Export of proteins from oocytes of Xenopus laevis

Cell, Vol. 17, 517426.

July 1979,

Copyright

Export of Proteins

0 1979

by MIT

from Oocytes

Alan Colman and John Morser Department of Biological Sciences University of Warwick Coventry CV4 7AL, England

Summary When human lymphoblastoid mRNA was microinjetted into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process. Introduction The oocytes of X. laevis provide a sensitive assay for human and mouse interferon messenger RNA (Reynolds, Premkumar and Pitha, 1975; Cavalier et al., 1977; Burke and Morser, 1978). Using this assay, Sehgal, Soreq and Tamm (1978) estimated that the functional half-life of polyadenylated human fibroblast interferon mRNA in oocytes was 6-8 hr. This result is surprising in view of the known stability of several other polyadenylated mRNAs in oocytes (Gurdon, Lingrel and Marbaix, 1973; Berridge and Lane, 1976; Huez et al., 1978). We wanted to see whether this instability was also shown by human lymphoblastoid interferon mRNA after injection into Xenopus oocytes. Preliminary experiments revealed that the amount of interferon extractable from injected oocytes reached a maximum value within 6 hr. Interferon was also found to accumulate in the incubation medium surrounding the oocyte. The scale of this external accumulation suggested that interferon, a secretory protein of the lymphoblast, might be specifically exported by the oocyte. It has already been shown that newly synthesized secretory proteins whose synthesis in oocytes is directed by injected mRNA are transferred across intracellular membranes into the lumen of the

of Xenopus

laevis

endoplasmic reticulum (Zehavi-Willner and Lane, 1977). This subcellular compartmentation is consistent with the known mechanisms by which secretory proteins are segregated within cells (Palade, 1975). Recent studies of the events involved in secretion have used cell-free translation systems supplemented with microsomal membranes (Blobel and Dobberstein, 1975a, 1975b; Rothman and Lodish, 1977; Lingappa, Devillers-Thiery and Blobel, 1977; Lingappa et al., 1978). Although important information has been derived from these studies, cell-free systems by their very nature cannot be used to study the ultimate cellular event, secretion. If the observed extracellular accumulation of interferon proves to apply generally to secretory proteins, then the oocyte will provide a complete system for the study of all the events associated with protein synthesis and secretion. This paper describes experiments designed to test the specificity of protein secretion by oocytes. These experiments, which involved the microinjection into oocytes of messenger RNAs encoding both secretory and nonsecretory proteins, unequivocally demonstrated the specificity of protein secretion by oocytes. Results Synthesis of Interferon in Oocytes Total polyadenylated mRNA from virus-induced lymphoblastoid cells (referred to as interferon mRNA) was purified by oligo(dT) chromatography and injected into batches of X. laevis oocytes. At various intervals, oocytes were homogenized and the homogenates were tested for interferon activity. The results shown in Figure 1 indicate that the interferon titer per oocyte reaches a maximum value within 2 hr of injection of the interferon mRNA. Subsequent experiments confirmed this rapid realization of a maximum titer, although the exact time of occurrence varied between 2 and 6 hr. These kinetics of accumulation could have occurred if either the interferon mRNA or interferon itself was degraded within oocytes. Alternatively, interferon could have been accumulating in the incubation medium surrounding the injected oocytes. We therefore analyzed this incubation medium and found that interferon accumulated in it for at least 45 hr (Figure 1). More importantly, the interferon titers in the incubation medium rapidly became higher than the titers within the oocyte. Consequently, when the aggregate interferon titer was plotted as a function of incubation time, it was evident that interferon was still being synthesized after 45 hr of culture (Figure 1). These observations indicate that, contrary to the results of others (Sehgal et al., 1978) interferon mRNA is relatively stable within the oocyte. Our results, however, do not exclude a small but constant degradation of newly synthesized interferon within the oocytes.

Cell 518

20 Figure

1. Time Course

Tme (h) of Interferon Synthesis

Oocytes were injected with 15 ng of interferon for various times in Earth’s solution. Oocytes were then assayed for interferon content. (u) incubation medium; (D--17) oocyte Approximately 20 oocytes were used for each

40 m Oocytes mANA and incubated and incubation media (M) Oocyte; + incubation medium. point.

