The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes

DEVELOPMENTAL

BIOLOGY

141,1-12 (1990)

The Secretory Pathway Is Blocked between the Trans-Golgi and the Plasma Membrane during Meiotic Maturation in Xenopus Oocytes DAVID S. LEAF, SUSANJ. ROBERTS,JOHN C. GERHART,AND HSIAO-PING MOORE Department

of Molecular

and Cell Biology, Division

of Cell and Developmental

Biology,

University of Calzfornia,

Berkeley,

Cahfmia

9.&W.?

Accepted May 4, 1990

Protein secretion is blocked in Xenopus oocytes arrested at second meitoic metaphase. In this report, we show that secretion becomes blocked coincident with germinal vesicle breakdown (GVBD). Transport through the metaphasearrested oocyte’s secretory pathway continues unimpeded until proteins reach the trans-Golgi. These conclusions are drawn from experiments using exogenous prolactin and vesicular stomatitus virus G protein (VSV G) encoded by SP6 transcripts and endogenous glycosaminoglycan (GAG) chains initiated on b-D-4-methylumbelliferyl-xyloside. From the initiation of maturation with progesterone until GVBD, secretion of prolactin synthesized before the start of maturation is comparable to secretion in immature oocytes, but after GVBD secretion of prolactin declines approximately 62% in the first hour. Not all steps in the secretory pathway are blocked when oocytes mature. Since VSV G protein acquires resistance to endo H digestion with equal efficiency in immature oocytes (arrested in first meiotic prophase) and matured oocytes (arrested in second meiotic metaphase), we conclude that transport of this protein from the ER to the Golgi is not inhibited at meiotic metaphase. Using [S6S]sulfate to label xyloside-initiated GAG chains we find that transport of GAG chains from the trans-Golgi to the cell surface is lbfold lower in matured oocytes than in immature oocytes. Examination of the size of GAG chains by SDS-PAGE and HPLC indicates that matured oocytes produce GAG chains significantly larger than GAG chains from immature oocytes. This increase in size suggests that GAG chains from matured oocytes have a longer residence time in the trans-Golgi than GAG chains from immature oocytes. Hence, part of the block to secretion in metaphase-arrested oocytes could be an inhibition of vesicle budding from the transGolgi. 0 2390 Academic Press, Inc.

and the vesiculation of nuclear envelope, endoplasmic reticulum (ER),’ and Golgi. Membrane traffic is regulated during the cell cycle of Effects of the cell cycle on membrane traffic can be higher eukaryotes. Receptor recycling, fluid phase pino- readily addressed in Xenopus oocytes. Immature stage 6 cytosis, adsorbtive pinocytosis, constitutive secretion, oocytes are arrested at first meiotic prophase. In vitro and regulated secretion are all inhibited in mitotic application of progesterone induces the resumption of mammalian cells (Warren et ab, 1984, 1983; Hesketh et meiosis through second meiotic metaphase. Appearance aZ., 1984; Featherstone et uZ., 1985; Berlin et aL, 1978; of a white spot in the animal hemisphere caused by the Berlin and Oliver, 1980; Oliver et aZ., 1985). Internal breakdown of the germinal vesicle signals the entry of membrane systems are dynamic during mitosis; the nu- the oocyte into the first meiotic metaphase. Since ooclear envelope and Golgi apparatus (and in some cases cytes are large and easily injected with RNA, secretion the endoplasmic reticulum) break down during pro- of exogenous proteins can be monitored in an individual metaphase and reassemble during telophase of the cell or in small groups of cells. This allows examination mammalian cell cycle (Burke et aL, 1982; Lucocq et aZ., of secretion in cells with well-defined and physiological 198’7; Lucocq and Warren, 1987; Zelligs and Wollman, cell cycle states. 1979). The Golgi apparatus fragments early in mitosis Xenopus oocytes undergoing meiotic maturation forming two populations; multivesicular clusters and share many similarities to mitotic cells. Entry into mifree vesicles (Lucocq et al, 1989). These clusters and vesi- tosis and meiosis is controlled by maturation-promoting cles appear to be important in partitioning the Golgi apparatus between daughter cells. Warren (1985) pro1Abbreviations used: MPF, maturation-promoting factor; ER, enposed that there is a fundamental interplay between doplasmic reticulum; GVBD, germinal vesicle breakdown; HA, hemagmembrane integrity and vesicular traffic. A general in- glutinin; VSV G, vesicular stomatitus virus glycoprotein; GAG, glycoshibition of vesicle fusion during mitosis was suggested aminoglycan; fl-D-xyloside, I-methylumbelliferyl /3-D-xyloside; endo to cause both the block of exocytic and endocytic traffic H, endoglycosidase H; CPC, cetyl pyridinium chloride. INTRODUCTION

1

0012-1606/90 $3.00 Copyright All rights

Q 1990 by Academic Press, Inc. of reproduction in any form reserved.

2

DEVELOPMENTAL BIOLOGY

factor (MPF) (Masui and Marker& 1971; Smith and Ecker, 1971; Miake-Lye et aZ.,1983; Gerhart et al, 1984; reviewed in Hunt, 1989). With the appearance of active MPF, the oocyte’s nuclear envelope (germinal vesicle) breaks down, chromosomes condense, and spindles form. Colman et al. (1985) demonstrated that the Golgi breaks down and protein secretion becomes blocked during meiotic maturation of Xenopus oocytes. They showed that dissolution of the Golgi correlated with germinal vesicle breakdown (GVBD). However, since they examined secretion in oocytes matured overnight and did not observe when GVBD occurred, they could not determine whether the block to secretion also correlated with GVBD. Recently, Ceriotti and Colman (1989) reported that the viral hemagglutinin protein (HA) was transported from the ER to the Golgi in mature oocytes, indicating that membrane traffic continues from ER to Golgi despite the vesiculation of the Golgi. In this report, we examine secretion in maturing oocytes using exogenously expressed prolactin and the vesicular stomatitus virus glycoprotein (VSV G), and endogenous glycosaminoglycan (GAG) chains initiated on 4-methylumbelliferyl P-D-xyloside. We have determined when after the administration of progesterone the block to secretion is established and whether it affects all membrane traffic in cells. We find that the block to secretion of prolactin is not established until shortly after GVBD. Similarly, VSV G protein appears on the cell surface when VSV G RNA is injected into immature oocytes, but not if VSV G RNA is injected into maturing oocytes after GVBD. However, there is no difference in the transport of VSV G from the ER to the Golgi in immature and mature oocytes as determined by the kinetics of acquisition of endoglycosidase H (endo H) resistance. The major effect of the block to secretion is an approximately 15-fold decrease in the rate of transport from the trans-Golgi to the plasma membrane as assayed by secretion of xyloside-initiated GAG chains. Thus the block to protein secretion during Xenopus meiotic maturation does not inhibit all intracellular membrane transport, but more specifically inhibits traffic between the Golgi and the cell surface. MATERIALS

