cells-The expression of the YP2 gene in D. melanogaster is stringently regulated with respect to sex, developmental stage, and tissues (see Refs. 24-28 and references therein). As was to be expected, transfection of mouse fibroblasts (Ltk- cells) with genomic DNA (2.2-kb Hind111 fragment; Ref. 10) containing the YP2 gene and 5'-regulatory sequences (27-30) did not result in any detectable expression of the YP2 protein (data not shown). In order to obtain expression of Drosophila YP2 in mammalian cells, the 5'- regulatory sequence of the YP2 gene was deleted, and the

resulting 5'-deleted, 1.8-kb YP2 gene, containing 48 bp of 5'- untranslated sequence, the entire translated sequence, a 68- bp intron, and the 3"untranslated sequence with two poly- adenylation signals, was cloned into the eucaryotic expression vector pSV2 (13) to give the construct pSV2-YP2 (Fig. 1).

Expression of Drosophila YP2 in Mammalian Cells-To monitor the expression of YP2, an antiserum was raised against yolk proteins purified from Drosophila flies, and anti- yolk protein antibodies were affinity-purified from the antiserum. The specificity of the antiserum and the affinity- purified antibodies was determined by two-dimensional immunoblotting. As reported previously (7), the three yolk proteins can be identified in a total fly homogenate by their migration in two-dimensional PAGE and their sulfate incorporation (Fig. 2, top and middle panels). The affinity-purified

14934 Tyrosine Sulfation of Drosophila Yolk Protein 2 in Mouse Fibroblasts

SDS PAGE

4 I E F

YP2 L . . :..*,.:

p ro te in staining

Fluorography

basic acidic FIG. 4. Drosophila YP2 is secreted by mouse fibroblasts

and comigrates with YP2 purified from flies on two-dimensional PAGE. Cells of L cell clone L-YP2-3 were labeled for 22 h with [3sS]methionine. Labeled YP2 was immunoprecipitated from the culture medium, mixed with unlabeled yolk proteins purified from Drosophila flies, and the mixture subjected to two-dimensional PAGE. The top panel shows the Coomassie Blue staining of the three yolk proteins of the fly. The positions of YP1, YP2, and YP3 are indicated by arrows. The bottom panel shows a fluorogram of the same gel, visualizing the [35S]methionine-labeled YP2 secreted by the L cells. The dotted line on the fluorogram indicates the position of the Coomassie Blue-stained YP2 spot. ZEF, isoelectric focusing.

psv2-YP2 L-YP2-3 pFG5

I I -m --- m " -

91-

67-

&.

43-

97 -

61 -

43-

lYP2 30-

+ + +

- - -

YP2

14- 30-

C M C M C M C M C M t o t a l immuno- immunoprecipitation

precipitation

FIG. 5. Mouse fibroblasts, transfected with pSV2-YP2, sulfate and secrete Drosophila YP2. Left panels, the L cell clone L- YP2-3 was labeled for 24 h with [3sS]sulfate. Aliquots (5%) of the total cell lysate ( C ) and the total medium (M) were analyzed by SDS- PAGE followed by autoradiography for 15 h (total). YP2 was immunoprecipitated from the remainder of the cell lysate and the culture medium and analyzed by SDS-PAGE followed by autoradiography for 48 h (immunoprecipitation). Right panel, cells were labeled for 24 h with [3sS]sulfate. ++, mixture of L cell clones obtained after co- transfection with pSV2-YP2 and the thymidine kinase-encodingplas- mid pFG5; +-, Ltk- cells transiently transfected with pSV2-YP2; - -, mock-transfected Ltk- cells. YP2 was immunoprecipitated from one-third of the cell lysate (C) and from the entire culture medium (M) and analyzed by SDS-PAGE followed by fluorography for 5 days. The position of YP2 is indicated by arrowheads. Molecular mass standards are given in kDa.