Stability of Interferon within the Oocyte Partially purified lymphoblastoid interferon injected into oocytes was rapidly inactivated. This inactivation occurred inside the oocyte, since the disappearance of interferon activity could not be accounted for by leakage or export into the incubation medium (Figure 2). First-order decay kinetics were not exhibited, so no half-life could be calculated. In six separate experiments, however, more than 99.9% of the interferon had been inactivated within 6 hr of injection. It is possible that interferon newly synthesized within the oocyte might be protected from degradation by subcellular compartmentation (see below), a phenomenon recently observed by C. Lane, S. Shannon and Ft. Craig (personal communication) with newly synthesized guinea pig milk proteins. We have therefore attempted to study the stability of newly synthesized interferon in the oocyte by adding cycloheximide to the incubation medium. Under these conditions protein synthesis is rapidly inhibited, yet the oocytes appear morphologically unaffected for up to 24 hr after administration of the drug (Koch, 1976; A. Colman, unpublished data). Oocytes were injected with interferon mRNA and incubated for 6 hr. The incubation medium was then removed and replaced with fresh medium containing cycloheximide at 100 pg/ml. Incubation was continued for a further 10 hr and the interferon titers in the oocytes and the medium were estimated. The results shown in Table 1 demonstrate that no interferon was detected within the oocytes after incubation with cycloheximide, but a quantity of interferon similar to that present in the oocyte after the 6 hr of initial incubation was found in the external medium. These results indicate that no significant degradation of interferon occurs during the cycloheximide treatment, and we conclude that newly synthesized interferon is protected from degradation while inside the oocyte.

10

20

Time (h) Figure

2. Time Course

of Injected

Interferon

Survival

in Oocytes

Oocytes were injected with 30 nl of partially purified interferon (7.2 x lo6 U/ml) and incubated for various times in Barth’s solution. Oocytes and incubation media were then assayed for interferon content. (o---O) oocytes; (u) mcubation media. Approximately 20 oocytes were used for each point.

Association of Newly Synthesized Interferon with Subcellular Membranes Zehavi-Willner and Lane (1977) recently demonstrated that vesicle fractions, presumably derived from the endoplasmic reticulum, could be obtained by centrifugation of oocyte homogenates on sucrose gradients. They also discovered that when the mRNAs specifying secretory proteins were injected into oocytes, the newly synthesized secretory proteins were found to be almost entirely associated with the vesicle fractions obtained after processing the oocytes. This association probably reflects compartmentalization of such proteins by membranes within the oocyte. Since interferon is a secretory protein known to be associated with cellular membranes (Falcoff et al., 1976; Abreu and Bancroft, 1978), we have investigated the location of newly synthesized interferon within the injected oocytes. The results shown in Table 2a indicate that newly synthesized interferon was found predominantly in the vesicle fraction, but that microinjetted interferon protein was not membrane-bound. Furthermore, only the newly synthesized interferon present in the vesicle fraction was significantly resistant to prolonged incubation with high concentrations of trypsin and chymotrypsin (Table 2b). This resistance was lost after disruption of the vesicles with detergent. [We attribute the residual activity remaining after enzyme digestion to the protective effect of the

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Table

of Proteins

1. Effect

from X. laevis Oocytes

of Cycloheximide

on Interferon Interferon

Incubation

Period

(Hr)

O-6 6-16 6-l 6 + cycloheximide

Oocyte 6 6 0

Synthesis

in Oocytes

Table

2. Fractionation

of Oocytes

Units per Oocyte Incubation Medium 2 12 6

Oocytes were each injected with 15 ng of interferon mRNA and incubated for 6 hr in Barth’s solution. 20 oocytes were then assayed for interferon while others (40) were incubated for a further 16 hr either in the presence or absence of 100 pg/ml cycloheximide. Oocytes and Incubation media were then processed, dialyzed and assayed for interferon.

large amount of oocyte protein present, since interferon alone is completely inactivated under these conditions (J. Morser, unpublished data).] These results confirm that the newly synthesized interferon is sequestered within the vesicles and not adventitiously adhering to them (Blobel and Dobberstein, 1975a). Thus we conclude that newly synthesized interferon, similar to other secretory proteins (Zehavi-Willner and Lane, 1977), occupies a specific subcellular location within oocytes. Export of Foreign Proteins from Oocytes Microinjection of an oocyte inevitably causes some damage to the cell. Hence it is conceivable that the observed accumulation of interferon outside the oocyte results from leakage of oocyte contents, although the scale of the accumulation makes this improbable; moreover, no significant leakage of interferon was observed after microinjection of the partially purified protein into oocytes (Figure 2). Alternatively, the export of secretory proteins specified by microinjected mRNAs might be a general phenomenon exhibited by oocytes. We distinguished between these possibilities by injecting mRNAs representing both secretory and nonsecretory proteins and then analyzing the incubation medium for each class of protein. A number of mRNAs including interferon mRNA, lactating guinea pig mammary gland mRNAs (encoding the secretory milk proteins) and rabbit hemoglobin mRNA were injected separately or together into oocytes. The oocytes were then labeled in incubation media containing ?S-methionine for 6 hr before electrophoretic analysis of oocyte homogenates and incubation media (Figure 3). Tracks l-4 show the electrophoretic profiles of oocytes injected with either hemoglobin mRNA (1). mammary gland mRNA (2), interferon mRNA (3) or injection medium alone (4). The electrophoretic profiles of the media surrounding these oocytes are shown in tracks 5-8, respectively. As shown in these profiles, only when mammary mRNAs were injected was there significant accumulation of protein in the