AND

METHODS

Supplies

Restriction enzymes, SP6 polymerase, ‘7-methyl GpppG cap analog, RNase-free DNase, endo H were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). T4 DNA ligase was purchased from New England Biolabs (Beverly, MA). Rabbit anti-sheep prolactin antiserum was obtained from U.S. Biochemicals (Cleveland, OH). Affinity-purified FITC goat antimouse IgG was purchased from Calbiochem (La Jolla, CA) and Bethesda Research Labs (Bethesda, MD).

VOLUME 141.1990

Ascites fluid of a mouse monoclonal antibody against VSV G was a kind gift of Dr. Leo LeFrancois, Upjohn Co, Kalamazoo, MI). [YS]methionine and [?S]sulfate were obtained from Amersham Inc., (Arlington Heights, IL). 4-methylumbelliferyl P-D-xyloside, diethylpyrocarbonate, progesterone, and pregnant mare serum gonadotropin were purchased from Sigma Chemical Co. (St. Louis, MO). Handling of Oocytes Xenw laevis females were injected with pregnant mare serum gonadotropin l-2 days before removal of ovaries. Ovary tissue was surgically removed from X laevis females anaesthetized with 0.12% benzocaine. Full grown stage 6 oocytes were manually dissected from the ovary. To induce meiotic maturation, immature stage 6 oocytes were incubated in 1 pg/ml progesterone in amphibian Ringer’s solution for l-2 hr. Plasmids A bovine prolactin cDNA clone in pSP64T was a kind gift from P. Walter (Dept. of Biochemistry and Biophysics, UCSF). A HindIII-BgZII fragment containing VSV G was originally subcloned from pSV2 G (Rose and Bergmann, 1982) into the HindIII-BamHI sites of Bluescript. In our hands T3 polymerase did not produce stable capped transcripts. Therefore we subcloned a HindIII-X&I fragment of VSV G from Bluescript into pSP64. SP6 transcripts were stable and translated in oocytes. In vitro Transcriptions SP6 transcripts capped with ‘I-methyl GpppG were generated according to Krieg and Melton (1986). Plasmid DNA was prepared from transformed JM103 cells by alkaline lysis and purified by cesium chloride banding according to Maniatis et al. (1982). pSP64T-prolactin DNA was linearized by EcoRI digestion and pSP64TVSV G DNA was linearized with a BamHI digestion. Template DNA (5 pg) was used for a transcription reaction which generates approximately the same mass of SP6 transcripts. After the transcription reactions, the template DNA was digested wtih RNase-free DNase, and extracted 3-5 times with equal volumes of phenol/ chloroform, 1 time with an equal volume of chloroform, and precipitated with + vol of 7 1Mammonium acetate, and 2 vol of ethanol. After pelleting and drying, the SP6 transcripts were reprecipitated. The pellet was resuspended in a final volume of 10 ~1 diethylpyrocarbonate-

LEAF ET AL.

Secretim Block during Xempus Meiotic Maturation

treated water. SP6 transcripts (40-50 nl) were injected per oocyte.

3

supernatant was removed and diluted with an equal volume of 2~ Laemmli sample buffer prior to polyacrylamide gel electrophoresis.

[85S]Methionine Labeling Immature and mature oocytes expressing prolactin or VSV G were labeled by either injection of 40-50 nl of [35S]methionine (approximately 0.6-0.75 &i), or by incubation for 30 min in [%S]methionine at a concentration of 1 mCi/ml. Thirty minutes after injection or incubation with [35S]methionine, the oocytes were transferred to chase medium consisting of Barth’s or Ringer’s medium with 10 mM methionine. [J5S]SuCfateLabeling of GAG Chains Oocytes incubated in 1 mM 4-methylumbelliferyl O-Dxyloside were injected with 40-50 nl of [YS]sulfate (approximately 1 &i per oocyte). Immunoprecipitations Immunoprecipitations were performed according to Moore and Kelly (1985). Oocytes were lysed in 100 ~1 (for individual oocytes) or 50 jJ/oocyte (for groups of oocytes) of NDETS (1% NP-40,0.4% deoxycholate, 66 mM EDTA, 10 mM Tris-HCl, pH 7.4, 0.1% SDS). Prolactin was immunoprecipitated one time using 1~1 rabbit antisheep prolactin antiserum per oocyte. VSV G was immunoprecipitated two times using l-2 gl of a rabbit polyclonal antiserum (Discotritium) per oocyte. The immunoprecipitates were eluted from Staphylococcus aureus cells by boiling in 30 ~1 of Laemmli sample buffer. VSV G immunoprecipitates were digested with endo H according to Rose and Bergmann (1982). Acetone Precipitations Media samples containing prolactin or xyloside-initiated GAG chains were precipitated by dilution in 4 vol of acetone overnight at -20°C. The precipitates were pelleted by a 15-min centrifugation at 14,000g and vacuum dried. For polyacrylamide gel electrophoresis, the acetone precipitates were resuspended in 15 ~1 of water, and 15 ~1 of 2~ Laemmli sample buffer. Preparation of Yolk-Free Cytoplasmic Fraction For Xyloside Studies Since yolk is the predominant cytoplasmic protein in oocytes, cell fractions containing yolk showed considerable distortion on polyacrylamide gels from overloading with yolk. We prepared a cytoplasmic fraction deenriched in yolk to examine xyloside-initiated GAG chains in oocytes. Oocytes were homogenized in 10 ~1 of phosphate-buffered saline (PBS) per oocyte, and the yolk pelleted by a 14,000g centrifugation for 5 min. The