Tyr(S1 Fly-YP2 L-YP2

0 0 0

FIG. 6. Drosophila YP2 secreted by mouse fibroblasts is sulfated on tyrosine. Cells of L cell clone L-YP2-3 were labeled for 18 h with ["S]sulfate. YP2 secreted by the L cells was immunoprecipitated from the culture medium, the immunoprecipitate subjected to SDS-PAGE, and the YP2 band subjected to tyrosine sulfate analysis (L- YP2) . For comparison, tyrosine [35S]sulfate standard (Tyr fS ) ) and purified [3sS]sulfate-labeled YP2 from flies (Fly-YP2) were also subjected to tyrosine sulfate analysis. An autoradiogram of the thin-layer cellulose sheet after electrophoresis a t pH 3.5 is shown. The dotted lines indicate the position of unlabeled tyrosine sulfate standard mixed to the sample prior to electrophoresis and detected by ninhydrin staining.

TABLE I The proportion of tyrosine ~5S]sul fa te per total ~ 5 S l s u l f a t e

in Drosophila Y P 2 secreted by mouse fibroblusts and YP2 purified from flies is the same

Purified [35S]sulfate-labeled YP2 from flies (Fly-YP2) and [35S] sulfate-labeled YP2 purified from the culture medium of L-YP2-3 cells (L-YP2) as described in the legend to Fig. 6, contained in gel pieces, were eluted by Pronase digestion, subjected to alkaline hydrolysis, and analyzed by cellulose thin-layer electrophoresis (see Fig. 6). The recovery of 35S radioactivity in alkali-stable form after hydrolysis and in the tyrosine sulfate spot after thin-layer electrophoresis, is given as percent of the starting material (Pronase eluate; Fly- YP2, 6618 cpm; L-YP2, 1768 cpm). Recoveries were corrected for that of tyrosine ["S]sulfate standard, which was 88% after alkaline hydrolysis and 71% after thin-layer electrophoresis.

Recovery of "S radioactivity

in alkali-stable form

Recovery of "S radioactivity

as tyrosine sulfate

% Fly-YP2 91 L-YP2 88

% 79 81

antibodies specifically recognized the three yolk proteins (Fig. 2, bottom panel). The same result was obtained with the antiserum (data not shown).

The suitability of the construct pSV2-YP2 to give expression of Drosophila YP2 in mammalian cells was examined after microinjection of the recombinant DNA into the nucleus of BHK cells. The presence of the YP2 protein in the microinjected cells was investigated by indirect immunofluorescence. A staining characteristic for the endoplasmic reticulum was observed 17 h after microinjection (Fig. 3B) , suggesting that YP2 was expressed and entered the secretory pathway. No staining was detected when BHK cells were microinjected


TABLE I1 ~ ~ s o p h ~ l ~ YP2 secreted by different mouse L cell clones is s ~ a t e d to the same degree

L cells were double-labeled for 20 h with [3H]tyrosine and [35S]sulfate. YP2 was immunoprecipitated from the culture medium and the immunoprecipitate after addition of unlabeled yolk protein marker subjected to two- dimensional PAGE. The 3H and "S radioactivity present in the Coomassie Blue-stained YP2 spot, which reflected YP2 synthesis and YP2 sulfation, respectively, was determined. The value obtained by dividing the 35S radioactivity by the 3H radioactivity reflected the relative degree of YP2 sulfation. Ltk-, untransfected Ltk- cells; clones 3, 18, 46, individual L cell clones obtained after co-transfection of Ltk- cells with pSV2-YP2 and pFGB; mixture of -30 clones, mixture of -30 individual L cell clones obtained after co-transfection.

