Proc. Nati. Acad. Sci. USA Vol. 76, No. 8, pp. 3800-3804, August 1979

Biochemistry

Immunological and chemical identification of a neurophysincontaining protein coded by messenger RNA from bovine hypothalamus* (biosynthetic precursor/cell-free translation/specific immunoprecipitation/polyacrylamide gel electrophoresis/peptide mapping)

LINDA C. GIUDICEt AND IRWIN M. CHAIKENt§ tClinical Endocrinology Branch and *Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205

Communicated by C. B. Anfinsen, May 30, 1979

ABSTRACT The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis. Poly(A-RNA was 'isolated from bovine hypothalamus and translated in a wheat germ system containing 5 or 3H-labeled amino acids. A number of unique [asS~cysteine- but few [35Smethionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of-purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neuro hysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [35S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested- with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [a5S]cysteine-labeled peptides (of the translation product) were identical to major cysteinecontaining peptides of 'authentic neurophysin. The data show that hypothalamic mRNA directs the translation of'several unique cysteine-rich roteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteinecontaining tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin. The peptide hormones oxytocin and vasopressin and their associated "carrier" proteins, the neurophysins, are cystine-rich constituents of peptidergic neurosecretory neurons in the mammalian hypothalamus (2, 3). In the cow, there are two chemically distinct and major neurophysins (I and II), both having a Mr of 10,000. These proteins are highly homologous in their primary structures and differ principally in amino acid sequences at their amino- and carboxyl-termini (4, 5). Each neurophysin is highly crosslinked by intrachain disulfide bonds and has about equal affinity for both of the neurohypophyseal peptide hormones (6, 7). From studies on vasopressin and neurophysin biosynthesis (8-10), it has been suggested that the hormones and their associated carrier proteins are synthesized as common precursors in the perikarya of the supraoptic and paraventricular nuclei in the hypothalamus. Precursor processing is believed to occur during intra-axonal transport to the posterior pituitary, where the hormone-neurophysin complexes are stored for subsequent release (2). No direct chemical evidence supporting this hypothesis, however, has been forth-

coming. Recently, high Mr (17,000 and 20,000) neurophysinimmunoreactive proteins have been identified in extracts of mouse hypothalamus (11). Also, in vivo and in vitro pulse-chase experiments have allowed identification of cysteine-containing rat hypothalamic proteins (Mr 20,000) that are apparently converted to Mr 17,000 and 12,000 polypeptides en route to the posterior pituitary (12, 13). Subsequently, these proteins were found to crossreact with anti-neurpphysin serum (14). In view of the available information, we have attempted to define the primary translated hypothalamic protein that ultimately gives rise to neurophysin and th obtain chemical evidence for this assignment. To do this, we have used previously prepared purified antibodies against neurophysin (15) to recognize translation products of hypothalamic mRNA in a cellfree biosynthetic system. The present communication reports immunological and chemical data showing that neurophysin II is synthesized as part of a 23,000-25,000 Mr protein. These results, when taken with previous data, suggest that neurophysin biosynthesis may conform to the signal hypothesis (16, 17) for the biosynthesis of secreted proteins. EXPERIMENTAL PROCEDURES

Materials Frozen bovine hypothalami and bovine posterior pituitaries were purchased from Pel-Freez and stored at -70°C. Oligo(dT)-cellulose was from P-L Biochemicals and nucleic acid grade phenol was from Bethesda Research Laboratories (Rockville, MD). Cowan I strain of Staphylococcus aureus (Pansorbin) and Trasylol (kallikrein inactivator) were obtained from Calbiochem. Reagents for gel electrophoresis were from Bio-Rad. Raw wheat germ was purchased from General Mills (Minneapolis, MN). Radiolabeled amino acids were from Amersham or New England Nuclear. Purified neurophysin antibodies were prepared by affinity chromatography on neurophysin-Sepharose as described (15). Other antibody preparations used were purified ribonuclease antibodies (15), antisera against oxytocin (Calbiochem), and nonimmune sera from rabbits prior to neurophysin immunization (15). Authentic bovine neurophysins I and II were isolated from posterior pituitaries essentially as described (15), with final purification by affinity chromatography on methionyltyrosylphenylalanyl-w-aminohexyl-agarose (18). Abbreviation: NaDodSO4, sodium dodecyl sulfate. * A preliminary account of this work has been presented at the annual meeting of the American Society of Biological Chemists in Dallas, TX, April 1979 (1). § To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3800