Containing Sample Interferon Protein

(a) Fraction incubation

Interferon mRNA

7

medium

Total oocyte

homogenate

Supernatant

(S)

Vesrcles

Interferon Injected

(H)

(V)

Pellet (P)

120

300 190

39

15

440

10

30 nl of either interferon (7.2 x lo6 U/ml) or interferon mRNA (1 mg/ml) were injected into batches of 25 oocytes. Oocytes were Incubated for 6 hr before fractionation and vesicle preparation by method 1 (see Experimental Procedures). Interferon assays were performed on each fraction and results are expressed as units of Interferon per fraction per 25 oocytes after correction for sampling. Approximately 5 x lo5 U of interferon were recovered from oocytes (25) assayed immediately after injection of interferon protein. Sample fb) Fraction Incubation

Injected

Interferon Protein

Interferon mRNA

3.2 x lo5

3785

medium

630

Total oocyte

homogenate

Supernatant

(S)

(H)

1.5 x 105

Pellet(P) Vesicles

(V)

Vesicles

+ enzymes

Vesrcles

+ NP-40

Vesicles

+ enzymes

+ NP-40

267

3000

119

3794

1695

672

1500

5352

1896

528

468

Oocytes (batches of 25) were each injected with 30 nl of interferon mRNA (1 mg/ml) or 30 nl of Interferon protein (7.2 x lo6 U/ml). Vesicles were prepared by method 2 (see Experimental Procedures) from RNA-injected oocytes after 6 hr of culture while protein-injected oocytes were processed immediately after injection. Vesicle fractions were incubated for 3 hr at 4OC with chymotrypsin + trypsin (both at 0.5 mg/ml) with or without NP-40 (1%). After the addition of 0.1 vol newborn calf serum (Flow), samples were dialyzed and assayed for interferon. Results are expressed as units of interferon per fraction.

incubation media. Our identification of the various milk proteins in the electrophoretic autoradiographs obtained using oocyte extracts is based upon the well documented data of others (Craig et al., 1976; ZehaviWillner and Lane, 1977; Craig, Mcllreavy and Hall, 1978). Extrapolation of this argument to the identification of proteins in the incubation media is justified in the case of the caseins A, B and C due to the marked similarity of the RNA-injected oocytes and incubation media. This identification has recently been confirmed by immune precipitation of the three caseins using specific antibodies (R. Craig, J. Morser and A. Colman, unpublished results). Although interferon was also secreted into the incubation medium by mRNA-injected oocytes (see below), we would not expect to detect it electrophoretically since total lymphoblastoid mRNA contains very little interferon

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Oocytes b 4 RNA(

;

Tracks

G+ 7

-

I 2 3 4

- MS4 ”

Cas A,

G,

Figure

3. Secretion

of Milk Proteins

by Oocytes

Oocytes were injected with the following solutions and immediately incubated for 6 hr in 2 mCi/ml ‘?,-methionine in Barth’s solution. Oocytes and incubation media were then processed for electrophoresis on a 12.5% polyacrylamide gel. (Track 1) 60 ng globin mRNA: (track 2) 20 ng mammary gland mRNAs; (track 3) 30 ng interferon mRNA; (track 4) distilled water. Processed incubation media from the oocytes shown in tracks (l-4) were run as follows: (track 5) globin mRNA; (track 6) mammary gland mRNA; (track 7) interferon mRNA; (track 8) distilled t+O. The arrows correspond to the positions of oocyte actin (A: 42,000 daltons). casein A (Gas A: 28,000 daltons), casein B (Gas B: 25,500 daltons). casein C (Gas C: 20,500 daltons) and globin (G: 16,000 daltons). The molecular weight of the milk protein M-54 (54,000 daltons) was estimated with reference to the positions of the external markers transferrin (76.600 daltons), glucose dehydrogenase (53,000 daltons), P-galactosidase (130,000 daltons) and phosphorylase (100,000 daltons). The radioactivity in selected bands was quantitated by excising the bands and counting in an NCS (Amersham-Searle)/toluene-based scintillant. Interferon mRNA-injetted oocytes and the surrounding incubation media were also