Cetyl Pyridinium

Chloride Precipitations

Cetyl pyridinium chloride (CPC) was used to precipitate xyloside-initiated GAG chains. To precipitate cell fractions, groups of nine oocytes were lysed in a total of 500 ~1 1% Triton X-100, IX PBS. The yolk fraction was pelleted by a 14,000g centrifugation for 5 min and the supernatant collected. A fraction of the supernatant was scintillation counted and a volume corresponding to a total of 1 X lo6 cpms (20 ~1for the immature oocytes, 24 ~1 for the mature oocytes) was diluted to a final volume of 500 ~1of 1% Triton X-100,1X PBS for the CPC precipitation. The media fractions were also diluted to a final volume of 500 ~1 of 1% Triton X-100, 1X PBS. Samples were digested overnight at 37°C with Pronase E at a final concentration of 1 mg/ml. GAG chains were precipitated at 37°C with CPC at a final concentration of 2% and with 0.13% chondroitin sulfate as a carrier. Precipitates were collected on Gelman metricell filters (0.45 pm), dried, resuspended in Ecolume (ICN) and counted on a Beckman LS-5 scintillation counter. HPLC Analysis of GAG Chain Length Media from immature and mature oocytes previously labeled with [35S]sulfate and incubated in 1 mM4-methylumbelliferyl @-D-xyloside were precipitated twice with acetone and then resuspended in 50 ~1 of 10 mM Hepes, pH 7.0. Samples were separated by HPLC on a Zorbax bioseries GF 250 column calibrated with proteins ranging from 670,000 to 17,000 kDa in molecular weight. Vitamin B-12 was included as a marker for the void volume. The column was eluted with 0.1 M potassium phosphate, pH 7.5, at 0.5 ml/min. Fractions (1.0 ml) were collected and processed for liquid scintillation counting. Indirect Immuno$uorescence of VSV G Immature and mature oocytes were injected with VSV G transcripts. At various times after transcript injection, oocytes were fixed in 4% paraformaldehyde in 1X PBS for 1 hr, embedded in O.C.T. compound (Miles Scientific), frozen, cryosectioned (15 pm) and stored at -20°C. The cryosections were postfixed for 1 hr in 1% paraformaldehyde, rinsed in IX PBS, and blocked in 1% bovine serum albumin (BSA), 50 mM glycine, 1X PBS for 20 min. The cryosections were then incubated for 2-4 hr in l/100 dilution of affinity-purified fluorescein isothiocyanate (FITC)-goat anti-mouse IgG, rinsed in 1X PBS, incubated for 1 hr in l/100 FITC rabbit antigoat IgG, rinsed in 1X PBS, and mounted in 25% glyc-

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DEVELOPMENTALBIOLOGY

VOLUME141.1390

erol with 1 mg/ml DABCO. Immunofluorescent staining was photographed with Ektachrome 400 film (Kodak) on a Zeiss Axiophot Microscope. Black and white photographs of the color plates were photographed with TMax 40 film (Kodak). RESULTS

Timing of the Block to Secretion Meiotic maturation can be induced in immature stage 6 oocytes by exposure to progesterone. During maturation, immature oocytes are released from a GB-prophase I arrest and enter M-phase as a result of the activation of MPF (reviewed in Hunt, 1989). The mature oocyte becomes physiologically arrested at metaphase II through the stabilization of MPF activity by cytostatic factor (Meyerhof and Masui, 197’7;Gerhart et d, 1984; Newport and Kirschner, 1984; Murray et al., 1989), recently identified as the proto-oncogene c-wws (Sagata et aZ.,1989). During maturation, the oocyte’s nuclear envelope fragments. This event, referred to as germinal vesicle breakdown (GVBD), is associated with the appearance of a white spot in the animal hemisphere caused by displacement of pigment by the surface migration of the meiotic spindle and release of germinal vesicle sap. A white maturation spot in the animal hemisphere is a clear morphological sign that GVBD has occurred. Two hours after GVBD, oocytes are capable of being fertilized or activated; we define these as “mature” oocytes. Oocytes in earlier stages of maturation are referred to as “maturing” oocytes. “Immature” oocytes are defined here as stage 6 oocytes arrested in GB/prophase I. To determine how soon the block to secretion becomes established after exposure to progesterone, we have followed the secretion of bovine prolactin. Oocytes injected with SP6 transcripts encoding prolactin efficiently express the protein allowing prolactin secretion to be examined in individual oocytes. Immature oocytes pulselabeled for 30 min with [?S]methionine will secrete labeled prolactin for at least 6 hr. The amount of prolactin secreted per hour varied between oocytes from approximately 1.5 to 7% of the total prolactin synthesized. Prolactin synthesized after GVBD is not secreted. First, we were interested in whether GVBD is a valid indicator of when secretion is blocked. Immature oocytes previously injected with prolactin RNA were incubated with or without progesterone. One hour after GVBD was observed, maturing oocytes were injected with [%S]methionine and cultured individually for 4 to 5 hr in chase medium containing 10 mM methionine. Immature oocytes were labeled identically. Figure 1 shows the secretion of prolactin from two immature and two maturing oocytes. Immature oocytes secrete prolactin slowly. Densitometric scans of the autoradiographs show that 1.5% of the labeled prolactin was secreted per

FIG. 1. Prolactin secretion in immature and mature oocytes. Immature oocytes were injected with prolactin SP6 transcripts, incubated in Ringer’s medium overnight and then cultured with or without progesterone. One hour after GVBD was observed the maturing oocytes (C, D) were injected with 40-50 nl of [Y!Jmethionine. Immature oocytes (A, B) were similarly injected with [S6S]methionine. Thirty minutes after injection, the oocytes were cultured individually in microtiter wells in Barth’s medium with 10 mM methionine. Media were collected hourly for 4 or 5 hr. After the last media collection, the cell fractions were immunoprecipitated with rabbit anti-sheep prolactin antiserum. The media samples were acetone precipitated. Immunoprecipitates and acetone precipitates were resolved on 15% SDS polyacrylamide gels and autoradiographed for 21-23 days.