Proc. Natl. Acad. Sci. USA 76 (1979)

Biochemistry: Giudice and Chaiken Table 1. Poly(A)-RNA yields from hypothalamustis and other tissues Tissue

Poly(A)-RNA, Aglg tissue*

164 Bovine pituitaryt 200 Mouse pituitaryt 188 Mouse pituitary tumort 5 Bovine hypothalamus 1.5 Solanum tuberosum * Based on the average yields from two preparations, which differed by less than 10%. t Data from Giudice et al. (19).

Methods Cell-Free Translation. Most of the general translation methods have been cited elsewhere (19). RNA was isolated from bovine hypothalami by using a modification (20) of the phenol extraction method of Aviv and Leder (21). mRNA was purified by affinity chromatography on oligo(dT)-cellulose (16), except that the final elution buffer was heated to 450C. Yields of hypothalamic mRNA ranged between 1 and 2% of the nucleic acids applied to the affinity columns. Wheat germ extracts for protein synthesis were prepared according to Roman et al. (22), and conditions for translations were as described (23). The optimal concentration of hypothalamic mRNA for translations was determined as discussed in Results; optimal Mg2+ and K+ concentrations were found to be 2 mM and 130 mM, respectively. Incubations were for 90 min at 270C, after which 5-yl aliquots were put on Whatman 3 MM filter paper disks to determine the incorporation of radiolabeled amino acids into hot trichloroacetic acid-insoluble material (24). Radioactivity was determined in a Packard Tri-Carb liquid scintillation counter (having 73% efficiency for '5S and 35% efficiency for 3H), using a toluene/Spectrofluor scintillant. Antibody Precipitation. Translated proteins that were precipitated with trichloroacetic acid (10% wt/vol) at 4°C were Z