mRNA. Considerable export of interferon into the incubation medium was demonstrated by bioassay, however (see legend to Figure 3). These results indicate that secretory proteins are selectively exported from oocytes; it could still be argued, however, that those oocytes injected with interferon or mammary gland mRNA were, for unspecified reasons, more leaky than the control and hemoglobin mRNA-injected oocytes. This possibility was excluded by the demonstration that after co-injection of globin, interferon and milk mRNAs, only the milk proteins (see below and Figure 4) and interferon (our unpublished results) accumulated in the incubation medium. Rate of Foreign Protein Export from Oocytes To establish whether different proteins are exported at different rates, we co-injected oocytes with mammary gland and hemoglobin mRNAs and electrophoretically analyzed the oocyte homogenates and incubation media as a function of time after addition of 35S-methionine to the media. Since injected mRNAs are only gradually “recruited” for translation as a consequence of the time required for mRNA diffusion throughout the oocyte, and since different mRNAs are recruited at different rates (Berridge and Lane, 1976). we allowed 24 hr to elapse between the time of injection and the beginning of labeling. This period is sufficient for both hemoglobin and the major mammary gland mRNAs to be fully recruited (Berridge and Lane, 1976; C. Lane, personal communication). This protocol also enables us to remove damaged oocytes before culture in 35S-methionine. The results of the experiment are shown in Figure 4 with the relevant quantitation displayed in Figure 5. It is evident from Figures 5a-5c that the amounts of caseins A, B and C within the oocyte were declining within 26 hr of incubation in 35S-methionine, in contrast with the continued accumulation within the oocyte of both globin and oocyte actin. More strikingly, after 26 hr caseins A and B were present in approximately equal amounts both inside and outside the oocyte. The secretion of casein C was evidently slower. A new protein which may be a derivative of casein B was also observed in the incubation medium, although not in the oocyte, after 26 hr. Nonspecific leakage as assayed by the presence of radioactivity in the actin region of the electrophoresed incubation medium was extremely low (Figures 5j and 5k), and it is possible that this radioactivity does not represent actin at all, but rather an oocyte secretory protein. Very little radioactivity accumulated in the globin region of the gel after 26 hr (Figure 5i), and the radioactivity that was present might reflect the presence of the milk processed for interferon assay; 10 U of interferon per oocyte were found inside the oocytes and 4 U of interferon per oocyte were found in the surrounding incubation media.

Export 521

of Proteins

from

X. laevis

Oocytes

Oocytes RNA+++++---+C++++---+C+C Tracks

1

2

3

4

5

6

-Incubation 7

8

9

lo

11

media12

13 14

15

16

17

18

40120 4089

4M54

Gas Bb

cascm

Figure

4. Time Course

of Milk Protein

Secretion

by Xenopus

Oocytes

Oocytes were co-mjected wrth 3 ng of globin mRNA and 10 ng of mammary gland mRNA, or wrth drstrlled water, and incubated for 24 hr before culturing m ?S-methionine at 1.6 mCi/ml in Barth’s solution. After various hmes most batches of oocytes and incubatron media were processed for electrophoresis on a 12.5% polyacrylamrde gel. Four batches of oocytes were removed from labeled media after 5 hr and each batch was left in 1 ml unlabeled Barth’s solution for 3 hr before further culture in 30 gl unlabeled media containing 10 mM methionine for 16 hr. Oocytes and the incubation media containing methionine were then processed for electrophoresis (see gel lanes marked with C). (Tracks l-5) mRNA-injected oocytes after 1, 3, 5. 8 and 26 hr; (tracks 6-8) &O-injected oocytes after 3. 8 and 26 hr; (track 9) mRNA-injected oocytes after methionine chase; (tracks 1 O-l 3) incubation media from mRNA-injected oocytes after 3. 5. 8 and 26 hr; (tracks 14-l 6) incubation media from H20-injected oocytes after 3, 8 and 26 hr: (track 17) incubation medium from mRNA-injected oocytes after 5 hr and prror to methionine chase; (track 16) incubation medium containmg 10 mM methionine used in methionine chase experiment. The arrows correspond to the positions of actin (A), caseins A, 6 and C 0s A, Cas B and Cas C) and globin (G). The molecular weights of oocyte proteins 0120 (120,000 daltons) and 089 (89.000 daltons) were estimated with reference to the posihons of the same external markers used in Frgure 3.

protein cr-lactalbumin (molecular weight 14,500) rather than globin (molecular weight 16,000). Another mammary gland protein of apparent molecular weight 54,000, designated M-54, accumulated quite rapidly in the incubation medium (see Figures 3, 4 and 5d)

but could not be detected at all in the oocyte (Figure 4) above the background of newly synthesized endogenous proteins. It is evident within the ooc! ‘e, however, in Figure 3, lane 2. These results suggest that, as with interferon, the