Relative degree of YP2 sulfation cells YP2 synthesis YP2 sulfation

(['Hltyrosine) ([36S]sulfate) ([35S]sulfate/[3H]tyrosine)

Ltk- Clone 3 Clone 18 Clone 46 Mixture of -30 clones

epm 0

847 400

0 100

cpm 0

937 352

0 94

TABLE I11 ~ r ~ o p ~ i ~ YP2 secreted by mowe fibroblasts contains close to 1 mol

of tyrosine sulfatelmol of polypeptide Cells of L cell clone L-YP2-3 were labeled for 20 b with [3H]

tyrosine. YP2 secreted by the L cells was immunoprecipitated from the culture medium, the immunoprecipitate after addition of unlabeled yolk protein marker subjected to two-dimensional PAGE, and the YP2 spot subjected to Pronase elution followed by alkaline hydrolysis. [3H]Tyrosine and [3H]tyrosine sulfate were determined after separation by two-dimensional cellulose thin-layer electropho- resislascending chromatography. The ['Hltyrosine value shown is the original counted value multiplied by a factor of 1.2 to account for the slightly lower recovery of [3H]tyrosine standard than [3H]tyrosine sulfate standard. The [3H]tyrosine and [3H]tyrosine sulfate values were used to calculate the percentage of ['Hltyrosine sulfate per total [3H]tyrosine (sum of [3H]tyrosine plus [3H]tyrosine sulfate). Multi- plication of this percentage by 17, the number of tyrosine residues per YP2 polypeptide (30), yields the number of tyrosine sulfate residues Der uolwevtide chain.

[3H1Tyrosine [3H]Tyrosine ['HITyrosine sulfate/ Tyrosine sulfate/ sulfate total [3H]tyrosine polypeptide

cpm cpm % mol/mol 7653 396 4.9 0.83

with DNA of a pSV2 construct containing the YP2 gene inserted in the opposite, anti-sense orientation (Fig. 3D).

Secretion of Drosophila YP2 from Mouse Fibroblasts-In order to perform biochemical studies on the YP2 protein expressed in mammalian cells, we isolated mouse fibroblast L cell clones after co-transfection of Ltk- cells with pSV2-YP2 and pFG5 which carries the thymidine kinase gene of Herpes simplex (16) and served as selectable marker for transfected cells. Of the over 100 L cell clones obtained, 50 were picked, and of these, 12 were labeled for 20 h with [35S]methionine followed by immunoprecipitation of the medium and cell lysate with anti-yolk protein antiserum. Fluorography after SDS-PAGE of the immunoprecipitates revealed that 8 of the 12 L cell clones expressed and secreted Drosophila UP2 (data not shown). Comparison of the amount of radioactive YP2 in the medium and cells of one L cell clone, referred to as L- YP2-3 and used for most of the subsequent studies, showed that 95% of the total YP2 recovered after 20 h of labeling was present in the medium. This suggested that YP2 was effi- ciently secreted from the cells, a conclusion that has recently been confirmed by pulse-chase experiments (35). Fig. 4 shows the two-dimensional PAGE of YP2 secreted from L-YP2-3 cells. [3sSS]Methionine-labeled YP2 (Fig. 4, bottom panel) co- migrated with unlabeled fly YP2 (Fig. 4, top panel). These data showed that mouse fibroblasts produced and secreted Drosophila YP2 and suggested that the post-translational

processing of this protein in the mammalian cell was very similar, if not identical, to that in the fly.

Sulfation of Drosophila YP2 by Mouse Fibroblasts-L-YP2- 3 cells were labeled for 24 h with [35S]sulfate and investigated for sulfation of Drosophila YP2. Immunoprecipitation revealed that these cells produced sulfated YP2 which accumulated in the medium (Fig. 5, left panel). The amount of sulfated YP2 in the medium was 97.5% of the total sulfated YP2 recovered in the medium and cells at the end of the 24- h labeling. Sulfated YP2 was only a minor component of the total sulfated proteins produced and secreted by the L cells as it was not detectable in fluorograms of SDS-gels of the total cell protein and the total medium (Fig. 5, left panel). As will be described below (Table 111), this was not due to a low degree of YP2 sulfation but reflected the low rate of YP2 synthesis relative to the total protein synthesis since similar results were obtained with [3H]tyrosine-la~led L-YP2-3 cells (data not shown).