C~

._I

0

20

a 0

.'tI

C

C

0

o.0

W

E M-

X Lo

cn o

:_x 0

E

.0

0Q-

-fra-ctionated with specific antibodies by using general procedures cited elsewhere (19). The acid precipitates were solubilized by brief sonication in 10 mM Tris-HCl buffer, pH 8.2, containing 1% sodium dodecyl sulfate (NaDodSO4), and the samples were boiled for 2 min. After cooling to 25°C, 9 vol of 20 mM Tris-HCl buffer, pH 7.5, containing Trasylol (100 units/ml), 10 mM EDTA, 150 mM NaCl, and 1% Triton X-100 was added. Antisera (10 ,ul), purified antibodies (20 ,ul of a solution of A280 = 0.057), or nonimmune sera (10 ,u) were added subsequently per 100,ul of translation mixture; in competition experiments unlabeled proteins or peptides (20 ,g per 100-Alj translation mixture) were added immediately before the antisera or antibodies. Antigen-antibody complexes were precipitated with Pansorbin by the procedure of Kessler (25). Analysis of Translation Products. Trichloroacetic acid precipitates or immunoprecipitates were reduced with dithiothreitol and alkylated with iodoacetamide (16) and analyzed by electrophoresis in polyacrylamide slab gels (1.5 mm thick) having a 5% acrylamide stacking gel (1 cm) and a 12-20% acrylamide gradient resolving gel (8.7 cm) in NaDodSO4 and buffers described by Maizel (26). After electrophoresis, gels were stained with Coomassie brilliant blue, destained (16), and prepared for fluorography (27). Peptide Mapping. Peptides of translation product and authentic bovine neurophysin II were analyzed after performic acid oxidation and trypsin digestion (28). The immunoprecipitate from a l/s-ml translation mixture was combined with 0.6 mg of neurophysin II. The lyophilized mixture was suspended in a 1.5-ml plastic Microfuge tube with 400,ul of performic acid (a mixture of 0.5 ml of 30% reagent grade hydrogen peroxide and 9.5 ml of 99% formic acid incubated in a glass-stoppered flask for 1 hr at room temperature and for a second hour in ice water). After performic acid oxidation for 3 hr in an ice-water bath, the sample was centrifuged, the pellet was resuspended with 200 ul of 99% formic acid and recentrifuged, and the combined supernatants (now containing oxidized neurophysin and solubilized, oxidized, radiolabeled translation products from the immunoprecipitate) were pooled and lyophilized. The oxidized protein mixture was dissolved in 5 ,ul of 0.25 M NH4HCO3, pH 8.5, containing 13 ,ug of trypsin treated with tosylphenylalanyl chloromethyl ketone (TPCK) (Worthington) and incubated at 37°C for 1 hr. Acetic acid was added to bring the pH to 3 and the sample was lyophilized. The dried peptide mixture was subjected to descending chromatography on Whatman 3 MM paper (16 hr, in upper phase of 1-butanol/ acetic acid/H20, 4:1:5 vol/vol) followed by electrophoresis at right angles (1500 V, 1 hr, in 0.6 M pyridine/acetate buffer, pH 3.6). The dried map was stained with a mixture of 100 ml of

C X

Table 2. Incorporation of radiolabeled amino acids into proteins synthesized in wheat germ extracts Specific Incorporation, activity, cpm/5 pl mRNAt Ci/mmol Amino acid* 45,000 1200 [35SJMethionine

0

00 0

CQ o 0

0 0

C

c

Poly(A)-RNA, ,g/100 ,u FIG. 1. Optimization of hypothalamic poly(A)-RNA-stimulated incorporation of radiolabeled amino acids into protein in a wheat germ cell-free synthesizing system. Poly(A)-RNA from bovine hypothalamus (amounts shown on abscissa) wasiincubated in translation mixtures'(100 Al) containing 2 mM Mg2+, 130 mM K+, and 20,qCi of [35S]methionine or 13 ,uCi of [3H]proline (1 Ci = 3.7 X 101° becquerels). Incubations were at 27°C for 90 min, after which incorporation of

[35S]methionine (0) or [3H]proline (O) into trichloroacetic-acidinsoluble material on filter disks was determined. Arrows indicate the endogenous translating capacity of the wheat germ extract in the absence of added mRNA.

3801

[35S]Cysteine

300

+

110,000

_

30,000 100,000

+

* Present at 1.8 ,M. +, Bovine hypothalamic mRNA at 70 Ag/ml; -, endogenous wheat germ activity. Determined on filter disks as trichloroacetic acid-insoluble material from a 5-pl aliquot of translation mixture at the end of incubation at 27°C. Initial cpm values on filter disks were 30,000 for [35Slmethionine and 20,000 for [35S]cysteine.

t

3802

Proc. Natl. Acad. Sci. USA 76 (1979)

Biochemistry: Giudice and Chaiken 2

A Mr X

4

3

x iO-3

10-3

94-

9468 = 60 433629-

21 1412

5

4

3

2

1

B

Mr

68-..

60 433629.,Is.

:. .:.f.

.1

21.518.51412-

OF

i.