hour. However, the total amount of prolactin secreted by maturing oocytes over 5 hr was less than 0.2% of the total amount of prolactin synthesized. Therefore the block to secretion of newly synthesized proteins is established within 1 hr after GVBD. Inhibition of prolactin secretion does not begin until GVBD. To determine if the block to secretion occurs earlier than GVBD, we took advantage of the slow half time of prolactin secretion. Immature oocytes injected with prolactin RNA were labeled for 30 min with [=S]methionine and subsequently chased with 10 mMmethionine. Progesterone was included in the chase medium to initiate maturation. After 30 min, the maturing oocytes were cultured individually and the medium was collected at hourly intervals for the next 6 hr. The timing of GVBD, as indicated by a white spot in the animal hemisphere, was noted for each oocyte. The secretion of prolactin from six individual oocytes is shown in Fig. 2. The oocytes are grouped according to when they underwent GVBD, which in this experiment varied from 3-5 hr after progesterone addition. Some oocytes did not develop a white maturation spot and these continued to secrete prolactin for 6 hr. In contrast, maturing oocytes which underwent GVBD showed a substantial decrease in secretion during the hour in which GVBD was observed or within the following hour. This correlation of the block to secretion with GVBD is apparent in Fig. 3, which shows the changes in the rate of prolactin secretion during meiotic maturation. This was tabulated from 20 oocytes from two batches of oocytes (including the oocytes shown in Fig. 2) in which oocytes were labeled 30 min with [36S]methionine,

LEAF ET AL. Time c&D None

1 24 kd

None

2

3hr.

3

3 hr.

4

5hr.

5

5 hr.

6

5

Secretion Block during Xerwpus Meiotic Maturation

FIG. 2. Prolactin secretion in maturing oocytes. Immature oocytes were injected with prolactin SP6 transcripts and incubated overnight in Ringer’s medium. Oocytes were incubated in 1 mCi/ml rS]methionine in Ringer’s medium for 30 min, and subsequently chased in Ringer’s medium with 10 mM methionine and 1 pg/ml progesterone. Oocytes were cultured individually in microtiter wells. Oocytes were examined every 20-30 min for GVBD. Media were collected hourly. Cells were lysed in 50 ~1 of Laemmli sample buffer at the end of the experiment. Media fractions were acetone precipitated. The acetone precipitated media samples, and l/20 of the cell fractions were resolved on 15% SDS polyacrylamide gels and autoradiographed for 4 days.

treated with progesterone, and cultured individually in 10 mM methionine. Because the time of GVBD varied from 1 to 5 hr after the chase began, we normalized the time points to GVBD to make them independent of when progesterone was added. Figure 3A shows that by 1 hr after GVBD, secretion decreased by approximately 63% compared to secretion during the hour in which GVBD occurred. Figure 3B shows when the block to secretion becomes established during oocyte maturation. The onset of the block to secretion is defined by the timepoint when an oocyte showed a 50% decrease or more in secretion compared to the previous timepoint. In 8/20 oocytes the onset of the block to secretion occurred during the hour in which GVBD was observed and in 11/20 oocytes it occurred in the hour after GVBD. Thus, the block to protein secretion occurs coincident with GVBD regardless of how long after progesterone addition the oocyte undergoes GVBD.

Taken together, these experiments define the time window when the block to secretion becomes instituted. The secretion of prolactin synthesized before maturation is not inhibited until GVBD. Prolactin synthesized after GVBD fails to be secreted. The block to secretion is not a direct response to progesterone since oocytes treated with progesterone,showed the same pattern of prolactin secretion as control immature oocytes for several hours before GVBD. It is apparent from Figs. 1 and 2 that there is more prolactin secreted after GVBD when prolactin is synthesized before compared to after GVBD. In order to examine the difference in prolactin secretion when it is synthesized before or at GVBD, we examined eight oocytes which underwent GVBD during the third hour after pulse labeling with [35S]methionine and compared them to 13 oocytes that underwent GVBD during the 30-min labeling period. We calculated the rate of prolactin secretion as a percentage of the total prolactin synthesized by the oocyte. In the 8 oocytes that underwent GVBD 3 hr after labeling, prolactin was secreted at a rate of 4.5 f 2.4% per hour during the 2 hr preceding

m .i 0.6 + ii z

0.6 0.4

2 - 0.2 4 ‘Z 1

d

-2

z g;, SC g.;

-1 GVBD 1 Time (Hours)

2

3

s 12 10 6

.; f-

6

g’

4

3: $

2 /---IL -4

-3

-2

-1 GVBD

1

2

”

Time (Hours)

FIG. 3. Correlation of GVBD and block to secretion. (A) The relative hourly rate of prolactin secretion during meiotic maturation. The time of GVBD varied from 1 to 5 hr after the start of the chase. In order to correlate secretion with GVBD, the hour in which GVBD occurred is set at time = GVBD. Secretion during the hgur before GVBD is set at time = -1. Secretion during the hour after GVBD is Act at time = +l. Secretion was examined from two batches of oocytes for a total of 20 oocytes. Oocytes were treated as described in Fig. 2. To quantify prolactin secretion, autoradiographs were scanned densitometrically, and the peaks of secreted prolactin were cut out and weighed. The rate of prolactin secretion at each hour was determined relative to the maximum hourly rate for each oocyte. (A) The average of these rates for the 20 oocytes before, during, and after GVBD. (B) A histogram plotting when oocytes first displayed the onset to the block to secretion during meiotic maturation. The onset to the block to secretion is defined as the time point at which an oocyte secreted less than 50% of the prolactin secreted in the previous time point.

6

FIG. 4. VSV G expression in immature and mature oocytes. Progesterone-treated oocytes were injected with VSV G SP6 transcripts 2 hr after GVBD. In the same interval, immature oocytes were injected with VSV G SP6 transcripts. At various times after injection oocytes were fixed, embedded in O.C.T., and frozen. VSV G was detected by indirect immunofluorescence employing a monoclonal antibody against VSV G as the

LEAF ET AI..