Cell 522

a) Cas. A

c) Gas.c

t$ Cas. B

4!Y

/ii!< 9)089

e) 0120

I) Globin

d)

M54

.,.<

/’

, , ,

h) 089

i) Actin 30 ‘0 X 22 0” 8 12 ci c;

5

lo

I5

2025

5

IO

IS

20

25

Time (h) Figure

5. Quantitation

of Protein

Export

from Oocytes

The samples shown in Figure 4 were electrophoresed on several 12.5% polyacrylamide gels. Milk protein bands, globin bands, actin bands and some oocyte protein bands were excised and counted as described in the legend to Figure 3. The corresponding regions of the control tracks were treated similarly. The incorporation attributed to the caseins. globin and the milk protein M-54 was obtained by subtracting control slices from sample slices. Since actin is an endogenous protein, synthesis occurred in both mRNA-injected and control oocytes. and no corrected value is obtainable. [Two-dimensional gel electrophoresis of ?G-methionine-labeled oocyte proteins, however, shows that >95% of the radioactivity migrating in the position of actin in this one-dimensional gel is oocyte actin (H. R. Woodland, personal communication; A. Colman, unpublished results)]. For the same reasons the secreted oocyte proteins 0120 and 089 are displayed uncorrected. The total incorporation of 35S-methionine into protein was estimated by precipitating 2 ~1 aliquots of centrifuged oocyte homogenates on Whatman GF/C filters in hot trichloroacetic acid (10%. w/v). (a) Casein A; (b) casein B; (c) casein C: (d) milk protein M-54; (e) oocyte protein 0120 in incubation media from mRNA-injected and (f) control oocytes; (g) oocyte protein 089 in incubation media from mRNA-injected and (h) control oocytes; (i) globin; (j) actin in mRNA-injected or(k) control oocytes. (M) Oocyte content; (U) incubation media; w) total acid-precipitable incorporation into mRNA-injected oocytes.

amounts of the milk proteins reach a maximum value within the oocytes, presumably as a consequence of continual rapid secretion into the incubation medium. A more critical quantitative analysis, however, is complicated by the fact that the specific activity of the oocyte’s methionine pool changes throughout the experiment, since this pool is very small (Eppig and Dumont. 1972; H. Woodland, personal communication) and the store of radioactive methionine in the

incubation medium is soon exhausted. This probably accounts for the apparent decrease in the rate of total protein synthesis shown in Figure 51. Independent evidence for the rapid secretion of the milk proteins was obtained when injected oocytes were first incubated in 35S-methionine for 8 hr before replacing the incubation medium with unlabeled medium. The results and exact conditions used in what constitutes a “methionine chase” experiment are displayed in Fig-

Export 523

of Proteins

from X. laevis Oocytes

Figure

6.

Tracks chase:

3, 9. 1 1 and 15 in Figure 4 were scanned (C) incubation media from mRNA-injected

Microdensitometry

Tracings

of Methionine

Chase

Experiment

in a Joyce-Loebl microdensitometer. (A) mRNA-injected oocytes; (D) from H,O-injected oocytes.

oocytes

before

and (B) after methionine

ures 4 and 6. Whereas the amounts of 35S-labeled oocyte actin and rabbit globin within the oocytes were unaffected by the “chase”, all detectable labeled milk protein disappeared. These proteins were not recovered in the incubation medium containing 10 mM methionine which surrounded the oocytes between 3 and 16 hr after the “chase” began. We presume that all the proteins were secreted into the large volume of unsupplemented incubation medium used in the initial 3 hr of the “chase”. Subsequent experiments (results not shown) demonstrated that milk proteins could be recovered from this particular fraction although the case was not always complete within the initial 3 hr period. Even so, we may conclude that secretion of milk proteins is rapid and that all the milk protein synthesized in the oocyte is available for secretion.

molecular weights of 120,000 and 89,000, respectively. The kinetics of accumulation of these two proteins in the incubation medium are shown in Figure 5. Similar kinetics are found in both RNA-injected and control oocytes, indicating that secretion of these proteins is not a consequence of mRNA injection. Neither of the two proteins is detectable within the oocyte against the background of newly synthesized proteins. The possibility that these proteins might have a bacterial or fungal origin was excluded by the observation that incubation of oocytes in chloramphenicol, gentamycin and mycostatin did not affect the outside accumulation of these proteins (A. Colman, J. Morser and C. Lane, unpublished results). Thus we conclude that endogenous proteins are secreted by the oocyte or its surrounding follicle cells.