Sulfation of Drosophila YP2 by L-YP2-3 cells was not a singular observation. As shown in the right panel of Fig. 5, a mixture of approximately 30 L cell clones obtained after co- transfection with pSV2-YP2 and pFG5 (lanes ++), as well as transiently transfected Ltk- cells (lanes +-), produced sulfated YP2 which accumulated in the medium as observed with L-YP2-3 cells. No sulfated band was observed in the position of YP2 when mock-transfected cells were analyzed (lanes - -1.

Drosophila YP2 Secreted by Mouse Fibroblasts fs Sulfated on Tyrosine-The nature of the sulfate linkage in Drosophila YP2 produced by mouse fibroblasts was determined (Fig. 6 and Table I). [35S]Sulfate-labeled YP2 secreted by L-YP2-3 cells and, for comparison, [3%]sulfate-labeled YP2 purified from flies was subjected to an alkaline hydrolysis procedure which is known to hydrolyze carbohydrate-linked [35S]sulfate and to preserve tyrosine-linked [35S]sulfate (22). Thin-layer electrophoresis revealed that the alkali-stable radioactive material was tyrosine [?3]sulfate in the case of L cell YP2 as well as fly YP2 (Fig. 6). For both L cell YP2 and fly YP2, about 80% of the total incorporated [35S]sulfate could by recovered as tyrosine [35S]sulfate after alkaline hydrolysis and thin-layer electrophoresis (Table I). Similar results were obtained with YP2 secreted from transiently transfected Ltk- cells and from a mixture of stable L cell clones (data not shown). These results confirmed previous data that most, if not all, of the sulfate in YP2 from Drosophila flies is bound to tyrosine (7,8) and showed that the same was the case for Drosophila YP2 secreted by mouse fibroblasts.

Drosophila YP2 Secreted by Different Mouse L Cell Clones

14936 Tyrosine Sulfation of Drosophila Yolk Protein 2 in Mouse Fibroblasts

L -YP2

Y I-

t

@-ab-@ FIG. 7. Drosophila YP2 secreted by

mouse fibroblasts and YP2 purified from flies yield very similar sulfopeptide maps. Purified [35S]sulfate-labeled YP2 from flies (Fly-YP2) and [35S]sulfate- labeled YP2 purified from the culture medium of L cell clones expressing YP2 (L- YP2) as described in the legend to Fig. 6, contained in gel pieces, were digested with proteases. Left panels, digestion with TPCK-trypsin, followed by separation of peptides on thin-layer cellulose sheets by electrophoresis at pH 3.5 in the first dimension and ascending chromatography (TLC) in the second dimension. Sulfopeptides were detected by autoradiography. L-YP2 was clone 9. Rightpanels, digestion with trypsin plus chymotrypsin, followed by separation of peptides by HPLC. The run of fly YP2 (top panel) contained 30% more radioactive material than that of L cell YP2 (clone 3, middle panel). For the mixing experiment (bottom panel), equal amounts of radioactivity of the fly-YP2 digest and the L-YP2 digest were mixed prior to HPLC. Sulfopep- tides were detected by liquid scintillation counting of the fractions.