Secretion Block during Xenopus Meiotic Maturation

GVBD. In the 2 hr after GVBD, this declined to 1.1 +- 0.9% per hour. However, this rate of prolactin secretion after GVBD is greater than that observed in 13 oocytes labeled at GVBD. Only one of these 13 oocytes showed any measurable prolactin secretion (in the first 2 hr of the chase, a single oocyte secreted 0.4% per hour). This suggests that prolactin secreted after GVBD in oocytes labeled before progesterone addition may come from a pool of prolactin that had already passed the blocking step(s) in the secretory pathway when the block became established. The block to secretion during meiotic maturation is not irreversible. We find that prolactin synthesized in immature oocytes and blocked in its secretion during maturation is secreted after mature oocytes are fertilized or activated (Roberts et al, in preparation). Location

of the Block to Secretion

7

detected on the cell surface by indirect immunofluorescence within 3 hr after RNA injection (data not shown). We examined whether or not meiotic maturation blocks the transport of VSV G to the plasma membrane. Immature and mature oocytes (2 hr after GVBD) were injected with VSV G RNA. Oocytes were processed for immunostaining 6 or 16 hr afterward. VSV G was detectable at the cell surface of immature oocytes within 6 hr after RNA injection (Fig. 4A). Mature oocytes did not have detectable staining of VSV G at this time (data not shown). Mature oocytes that were fixed 16 hr after injection of RNA showed an intracellular accumulation of VSV G, but none at the cell surface. The location of the intracellular staining varied. In some mature oocytes, pun&ate staining was evident in the cortex 0.1-0.2 pm from the plasma membrane (Fig. 4B). In other oocytes, the intracellular staining was deeper (3.8 pm from the cell surface) and appeared to circumscribe yolk platelets (Fig. 4C). The deep intracellular staining of VSV G was observed to extend several hundred micrometers into mature oocytes (data not shown). We do not know the identity of the intracellular structures stained with the VSV G antibody, but the staining is specific to oocytes injected with VSV G RNA. The lack of staining of VSV G at the cell surface of mature oocytes demonstrates that the transport of VSV G to the plasma membrane is blocked after GVBD, consistent with the time at which prolactin secretion is blocked.

In the experiments described above, we show that the block to prolactin secretion during meiotic maturation is correlated with GVBD. Since prolactin is not glycosylated, it is not possible to biochemically trace the protein through the secretory pathway by assaying carbohydrate modifications specific to various compartments of the ER and Golgi. To monitor ER to Golgi traffic in Xenopus oocytes, we assayed the endo H resistance of the well characterized vesicular stomatitus glycoprotein (VSV G). In mammalian cells, the cell surface exER to Golgi trafic is not blocked in mature oocytes. pression and ER to Golgi transport of VSV G is blocked Since the transport of VSV G to the cell surface is during mitosis (Warren et al., 1983; Featherstone et al., blocked in mature oocytes, we tested whether all steps in the secretory pathway are equally affected by matura1985). In addition, we used 4-methylumbelliferyl @-D-xylo- tion. To monitor ER to Golgi traffic, we assayed the side, an acceptor for glycosaminoglycan (GAG) chain acquisition of endo H resistance of VSV G in immature elongation, to assay membrane traffic between the and maturing oocytes. In mammalian cells, glycoprotrans-Golgi and the cell surface. The xyloside-initiated teins become endo H resistant due to carbohydrate modiGAG chains (referred to as GAG chains) are sulfated fications which occur in the medial compartment of the and constitutively secreted (Burgess and Kelly, 1984; Golgi (Kornfeld and Kornfeld, 1985). Brion and Moore, submitted for publication). The proImmature oocytes were injected with VSV G RNA cess of sulfation occurs in the trans-Golgi of mamma- and incubated with or without progesterone. Two hours lian cells (Iozzo, 1987). Hence, the secretion of GAG after GVBD occurred in the progesterone-treated oochains labeled with [YS]sulfate provides a convenient cytes, both groups of oocytes were labeled by incubation measure of traffic between the trans-Golgi and the cell in [35S]methionine for 30 min and chased with 10 mM surface. As opposed to the analysis of protein secretion methionine for 225 min. At various times during the in Xenopus oocytes, the secretion of GAG chains is inde- chase, VSV G was immunoprecipitated from homogependent of protein synthesis and reflects the secretion nates of oocytes, digested with endo H, and displayed on of endogenous material. SDS gels (Fig. 5). Densitometric scans of the autoradioCell surface expression of VSV G is blocked in mature graphs were used to quantify the fraction of VSV G oocytes. When injected with VSV G RNA, immature which was endo H resistant. At 15 min after the start of Xenopus oocytes rapidly transport the newly synthethe chase, 35% of VSV G was endo H resistant in immasized VSV G protein to the plasma membrane. VSV G is ture and mature oocytes. By 45 min of the chase, 85% of primary antibody. (A) An immature oocyte fixed 6 hr after injection. (B and C) Mature oocytes fixed 16 hr after injection. The oocytes in A and B were isolated from the same ovary. The arrows in A, B, and C show the cell surface of the oocytes. The arrowheads in B and C show the intracellular staining of VSV G in mature oocytes. (Magnification, 5126X).

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VOLUME 141,199o

Mature oocytes

Immature oocytes

sults clearly show that transport of VSV G from the ER to the Golgi is not blocked in mature oocytes. We conclude that the block to secretion during meiendoH + ++ + ++ otic maturation does not universally affect membrane traffic. Rather, the analysis of VSV G suggests that the 66kd block to secretion is imposed after the medial Golgi where glycoproteins are modified to their endo H-resistant form. This contrasts with the block to secretion in mitotic mammalian cells in which traffic of VSV G from 1 2 3 4 5 6 7 6 9 10 11 12 ER to Golgi is blocked (Featherstone et al, 1985). The continuation of ER to Golgi traffic after GVBD taken FIG. 5. ER to Golgi transport of VSV G. Immature oocytes were injected with VSV G SP6 transcripts, incubated overnight and cul- together with the observation that Golgi dissolution octured with or without progesterone. Two hours after GVBD, 15 ma- curs at GVBD (Colman et CAL, 1985), implies that memture oocytes were incubated in 1 mCi/ml $S]methionine for 30 min; 15 brane traffic is not functionally coupled to the morphoimmature oocytes were similarly labeled. After a 30-min pulse, oological state of Golgi in mature oocytes. The acquisition cytes were chased in Barth’s medium containing 10 mM methionine for 225 min. Groups of five oocytes were lysed at 15,45, and 225 min of of endo H resistance in mature oocytes also suggests the chase. The samples were immunoprecipitated with a rabbit antithat morphologically intact Golgi are not required for VSV G antiserum and one-half of the immunoprecipitate was treated carbohydrate modifications of glycoproteins in the sewith endo H. The immunoprecipitates were resolved on a 10% SDScretory pathway. polyacrylamide gel and autoradiographed (lanes 1-6,11-12,2-day exTrans-Golgi to cell surface transport of GAG chains is posure; lanes 7-10, 4-day exposure). Lanes 1-6, immature oocytes; inhibited in mature oocytes. Having established that lanes 7-12, mature oocytes; lanes 1,2,7,8,15 min chase; lanes 3,4,9, lo,45 min chase; lanes 5,6,11,12,225 min chase; lanes 1,3,5,7,9,11, proteins in the secretory pathway are transported from chase

15 min.