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.~W

-.4

IF

FIG. 2. Proteins synthesized in wheat germ system supplemented with hypothalamic mRNA and identification of a neurophysin-immunoreactive protein. Analysis was by NaDodSO4/polyacrylamide gel electrophoresis and subsequent fluorography. (A) [35S]Methionine-labeled proteins translated in the presence or absence of added mRNA. Lane 1, in the absence of added mRNA; lane 2, in the presence of bovine hypothalamic mRNA at 70 jg/ml; lane 3, as in lane 2 and immunoprecipitated with purified antibodies to neurophysin II; lane 4, as in lane 2 and nonspecifically immunoprecipitated with nonimmune serum. (B) [35S]Cysteine-labeled proteins translated in the presence or absence of added mRNA. Lane 1, in the absence of added mRNA; lane 2, in the presence of hypothalamic mRNA at 70 ,ug/ml; lane 3, as in lane 2 and immunoprecipitated with antibodies to neurophysin II; lane 4, as in lane 2 and immunoprecipitated with antibodies to ribonuclease; lane 5, as in lane 2 and immunoprecipitated with nonimmune serum. The positions of Mr standards were detected by Coomassie brilliant blue stain. Standards include phosphorylase b (94,000), bovine serum albumin (68,000), catalase subunit (60,000), ovalbumin (43,000), lactate dehydrogenase subunit (36,000), carbonic anhydrase (29,000), soybean trypsin inhibitor (21,500), ferritin subunit (18,500), lysozyme (14,000), and cytochrome c (12,000).

0.1% ninhydrin in 95% ethanol (vol/vol) plus 4 ml S-collidine. Radioautography of this stained map was effected with Kodak X-Omat R x-ray film.

RESULTS Translation and Immunological Characterization. The yield of poly(A)-RNA from hypothalamus, shown in Table 1, was found to be particularly low compared to yields of mRNA from tissues specialized in the synthesis and secretion of proteins. Nonetheless, when hypothalamic mRNA was translated in a wheat germ cell-free biosynthetic system, radiolabeled amino acids were incorporated into protein (Fig. 1). Maximum incorporation was achieved with 70Og of poly(A)-RNA per ml of translation, regardless of the labeled amino acid present. Optimal hypothalamic mRNA-directed protein synthesis was, however, only 2- to 3-fold greater than the wheat germ endogenous translating capacity (i.e., in the absence of added mRNA). This result is in marked contrast to the translation of other mRNAs-e.g., pituitary mRNA (19)-for which 10- to 20-fold incorporation of radiolabeled amino acids into protein is typically found (19, 29, 30). Similar low incorporations were found for the hypothalamic mRNA when the translations were performed in the rabbit reticulocyte lysate cell-free system or in the wheat germ system under a variety of conditions, but the reason for the apparent inefficient translation of brain mRNA in cell-free systems remains unclear. Incorporation of [%5S]cysteine into protein was high compared to [35S]methionine (Table 2). The results in Fig. 2A, lane 2, show that hypothalamic mRNA does not direct the synthesis of any outstanding methionine-rich proteins other than a 12,000 Mr species that probably is an endogenous wheat germ product (compare with lane 1). Nevertheless, a minor methioninecontaining protein (Mr 23,000-25,000) was immunoprecipitated from translations with purified neurophysin II antibodies (Fig. 2A, lane 3). When translations were carried out in the presence of [a5Sicysteine, several major cysteine-containing

proteins were detected (Fig. 2B, lane 2). Here, neurophysin II antibodies immunoprecipitated a protein, again of Mr 23,000-25,000, that comigrated in NaDodSO4/polyacrylamide 1

2

3

4

5

6

7 x

10-3

-94 -- 68 -60 -43 36 29

-

-21.5 -18.5 -12

FIG. 3. Specificity of immunoprecipitation of [35S]cysteinelabeled neurophysin translation product. Analysis was by NaDodSO4/polyacrylamide gel electrophoresis and subsequent fluorography, with conditions of translation as defined in the legend to Fig. 2B. Other details are in Methods. Specificity of immunoprecipitation of 23,000-25,000 Mr protein with neurophysin II antibodies (lane 1) is shown by competition with authentic neurophysin II (lane 4) and authentic neurophysin I (lane 3) and no competition with ovalbumin (lane 5) or oxytocin (lane 6). Nonspecific immunoprecipitation by two different nonimmune sera is shown in lanes 2 and 7. Molecular weights shown are as in the legend to Fig. 2.