300

E $ 200

100

250

200

150 4 U

100

50

250

200

E 150 4 U

100

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L-YP2

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40 7 ID 3 t

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0

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20 5 1. c ID

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40 50


Is Sulfated to the Same Degree-The relative degree of SUI- fation of Drosophila YP2 produced by different L cell clones was determined by comparing the incorporation of a radioactive amino acid ([3H]tyrosine) to that of [35S]sulfate in cellular YP2 (not shown) and secreted YP2 (Table 11). (This relative degree of YP2 sulfation provided no information on the absolute stoichiometry of YP2 sulfation.) When untransfected Ltk- cells were analyzed, no radioactivity was found in the gel in the position of unlabeled marker YP2. Analysis of three individual L cell clones revealed that two clones (clones 3 and 18) expressed YP2, whereas one clone (clone 46) did not, as monitored by [3H]tyrosine incorporation. The two positive clones produced different amounts of YP2. Both of these clones produced sulfated YP2, as shown by ["SI sulfate incorporation. The ratio of i3%]sulfate to @i]tyrosine incorporated into YP2 was almost identical for the two clones. The same ratio was obtained when a mixture of about 30 randomly chosen aminopterin-resistant L cell clones was analyzed. (The amount of [3H]tyrosine-labeled YP2 produced by the mixture of clones was lower than that produced by individual YP2-positive clones, presumably because of the presence of YP2-negative cells.) These results showed that different L cell clones producing YP2 sulfated the protein to the same relative degree.

The Stoichiometry of Tyrosine Sulfation of Drosophila YP2 Produced by Mouse F i b ~ o b ~ ~ t s Is the Same as in the Fly- Previous experiments have shown that YP2 from Drosophila flies is sulfated with an average stoichiometry of 0.9 mol of tyrosine sulfate/mol of polypeptide (7). It was important to determine whether the stoichiometry of tyrosine sulfation of Drosophila YP2 was similar when this protein was produced by mouse fibroblasts. Quantitation of the amount of unmod- ified [3H]tyrosine and [3H]tyrosine sulfate in an alkaline hydrolysate of YP2 secreted by L-YP2-3 cells showed that approximately 5% of the 17 tyrosine residues in this polypeptide were sulfated (Table 111). This equaled an average stoichiometry of 0.83, which was a very similar value to that found in flies (7).

The Site of Tyrosine Sulfation of Drosophila KP2 Produced by Mouse Fibroblasts Is the Same as in the Fly-We have shown in the preceding paper (8) that YP2 from Drosphila flies is sulfated at one tyrosine residue which is located at sequence position 172 of the preprotein. In this study, we investigated whether the same site is sulfated when Drosophila YP2 is produced by mouse fibroblasts. After extensive tryptic digestion of [35S]sulfate-labeled YP2 secreted by L cells, the pattern of the resulting sulfopeptides was very similar to that obtained with [35S]sulfate-labeled YP2 from flies (Fig. 7, left panel). The two sulfopeptides observed in both cases probably corresponded to the sulfopeptides A and B detected after HPLC in the preceding paper (Ref. 8, Fig. 2 of Miniprint). The generation of two sulfopeptides by tryptic digestion of single site-sulfated YP2 can be explained by the sequence on the COOH-terminal side of tyrosine 172 (see preceding paper, Ref. 8).

The smallest sulfopeptide obtained after tryptic digestion of YP2 from flies contains not only tyrosine 172 but also two other tyrosine residues (see Fig. 1 of Ref. 8). To demonstrate that neither of these other 2 tyrosine residues were sulfated in Drosophila YP2 produced by mouse fibroblasts, [35S]su1- fate-labeled YP2 secreted by L-YP2-3 cells waR double-digested with trypsin and chymotrypsin, which in the case of fly YP2 yields a sulfopeptide containing only 1 tyrosine residue, the sulfated tyrosine 172 (see Fig. 1 of Ref. 8). As shown in Fig. 7, right panel, a single sulfopeptide peak was observed after HPLC of a t~tic/chymotryptic digest of YP2 from L-

YP2-3 cells. The elution time of this peak was very similar to that of the single sulfopeptide peak obtained after tryptic/ chymotryptic digestion of YP2 from flies. These results strongly suggest that the same tyrosine residue of YP2 (i.e. tyrosine 172) was sulfated when the protein was produced in mouse fibroblasts instead of flies.