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225 min.

15 min.

45 min.

225 min.

mock endo H digestion; lanes 2,4, 6, 8,10,12, endo H digestion. Immature Oocyte Medium

VSV G was resistant to endo H digestion in immature and mature oocytes, and by 225 min of the chase 90% of VSV G was endo H resistant in both groups. These re-

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600 $ 0 400

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FIG. 6. GAG chain secretion in immature and mature oocytes. Im-

mature oocytes were incubated in 1 mM4-methyl umbelliferyl b-D-Xyloside with or without progesterone. Two hours after GVBD, 15 mature oocytes were injected with 40-50 nl [86S]su1fate.In addition, 15 immature oocytes were injected with [“Slsulfate. Media were collected 4.5 hr and 18 hr later. The media fractions were acetone precipitated. One-fifth of a yolk-free cell fraction and the acetone precipitates of the media were resolved on a 18% SDS-polyacrylamide gel and autoradiographed (lanes 2,3,5,6, l-day exposure; lanes 1,4,5-day exposure). Lanes l-3, immature oocytes; lanes 4-6, mature oocytes; lanes 1,4, one-fifth of the yolk-free cell fraction; lanes 2,5,0 to 4.5-hr media; lanes 3, 6,4.5 to 18-hr media.

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50

FIG. 7. HPLC analysis of GAG chain length. Media from incubations of stage 6 immature and mature oocytes were acetone precipitated and resuspended in 50 pl of 10 mMHepes, pH 7.0. Samples were separated by HPLC on a gel-filtration column calibrated during each run with the following standards: thryoglobulin, 67O,ooO,gamma globulin, 158,ooO;ovalbumin, 44,000, and myoglohin, 17,000.Arrows above the elution profile indicate the position of these proteins as they were eluted from the column. For the immature medium, 1 X lo6 cpms were loaded onto the column and for the mature medium, 3 X 10’ cpms were loaded.

LEAF ET AL. A

Secretion Block during Xenopus Meiotic Maturation

9

chains appeared as a smear on the autoradiographs of SDS gels (Fig. 6). Secretion of GAG chains is signifi60000 cantly reduced in mature oocytes. Mature oocytes pro'B m duced GAG chains apparently larger than GAG chains 5 s 60000 from immature oocytes. -2 The apparent increase in size of GAG chains in ma6 40000 3 ture oocytes was confirmed by gel filtration HPLC (Fig. (7 I 20000 7). Immature and mature oocytes were cultured in 1 mM c P-D-xyloside and injected with [?S]sulfate. Samples of acetone-precipitated media from immature and mature Immature Mature oocytes were separated on a column calibrated with protein molecular weight standards. The elution profiles were determined by scintillation counting of the fracB 60000 3 tions (Fig. 7). The elution profiles indicated that the k u 50000 secreted sulfated GAG chains in mature oocytes range 2 3 40000 l Immature from 670-17 kDa while in immature oocytes the major ‘0 2 30000 0 Mature peaks are between 44 and 17 kDa. There was also a large 0 z 20000 peak of [“S] of unknown composition eluting with the b 10000 void volume (cl.3 kD) in the medium from mature oo8 cytes. The difference in molecular weight between me0 0123456 dia from mature and immature oocytes is larger than Time (hrs) that predicted from the SDS gels and the discrepancy FIG. 8. Rate of GAG chain transport. Immature oocytes were incu- might be due to nonlinear resolution of sulfated GAG bated in 1 mM 4 methyl umbelliferyl P-D-xyloside with or without chains on SDS gels. progesterone. Mature oocytes were injected with 40-50 nl [Tl]sulfate 2 In order to quantatively assay secretion of GAG hr after GVBD. At the same time, immature oocytes were injected chains, we utilized cetyl pyridinium choride (CPC) to with [%]sulfate. Immature and mature oocytes were cultured in groups of nine. Media were collected hourly for 5 hr. Cell and media precipitate GAG chains. Groups of immature oocytes were incubated with xyloside with or without progestersamples were digested overnight with pronase E and precipitated with cetyl pyridinium chloride (CPC). Precipitates were collected on one. Mature oocytes (1 hr after GVBD) and immature filters and scintillation counted. (A) CPC precipitable ?S-GAG chains oocytes were injected with [35S]sulfate. The immature in the cell and media fractions from mature and immature oocytes. and mature oocytes were cultured in groups of nine. Me(B) Cumulative CPC precipitable ?3-GAG chains secreted plotted dia were collected at hourly intervals and at the end of versus time. Immature oocytes (0); mature oocytes (0). the experiment the cells were homogenized. Media and cell fractions were precipitated with CPC, and the the ER to the Golgi in maturing oocytes blocked in pro- amount of [?S] in the precipitates was determined by tein secretion, we asked whether traffic from the trans- scintillation counting. Figure 8A shows that 5 hr after the injection of [?S]sulfate, immature and mature ooGolgi to cell surface is affected during meiotic maturation. This was examined by the analysis of secretion of cytes contained approximately the same number of CPC precipitable cpms in the cell. However, there was a subGAG chains initiated on 4-methylumbelliferyl P-D-xystantial difference in the number of CPC precipitatible loside. Xyloside-initiated GAG chains secreted from mam- cpms secreted. Consistent with the SDS gel analysis, malian cells resolve as a ladder on SDS gels (Burgess CPC precipitations showed that the secretion of GAG and Kelly, 1984; Brion and Moore, submitted for publi- chains is greatly reduced, but not completely inhibited cation) presumably reflecting heterogeneity in GAG in mature oocytes. The difference in the rates at which immature and chain length. Xenopus oocytes secrete GAG chains with similar characteristics (Fig. 6). Immature and mature mature oocytes secreted GAG chains is evident in Fig. 8B. The relative rates of secretion of mature and immaoocytes (2 hr after GVBD) were incubated in 1 mMP-Dxyloside for 3 hr, and then injected with [35S]sulfate. Oo- ture oocytes can be estimated by comparing the slopes of cytes were injected with [%]sulfate to insure that the the cumulative amount of GAG chains secreted over labeled GAG chains were secreted exclusively from the hour intervals. The slope describing secretion from the oocytes and not from the single layer of follicle cells still immature oocytes is estimated to be 15-fold greater surrounding the oocytes. Afterward, media from groups than that for mature oocytes. Therefore, a major compoof oocytes were collected at 4.5 and 18 hr. GAG chains nent of the block to secretion is an approximately 15from media samples were acetone precipitated and re- fold inhibition of transport between the trans-Golgi and solved on SDS gels along with a fraction of the oocytes the cell surface. If the processes of sulfation and chain from the 18 hr-incubation. The [%]sulfate-labeled GAG elongation occur concomitantly, the increase in size of 3