Biochemistry:

Giudice and Chaiken

Proc. Natl. Acad. Sci. USA 76 (1979)

A

3803

B Origin

o -*--- Electrophoresis

OT 4

0 (0

Origin

0

OT-1

., I

OT-3

0fi) \ .e \

>I

0)~

cu 0 an Eor 0

is

0:C.

00

:0 I.

I

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..

FIG. 4. Comparison of peptides of authentic neurophysin II and the neurophysin translation product. [35S]Cysteine-labeled translation product and authentic neurophysin II were mixed, oxidized with performic acid, digested with trypsin, and fractionated by two-dimensional paper chromatography/paper electrophoresis. (A) Ninhydrin-stained map; (B) autoradiograph of the same map. Neurophysin cysteine-containing peptides OT-1, OT-3, and OT-4 are identified on the basis of separate experiments on these authentic neurophysin II peptides (W. M. McCormick, personal communication). The fourth expected cysteine-containing peptide, OT-2, was not detected strongly in the map. The two components denoted "Y" in A initially stain yellow with collidine/ninhydrin before turning blue with time. The dashed arrow in B indicates a major nonneurophysin [35S]cysteine-labeled component, which has a mobility similar to that of free cysteic acid.

gels with one of the major [a5S]cysteine-labeled translation products (Fig. 2B, lane 3). The relative incorporations of 35S from cysteine and methionine indicate that the 23,000-25,000 Mr protein immunoprecipitated from translations is a cysteine-rich, methionine-poor protein. The neurophysin specificity of the immunoprecipitation of the 23,000-25,000 Mr protein was shown in a series of assays. Authentic neurophysin II and structurally homologous neurophysin I, when added in saturating amounts, competed against the translation product for neurophysin antibodies (Fig. 3, lane 1 versus lanes 3 and 4). However, structurally unrelated ovalbumin did not compete (lane 5), nor did the nonapeptide hormone oxytocin (lane 6). Also, there was no crossreactivity of the 23,000-25,000 Mr species with purified antibodies to ribonuclease (Fig. 2B, lane 4) or with nonimmune serum (lane 5). Furthermore, when translations were carried out in wheat germ but with mRNA from mouse pituitary tumor cells (19), neurophysin antibodies did not immunoprecipitate the 23,000-25,000 Mr species (data not shown). Biochemical Evidence. While the immunological data suggest that the 23,000-25,000 cysteine-rich hypothalamic protein is structurally homologous with authentic neurophysin, more definitive proof for the presence of the neurophysin sequence in the high molecular weight protein was obtained from a comparison of tryptic peptides of authentic neurophysin II and the translation product. In the tryptic map of a mixture of 0.6 mg of authentic neurophysin II and pg amounts of The nomenclature used for tryptic peptides is that of Wuu and Crumm (28).

23,000-25,000 Mr species, three major, cysteine-containing peptides-OT-1, OT-3, and OT-41-were observed upon ninhydrin staining (Fig. 4A). An autoradiograph of the tryptic map to detect peptides derived from the [-3S]cysteine-labeled translation product (Fig. 4B) shows at least four major [35S]cysteine-containing peptides, three of which overlap precisely with major cysteine-rich peptides of authentic neurophysin II. The identity of a fourth major labeled component in the precursor preparation with high anodal mobility at pH 3.6, as well as that of a possible fifth peptide running just above OT-1, has nQt yet been ascertained.