DISCUSSION

We have shown that an insect tyrosine-sulfated secretory protein undergoes sulfation and secretion after expression in a m ~ m a l i a n cell. Analysis of several mouse fibroblast clones expressing Drosophila YP2 revealed that all features of the sulfation of Drosophi~ YP2 in flies (7, 8) were observed in the mammalian system: (i) sulfate was linked largely, if not exclusively, to tyrosine; (ii) the stoichiometry of tyrosine sulfation was close to 1; (iii) tyrosine 172 was the single site of sulfation. The latter finding has recently been supported by the observation that YP2, in which tyrosine 172 was mutated to phenylalanine, is produced by mouse fibroblasts in completely unsdfated form (35).

These results lead to two conclusions about the tyrosine sulfation of an insect substrate protein by a mammalian tyrosylprotein sulfotransferase in intact cells. First, the recognition of the insect protein substrate by the mammalian enzyme is highly specific since none of the other 16 tyrosine residues in Drosophila YP2 served as sulfate acceptor. Second, the efficiency of the reaction between the insect protein substrate and the mammalian enzyme is high since sulfation of tyrosine 172 approached a stoichiometric level, Thus, essential parameters of the enzyme-substrate interaction in the tyrosine sulfation reaction in intact cells appear to be highly conserved between insects and mammals.

In addition, other forms of post-translational processing of Drosophila YP2 in mouse fibroblasts (such as signal peptide cleavage, N-glycosylation, and phosphorylation) were indis- tinguishable from those in flies. We found that the mobility in two-dimensional PAGE of Drosophila YP2 produced by mouse fibroblasts was very similar, if not identical, to that produced by flies. This in general indicates that the post- translational processing of Drosophila YP2 in fly and mouse cells does not differ significantly with respect to isoelectric point and apparent molecular weight of the final protein product. These results are also consistent with the previous conclusion (31) that signal peptide cleavage is conserved between insects and mammals. In addition, we have noticed (i) that Drosophila YP2, which contains a potential N-glycosylation site at asparagine 25 (30), lacks N-linked oligosac- charides not only in flies (32) but also in mouse fibroblasts since the electrophoretic mobility of the protein did not change after tunicamycin treatment of L-YP2 cells (data not shown) and (ii) that Drosop~~la YP2 is phosphorylated not only in flies (32, 33) but also in mouse fibroblasts (data not shown). Finally, the identical ele~rophoretic mobility of Dro- sophila YP2 from L cells and from flies also indicates that the splicing of the single intron (30), present in the fly genomic DNA used for transfection, occurred correctly in the mammalian cell.

The secretion of Drosophila YP2 by mouse fibroblasts ap- peared to be an efficient process since after a 20- to 24-h labeling period most of the YP2 was found in the medium (97.5% of sulfate-labeled YPZ; 95% of amino acid-labeled YP2). We have previously hypothesized that tyrosine sulfation might be important for the intracellular transport of certain secretory proteins. The conserved, stoichiometric, single-site sulfation of Drosophila YP2, t,ogether with the avail- ability of cloned YP2 DNA, makes this protein a suitable

14938 Tyrosine Sulfatwn of Drosophila Yolk Protein 2 in Mouse Fibroblasts

object to test this hypothesis. In studies to be reported else- where (35), using mouse fibroblasts producing Drosophila YP2, we have found that tyrosine sulfation promotes the rapid secretion of this protein.

Acknowledgments-We thank Beate Timm for help with microinjection of BHK cells and with immunofluoresence, Dr. Uli Bernard for advice in transfecting Ltk- cells, Christof Niehrs and Jane Stinchcombe for helpful comments on the manuscript, and Petra Riedinger and Rachel Wainwright for help in the preparation of the figures and the text, respectively.