k u T) 8

100000

DEVELOPMENTALBIOLOGYVOLUME141,1990

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GAG chains from mature oocytes suggests that GAG chains have a longer residence time in the trans-Golgi of mature oocytes. Possibly, one cause of the block to secretion is an inhibition of budding from the trans-Golgi. DISCUSSION

Timing of the Block to Secretion The process of meiotic maturation generally takes 6-10 hr and can be divided into several temporal phases culminating in germinal vesicle breakdown (Maller, 1985). Early events initiated by progesterone binding at the cell surface lead to the activation of MPF. Late events including germinal vesicle breakdown, chromosome condensation, and spindle formation depend upon the activity of MPF. The breakdown of the Golgi is another late event in maturation, since Golgi dissolution is concomitant with GVBD. After GVBD, the oocyte extrudes the first polar body and enters second meiotic metaphase, cortical granules are displaced to the outer edge of the cell cortex and changes in the cortical ER make the oocyte capable of fertilization-related responses such as the calcium-dependent depolarization and activation (Campanella et al. 1984; Charbonneau and Grey, 1984). It is within this temporal framework of meiotic maturation that we assign the block to protein secretion. Colman et al. (1985) reported that protein secretion was inhibited at an unspecified time after GVBD, as observed in oocytes cultured overnight in progesterone. We have defined the onset of the block to secretion more closely by examining prolactin secretion during the period leading up to GVBD. The analyses of prolactin secretion during maturation suggest that GVBD is a landmark for when the block to secretion becomes established. If prolactin is synthesized during the first hour after GVBD, it fails to be secreted but if prolactin is synthesized before progesterone treatment, its secretion does not decrease until approximately 1 hr after GVBD. These data suggest that the block to secretion is not an immediate response to progesterone binding to the cell surface. Rather, the cessation of secretion occurs at or shortly after GVBD and thus may be the result of MPF action. Membrane Trafic in Mature Oocytes What steps in vesicular transport are inhibited in mature oocytes? We do not think that the block to secretion is due to inhibition of a general process required for vesicular transport. Traffic from the ER to the cisGolgi, and traffic between the cis and medial compartments of the Golgi, still operate in mature oocytes. Our analysis of GAG chain secretion suggests that the major effect of the block to secretion is a l&fold inhibition of transport from the trans-Golgi to plasma membrane.

Therefore, inhibition of one or more of the processes of vesicle budding from the trans-Golgi, vesicle targeting to the plasma membrane, or fusion with the plasma membrane may cause the block to secretion in mature oocytes. Colman et al. (1985) suggested that the block to secretion during meiotic maturation does not result from a failure of vesicle fusion with the plasma membrane. They predicted that if secretion were blocked at the plasma membrane, more immunofluorescent staining of ovalbumin would be found in the cortex of the mature oocyte due to the accumulation of secretory vesicles at the cell surface. Both immature and mature oocytes showed staining in the interior of the cell; the major difference between them was a more reticular staining in the immature oocyte. The comparison of prolactin secretion in oocytes labeled before or after GVBD also suggests that fusion at the plasma membrane is not the primary cause for the block to secretion. The block to secretion is apparently more effective on newly synthesized proteins than on proteins already in the secretory pathway. Oocytes pulse-labeled before progesterone administration continued to secrete small quantities of prolactin in the first hour following GVBD while oocytes labeled during or 1 hr after GVBD did not secrete a significant amount of prolactin. We suggest that prolactin secreted after GVBD had already passed the point where secretion is blocked by the time the block became established. Since vesicle fusion with the plasma membrane is the final step in the secretory pathway, it would appear that this step is not significantly affected during maturation. If fusion of secretory vesicles at the plasma membrane is not the primary cause of the block to secretion, where else might the block occur? Inhibition of budding from the trans-Golgi could explain the larger size of GAG chains produced by mature oocytes, and be partially if not fully responsible for the 15-fold reduction in GAG chain secretion in mature oocytes. The apparent increased size of the GAG chains indicates that mature oocytes have GAG chains as much as 10 times longer than GAG chains produced in immature oocytes. The precise site of GAG chain elongation within the Golgi apparatus is not known. If that process is concomitant with sulfation as suggested by Dietrich et al. (1988), the larger size of the GAG chains may result from longer residence time in the trans-Golgi, the site of sulfation. A longer residence time implies that the process of vesicle budding from the trans-Golgi is inhibited in mature oocytes. Does Vesicular Trafic Control Golgi Integrity? In a model to explain how membrane systems break down during mitosis, Warren (1985) proposed that the

LEAF ET AL.