Oocyte Proteins Are Also Secreted by Oocytes Examination of the relevant gel tracks in Figure 4 (1 O16) reveals that most oocyte proteins are not secreted; some oocyte proteins, however, are selectively exported from oocytes. The most obvious of these proteins, designated O-120 and O-89, have apparent

Discussion The faithful translation of messenger RNA microinjetted into oocytes of X. laevis was first reported by Gurdon et al. (1971). This procedure has subsequently been used to investigate the biological prop-

Cell 524

erties of different RNAs within a living cell. For example, several investigators have measured the stability of exogenous mRNAs in the oocyte (Gurdon et al., 1973; Berridge and Lane, 1976; Huez et al., 1978; Sehgal et al., 1978) and studied how polyadenylation (Huez et al., 1974, 1978; Sehgal et al. 1978) or capping (Furuichi, La Fiandra and Shatkin, 1977; Lockard and Lane, 1978) affect this stability. It also appears that most of the usual post-translational modifications of various proteins (such as polypeptide cleavage, N terminal acetylation, phosphorylation, and so forth; for review see Lane and Knowland, 1975) will occur normally within the oocyte after injection of the purified messenger RNA, indicating that all the necessary information for modification is present in the mRNA sequence. More recently, ZehaviWillner and Lane (1977) have demonstrated that secretory proteins, translated in the oocyte from injected mRNAs, are compartmentalized inside the oocyte. This compartmentation involves membrane transfer of the newly synthesized proteins, and again it appears that the nucleotide sequence alone contains all the necessary information for this transfer, a result in agreement with the in vitro findings of others (Blobel and Dobberstein, 1975a, 1975b; Lingappa et al., 1978). An implicit assumption in many of the above studies was that the proteins translated on injected mRNAs remained within the oocyte. It is our contention (see below) that for secretory proteins this is not the case. Indeed, we show that far more interferon accumulates in the incubation medium surrounding RNA-injected oocytes than remains within the oocytes themselves (Figure 1). Quantitation of total interferon produced indicates that human interferon messenger RNA is functionally stable within the oocyte for at least 45 hr. This value contrasts with the much shorter values of 6-8 hr for the functional half-life of human interferon mRNA reported by Sehgal et al. (1978); however, it is not clear from their report whether the incubation media were assayed for interferon activity. Lebleu et al. (1978) also observed the appearance of small amounts of mouse interferon in the incubation medium surrounding messenger-injected oocytes. The observation of interferon accumulation outside the oocyte, however, does not demonstrate per se that secretion is occurring, since nonspecific leakage could account for such accumulation. This paper presents evidence that proteins can be secreted by oocytes and argues that the information specifying secretion must also be encoded in the mRNA molecule in a way that can be interpreted by the amphibian oocyte. Our conclusion is based on the following experimental observations. Human interferon (Figure 1) and the guinea pig caseins A, B and C (Figures 3 and 4) accumulate demonstrably in the incubation media surrounding messenger RNA-injected oocytes; all these proteins are

secretory. Such accumulation was not observed for rabbit globin even when hemoglobin mRNA was coinjected with the mammary gland mRNAs; nor was it observed for actin, one of the major endogenous proteins synthesized by the oocyte. Neither globin nor actin are secretory proteins. The highly sensitive biological assay for interferon has enabled us to calculate the absolute rate of interferon secretion to be 1 unit per oocyte per hr. Assuming a specific activity for purified leukocyte interferon of 4 x 10’ U/mg (Rubinstein et al., 1978) this rate of secretion is equivalent to 2.5 pg interferon per oocyte per hr. No such computation of absolute rates can be obtained with the mammary gland proteins because of the changing specific activity of the radioactive methionine pool within the oocyte during the experiment. Comparison of the relative rates of secretion of the three caseins into the incubation medium, however, reveals that caseins A and B accumulate faster than casein C even though similar amounts of 35Smethionine are incorporated into all three proteins within the oocyte (see Figure 5). A further impediment to accurate quantitation is examplified by the secretion of the mammary gland protein, M-54. This protein was very difficult to distinguish within the oocyte against the background of newly synthesized endogenous proteins (Figures 3 and 4). Milk proteins synthesized from injected messenger RNAs inside oocytes are associated with membranes (Zehavi-Willner and Lane, 1977). Injected milk proteins, however, do not associate with membranes (C. Lane, S. Shannon and R. Craig, personal communication). In agreement with these results we find that newly synthesized interferon but not injected interferon protein associates with membranes and only newly synthesized interferon is secreted. This result implies that in this heterologous system, membrane association is a mandatory intermediate step in secretion (see Palade, 1975). The methionine chase experiment indicates that all the newly synthesized milk proteins are available for secretion, ruling out the possibility that secreted milk proteins derive from a small membrane-free intracellular pool and apparently arguing against the view that subcellular compartmentation in oocytes exclusively fulfills a storage function as originally proposed by Zehavi-Willner and Lane (1977). The actual process of secretion from the oocyte remains a mystery. Amphibian oocytes are surrounded by three layers of follicle cells in close apposition (Wallace and Dumont, 1968). The role of these follicle cells remains obscure, although they are known to synthesize a progesterone-like molecule responsible for oocyte maturation in vivo (Masui, 1967) and are also implicated in oocyte uptake of the blood-borne yolk protein (Wallace and Dumont, 1968; Wallace, Jared and Nelson, 1973). Gap junctions