REFERENCES

1. Huttner, W. B., and Baeuerle, P. A. (1988) Mod. Cell BioL 6,97-

2. Huttner, W. B. (1987) Trends Biochem. Sci. 2,361-363 3. Huttner, W. B. Baeuerle, P. A., Benedum, U. M., Friederich, E.,

Hille, A., Lee, R. W. H., Rosa, P., Seydel, U., and Suchanek, C. (1986) Colloq. INSERM (Znst. Natl. Sante Rech. Med.) 139,

4. Lee, R. W. H., and Huttner, W. B. (1985) Proc. Natl. Acad. Sci.

5. Baeuerle, P. A., and Huttner, W. B. (1987) J. Cell BioL 105,

6. Hortin, G., Folz, R., Gordon, J. I., and Strauss, A. W. (1986)

7. Baeuerle, P. A., and Huttner, W. B. (1985) J. Bwl. Chem. 260,

8. Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. BioL Chem. 263,14925-14929

9. Maniatis, T., Fritsch, E. G., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manuul, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

10. Hovemann, B., Galler, R., Walldorf, U., Kiipper, H., and Bautz, E. K. F. (1981) Nucleic Acids Res. 9,4721-4734

11. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12,7035-7056

12. Timm, B., Kondor-Koch, C., Lehrach, H., Riedel, H., Edstriim, J.-E., and Garoff, H. (1983) Methods EnzymL 96,496-511

140

199-217

U. S. A. 82,6143-6147

2655-2664

Biochem. Bwphys. Res. Commun. 141,326-333

6434-6439

13.

14. 15.

16.

17.

18.

19. 20.

21.

22. 23. 24. 25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Garoff, H., Kondor-Koch, C., Pettersson, R., and Burke, B. (1983)

Laemmli, U. K. (1970) Nature 227,680-685 Sussman, D. J., and Milman, G. (1984) Mol. Cell. Bwl. 4, 1641- 1643

ColbBre-Garapin, F., Chousterman, S., Horodniceanu, F., Kour- ilskv. P.. and Garauin. A.-C. 11979) Proc. NatL Acad. Sci. U. S.

J. Cell Bwl. 97,652-658

. I

A. ?e, 3755-3759 '

Gotoh, T., and Schutz, G. (1983) EMBO J. 2,1487-1492 Matthias, P. D., Bernard, H. U., Scott, A., Brady, G., Hashimoh-

Graham, F. L., and van der Eb, A. J. (1973) Virology 52, 456-

OFarrell, P. H. (1975) J. BwL Chem. 250,4007-4021 O'Farrell, P. Z., Goodman, H. M., and OFarrell, P. H. (1977) Cell

Lee. R. W. H., and Huttner. W. B. (1983) J. Biol. Chem. 258,

467

12,1133-1142

11326-11334 . .

Huttner. W. B. (1984) Methoap EnmmL 107. 200-223 Erikson; E., andErikeon, R. L. (198i)) Cell 21; 829-836 Postlethwait, J. H., and Jowett, T. (1980) Cell 20,671-678 Isaac, P. G., and Bownes, M. (1982) Eur. J. Biochem. 123,527-

Belote, J. M., Handler, A. M., Wolfner, M. F., Livak, K. J. and

Garabedian, M. J., Hung, M.-C., and Wensink, P. C. (1985) Proc.

Hung, M.-C., Barnett, T., Woolford, C., and Wensink, P. C.

Hovemann, B., and Galler, R. (1982) Nucleic Acids Res. 10,2261-

Hung, M.-C., and Wensink, P. C. (1983) J. Mol. Bwl. 164,481-

Brennan, M. D., Warren, T. G., and Mahowald, A. P. (1980) J.

Minoo, P., and Postlethwait, J. (1985) Arch. Insect. Biochem.

Brennan, M. D., and Mahowald, A. P. (1982) Insect Biochern. 12,

Garabedian, M. J., Shepherd, B. M., and Wensink, P. C. (1986)

Friederich, E., Fritz, H.-J., and Huttner, W. B. (1988) J. Cell

534

Baker, B. S. (1985) Cell 40,339-348

Natl. Acad. Sci. U. S. A. 82,1396-1400

(1982) J. Mol. BioL 154,581402

2274

492

Cell Bid. 87,516-520

PhySioL 2.7-21

669-673

Cell 45,859-867

Bwl., in press

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Expression, Tyrosine Sulfation, and Secretion of Yolk Protein 2 of