Secretion Block during Xenopus M&tic

cessation of membrane traffic and the breakdown of the nuclear envelope, ER and Golgi are functionally related. The block to secretion was perceived as a consequence of a mechanism which breaks down the Golgi apparatus and allows for its equitable distribution between daughter cells. An activity required for all vesicle fusion events was postulated to be inhibited during mitosis, while vesicle budding remained unaffected. The lack of vesicle fusion would result in the inhibition of membrane traffic during mitosis. The breakdown of the Golgi and the nuclear envelope during mitosis was proposed to be due to continued vesicle budding and failure of vesicle fusion. Colman et al (1985) reported that like mitotic cells, Golgi breakdown in mature oocytes occurred coincident with breakdown of the nuclear envelope. Intact Golgi were not found in mature oocytes that were fixed after GVBD. We find that VSV G is still transported from the ER to Golgi in mature oocytes. VSV G in immature and mature oocytes becomes approximately 85% endo H-resistant by 45 min of the chase. Ceriotti and Colman (1989) have recently reported similar results regarding the transport of the viral hemagglutinin protein HA. They found that 66% of HA in immature oocytes and 49% of HA in mature oocytes became resistant to endo H digestion within 6 hr of the chase. Although VSV G and HA greatly differ in their rates of transport from the ER to Golgi, both membrane proteins continue to be transported to the Golgi in mature oocytes. The different rates of acquisition of endo H resistance argues against the possibility that both HA and VSV G are retained within the ER and modified by newly synthesized Golgi enzymes. For the same reason it is implausible that the disappearance of the Golgi apparatus during maturation is due to movement of Golgi elements into the ER as in cells treated with Brefeldin A (Lippincott-Schwartz et czb,1989; Doms et aZ., 1989). These results do not support the proposition that an activity required for all vesicle fusion events is lacking at M phase (Warren, 1985), since vesicles budded from the ER must still be capable of fusing with the cis-Golgi in mature oocytes. We suggest that vesicular traffic is not functionally coupled to Golgi dissolution. Membrane traffic in meiotic oocytes contrasts with traffic in mitotic mammalian somatic cells. In CHO cells arrested in mitosis by nocadozole, the transport of VSV G from the ER to the Golgi is blocked (Featherstone et aL, 1985) while GAG chains continue to be transported from the trans-Golgi to the surface (Kreiner and Moore, 1990). This may reflect an inherent difference in membrane traffic between mammalian cell mitosis and Xenopus oocyte meiosis. It does not seem likely that the inhibition of ER to Golgi transport during mitosis is a result of the prolonged nocodazole treatment used to

Maturation

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arrest cells in metaphase. Mature oocytes incubated in nocodazole are still competent in transport of VSV G from ER to Golgi (data not shown). Also, yeast cells arrested in mitosis by nocodazole are capable of ER to Golgi transport (Makarow, 1988) as well as protein secretion. Thus Xenw oocytes blocked in meiotic metaphase differ from mammalian cells blocked in mitosis in these important aspects of membrane traffic. The reason for these differences is not understood and is currently being examined. Meiotic maturation prepares the oocyte for fertilization and subsequent development. During embryogenesis large amounts of stored membranes are inserted into the cleavage furrow and membrane traffic becomes polarized (Roberts et aL, in preparation). Perhaps in mature oocytes, ER to Golgi traffic continues while transport to the surface is blocked to allow storage of newly synthesized membranes in preparation for insertion into cleavage furrows of the future embryo. We thank David Schneider, Mike Ellis, and Matt Singer who participated in the initial phase of this work; Mike Wu, for defolliculating many oocytes; Dr. Mary Sue Lowery for her assistance with the HPLC analysis of GAG chains; and the members of the Gerhart and Moore labs for stimulating discussions. This work was supported by PHS Grants GM 18363to J.C.G., and GM 35239to H.P.M.; NSF Presidential Young Investigator Award DCB 8451636,Juvenile Diabetes Foundation Research Grant 187381,and an Alfred P. Sloan Research Fellowship (BR2497) to H.P.M.; and NIH 6-doctoral fellowships GM 12019to D.S.L. and GM 06965 to S.J.R. REFERENCES BERLIN, R., OLIVER, J. M., and WALTER, R. W. (1978). Surface functions during mitosis. I. Phagocytosis, pinocytosis and mobility of surface-bound Con A. Cell 15,327~341. BERLIN, R. D., and OLIVER,J. M. (1980). Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique. J. CeUBid 85, 660-671. BURGESS,T. L., and KELLY, R. B. (1984). Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan. J. CeU Bid 99,2223-2230. BURKE, B., GRIFFITHS,G., REGGIO,H., LOWARD, D., and WARREN,G. (1982). A monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1,1621-1628. CAMPANELLA, C., ANDREUCCETTI, P., TADDEI, C., and TALEVI, R. (1984). The modifications of cortical endoplasmic reticulum during in vitro maturation of Xenopu.s Levis oocytes and its involvement in cortical granule exocytosis. J. Ezp. Zool. 22,283-293. CERIOTTI,A., and COLMAN,A. (1989). Protein transport from endoplasmic reticulum to Golgi complex can occur during meiotic metaphase in Xeop oocytes. J. Cell BioL 109,1439-1444. CHAREONNEU,M., and GREY, R. D. (1984). The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laaris Deu. Biol. 102,90-97. COLMAN,A., JONES,E., and HEASMAN,J. (1985). Meiotic maturation in Xerwpus oocytes: A link between the cessation of protein secretion and the polarized disappearance of Golgi apparati. J. CeUBiol 101, 313-318.

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DIETRICH, C. P., NADER, H. B., BUONASSIS,V., and COLBURN,P. (1988). Inhibition of synthesis of heparan sulfate by selenate: Possible dependence on sulfation for chain polymerization. FASEB J. 2,56-59. DOMS,R. W., RUSS,G., and YEWDELL,J. W. (1989). Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J. CeUBioL 109.61-72. FEATHERSTONE, C., GRIFFITH% G., and WARREN,G. (1985). Newly synthesized G protein of vesicular stomatitus virus is not transported to the Golgi complex in mitotic cells. J. Cell BioL 101,2036-2046. GERHART, J., WV, M., and KIRSCHNER,M. (1984). Cell cycle dynamics luevis oocytes of an M-phase specific cytoplasmic factor in Xenw and eggs. J. Cell BioL 98,1247-1255. HESKETH, T. R., BEAVEN, M. A., ROGERS, J., BURKE, B., and WARREN, G. B. (1984). Stimulated release of histamine by a rat mast cell line is inhibited during mitosis. J. Cell BioL g&2250-2254. HUNT, T. (1989). Maturation promoting factor, cyclin and the control of M-phase. Cum Opir~ Cell BioL 1,268-274. IOZZO,R. V. (1987) Turnover of heparan sulfate proteoglycan in human colon carcinoma cells. A quantitative biochemical and autoradiographic study. J. BioL Chem 262,1888-1900. KORNFELD, R., and KORNFELD, S. (1985). Assembly of asparaginelinked oligosaccharides. Annu. Rev. B&hem 54,631-664. KREINER, T., and MOORE,H.-P. H. (1990). Membrane traffic between secretory compartments is differentially affected during mitosis. Cell Reg., 1,415-424.

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