Export 525

of Proteins

from X. laevis Oocytes

existing between the follicle cells and oocytes in hormone-stimulated ovaries have recently been described (Brown, Wiley and Dumont, 1979). Although such junctions allow direct cell-to-cell communication, the transfer of molecules as large as proteins via such junctions has not been demonstrated. Since many channels exist between the cells of the follicular epithelium (Wallace and Dumont, 1968; Dumont and Brummett, 1978), however, it is possible that both protein uptake and secretion occur by an extracellular route. Nonetheless the integrity of the follicular epithelium is necessary for selective protein uptake (Wallace et al., 1973), and we are currently investigating its role in secretion. Finally, it is pertinent to ask what kind of recognition mechanisms are involved in the process by which an amphibian oocyte will translate the messenger RNA for a foreign secretory protein and then secrete that protein. To our knowledge no previous report ascribing a secretory function to the oocyte exists, and unless the oocyte does indeed have a physiologically important secretory function it is not at all obvious why oocytes should be capable of secretion. It is possible, however, that the ability to secrete certain proteins is a general cellular property manifested only when a secretory protein is synthesized in the cell [in regard to the latter point, it is interesting to note that bacteria harboring a recombinant plasmid which contains chick ovalbumin cDNA will both synthesize and secrete ovalbumin (Frazer and Bruce, 197811. According to the “signal” hypothesis (Blobel and Dobberstein, 1975a. 1975b; Blobel, 19771, the segregation of secretory proteins into the intracisternal space of the endoplasmic reticulum is due to a signal peptide in the nascent polypeptide chain. This sequestration occurs both in vitro (Blobel and Dobberstein, 1975a, 1975b; Longappa et al., 1978) and in vivo (ZehaviWillner and Lane, 1977) and is not species-specific. Many proteins transferred into the lumen of the endoplasmic reticulum during or after translation are not secreted, however, but become incorporated into other intracellular organelles such as peroxisomes, lysosomes, membranes, and so forth (Katz et al., 1977; Goldman and Blobel, 1978). It is not known how these various proteins are sorted according to their final destination. It is possible that the spatial organization of the endoplasmic reticulum automatically determines the destination of proteins transferred into it, in which case no further “signal” designating the final address of the protein seems necessary. It is equally possible that the ultimate discrimination between these proteins may require a further “signal” which may or may not involve specific cellular recognition mechanisms. The oocytes of X. laevis offer an attractive approach to these problems since for one group of proteins-secretory proteins-translation, compartmentation and secretion can be studied concurrently within a foreign living cell.

Experimental

Procedures

Animals Adult X. laevis obtained from the South African Snake Hoek, Cape Province, South Africa) were kept at 19°C.

Farm

(Fish

Chemicals Except where otherwise mentioned, all chemicals were of analytical grade and were purchased from British Drug Houses Ltd. (Poole. UK). ?S-methionine (150-300 Ci/mmole) was supplied by the Radiochemical Centre (Amersham, UK). Chloramphenicol and cycloheximide were obtained from Sigma Ltd. Preparation of Messenger RNAs Total poly(A)+ mRNAs were isolated by the methods of Morser et al. (1979) from Namalwa cells (a human lymphoblastoid line) which had been induced to produce interferon with Sendai virus. Lactating guinea pig mammary gland mRNAs were prepared as described by Craig et al. (1976) and were a gift from R. Craig; the principal milk proteins encoded in this preparation were caseins A, B and C and a-lactalbumin. which have molecular weights of 28,000, 25.500. 20.500 and 14.500 daltons. respectively (Craig et al., 1978). Rabbit globin mRNA was a gtft from H. R. Woodland. Preparation and Microinjection of Oocytes Large X. laevis females were killed by the injection of barbiturate and the ovary was removed, divided into small clumps of oocytes and stored in modified Barth’s solution (Gurdon. 1968) containing sodium penicillin and streptomycin sulphate, both at 10 pg/ml, and mycostatin at 20 U/ml. Microinjection of oocytes with approximately 30 nl aliquots of mRNAs in distilled water was performed as described by Gurdon (1974). Unless otherwise indicated, injected oocytes were first cultured for 24 hr in Barth’s solution (50 oocytes per 5 ml). Any damaged oocytes were discarded and the remainder were cultured in small wells containing 30 ~1 of 2 mCi/ml ?S-methionine in Barth’s solution, with five oocytes per well. All incubations were performed at 21°C. At the end of the incubation period oocytes and incubation media were frozen separately at -70°C before further analysis. Incubation medium was only analyzed from those wells in which all the oocytes appeared morphologically healthy. Interferon Assay Oocyte samples for interferon assay were homogenized in 2% Glasgow modified minimal essential medium (Flow Laboratories Ltd.; Irvine. UK) at 100 PI per oocyte and centrifuged for 5 min in an Eppendorf microcentrifuge at 10,000 X g at 4°C. The resulting supernatant was retained for assay. Oocyte supernatant. oocyte fractions or incubation media that had been treated with detergents or that contained 35S-methionine or cycloheximide were exhaustively dialyzed against phosphate-buffered saline (pH 7.6) before assay. Otherwise incubation media and oocyte supernatants were assayed directly for interferon by the method described by Atherton and Burke (1975) using MDBK cells. In this assay 10 units are equivalent to 1 reference research unit (BS 69/i 9) obtained from the National Institute of Biological Standards and Control. All results are given in reference research units (the actual results observed were drvrded by 10 to convert to reference units). Polyacrylamide Gel Electrophoresis Oocyte samples for gel electrophoresis were homogenized at 5 oocytes per 200 ~1 in 1% sodium dodecylsulphate, 1% Z-mercaptoethanol, 0.0625 M Tris-HCI (pH 6.8) containing 1 mM phenylmethylsulfonylfluoride (sample buffer). After centrifugation for 5 min at room temperature in an Eppendorf microcentrifuge at 10,000 X g. the supernatant was removed and diluted with 0.2 vol 60% (w/v) sucrose, 0.06% bromophenol blue in sample buffer. Samples of incubation media were precipitated by addition of 0.2 vol ice-cold 50% (w/v) trichloroacetic acid in the presence of 2 pg bovine serum albumin. The precipitates were collected by centrifugation and washed twice with 1 ml ice-cold acetone before air-drying and resuspensron in sample buffer containing sucrose and bromophenol blue

Cell 526

(50 ~1 for every 30 pl of incubation medium precipitated). 10 pl aliquots of oocyte samples and 20 pl aliquots of incubation media samples were electrophoresed overnight on 12.5% polyacrylamide gels (Laemmli. 1970) at 8 mA and then fixed in 45% methanol, 10% acetic acid before drying down and exposing to Kodirex X-ray film (Kodak Ltd). Preparation of Vesicle Fraction from Oocytes After some experiments, unfrozen oocytes were homogenized in batches of 30 in 0.3 ml T buffer [50 mM KCI. 10 mM magnesium acetate, 20 mM Tris-HCI (pH 7.6) containing 1 mM phenylmethylsulphonylfluoride] at 0°C and processed by one of the following methods [modified after the procedure of Zehavi-Willner and Lane (1977) and C. Lane, personal communication]: Method 1 The homogenate 0-l) was layered onto 1 ml 20% (w/v) sucrose in T buffer and centrifuged at 10,000 x g in an Eppendorf microcentrifuge for 15 min at 0°C. The supernatant (S) was removed, and the vesicles present in the pellet were solubilized in 0.2 ml 1% NP-40 in phosphate-buffered saline (pH 7.6) and centrifuged at 10,000 X g as above for 2 min. The resulting supernatant (V) and pellet (P) were retained for assay. Method 2 The homogenate 6-f) was layered onto a “step” gradient consisting of 1 ml 20% (w/v) sucrose in T buffer above 1 ml 50% (w/v) sucrose in T buffer, and centrifuged at 10,000 X g in an M.S.E. 8 X 5 ml rotor (HS 18) for 15 min at 0°C. The vesicle fraction (V) was then removed from the 20%/50% interface and retained along with the remaining supernatant (S) and pellet (P) for assay.

The authors are grateful for support to the Cancer Research Campaign (A.C.) and to the Medical Research Council (J.M.) for a program grant to Derek Burke. We thank J. Flint and A. Lyons for excellent technical assistance. We thank Ft. Old, D. C. Burke and H. R. Woodland for helpful comments on the manuscript. Guinea pig mammary gland RNA and human interferon were generous gifts from R. K. Craig and K. Fantes. respectively. We thank C. D. Lane for useful experimental suggestions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

March

14, 1979;

revised

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