DISCUSSION The data presented here show that hypothalamic mRNA directs the synthesis of several major cysteine-containing proteins in a cell-free system. Although the identities of most of these proteins remain obscure, the data in Figs. 3 and 4 demonstrate that one cysteine-rich protein of Mr 23,000-25,000 is a high molecular weight form of neurophysin. This identification is supported by clearcut specific immunological recognition of the protein by purified antibodies against authentic neurophysin as well as by the correspondence of chromatographic and electrophoretic mobilities of several component cysteinecontaining peptides. In view of the origin of the 23,000-25,000 Mr species from cell-free translation, it is assumed that all of the apparent molecular weight corresponds to protein sequence and not to nonprotein moieties that could be incorporated by posttranslational processing. Although our data identify the 23,000-25,000 Mr species as a neurophysin-containing form, the definition of this as a

3804

Proc. Natl. Acad. Sci. USA 76 (1979)

Biochemistry: Giudice and Chaiken

neurophysin precursor can be made only hypothetically at present. The translations described here are conducted in the absence of microsomal membranes, which normally process proteins destined for secretion (16, 17). Thus, some of the products coded by hypothalamic mRNA could represent "pre" or "pro" proteins,t1 including possible neurophysin precursors. In this context, it is of interest that neurophysin-immunoreactive proteins of approximately 20,000 Mr found in extracts of hypothalamus (11) and in pulse-chase biosynthetic studies (14) have been suggested as "pro-neurophysins." The size of the 23,000-25,000 Mr neurophysin-containing translation product (3000-5000 larger than putative pro-neurophysins) would be consistent with its being "pre-pro-neurophysin." Much indirect evidence has suggested that the neurophysins and the neurohypophyseal hormones are synthesized as common precursors (10). Nonetheless, no data have shown directly, for example, that the precursors of neurophysin and vasopressin are the same molecule. Our present immunological analyses (unpublished) have detected no reactivity of antisera to oxytocin and vasopressin with the neurophysin precursor. It is possible that the neurohyophyseal hormones are not a part of the linear sequences of neurophysin precursors. Alternatively, the hormones could be a part of a precursor, but not in a conformational state that can be recognized by antibodies directed against the isolated form. In support of the latter possibility are some recent data on the isolation of a 20,000 Mr protein from dog pituitaries, which exhibited crossreactivity with anti-vasopressin serum only after limited proteolysis (32). The existence of small biologically active peptides within larger precursor proteins is not unprecedented, as shown recently for the opiate peptides, the endorphins (33-36). However, whether a similar mode of synthesis has been adapted for vasopressin and oxytocin remains to be defined chemically. We are grateful to William M. McCormick for hishelpful discussions and suggestions and to Donna A. Sobieski for her excellent technical assistance. One of us (L.C.G.) was supported by the National Research Service Award No. 5F32AM05790-02.

l Pre proteins contain an amino-terminal ("signal") peptide that is cleaved cotranslationally by microsomal proteases; pro proteins contain additional amino-terminal, carboxyl-terminal, or intrachain sequences that are cleaved posttranslationally by intra- or extracellular proteases (16, 17, 31). 1. Giudice, L. C. & Chaiken, I. M. (1979) Fed. Proc. Fed. Am. Soc. Exp. Biol. 38,621 (abstr. 2075). 2. Lederis, K. (1974) in Handbook of Physiology, eds. Greep, R. 0. & Astwood,.E. B. (Amer. Physiol. Soc., Washington, D C), Vol. 7, pp. 81-102. 3. Scharrer, B. (1977) in Peptides in Neurobiology, ed. Gainer, H. (Plenum, New York), pp. 1-8. 4. Chauvet, M. T., Chauvet, J. & Acher, R. (1976) Eur. J. Biochem.

69,475-485.

5. Schlesinger, D. H., Audhya, T. K. & Walter, R. (1978) J. Biol. Chem. 253, 5019-5024. 6. Breslow, E. (1975) Ann. N. Y. Acad. Sci. 248,423-441. 7. Cohen, P., Camier, M., Wolff, J., Alazard, R., Cohen, J. S. & Griffin, J. H. (1975) Ann. N. Y. Acad. Sci. 248,463-479. 8. Sachs, H. & Takabatake, Y. (1964) Endocrinology 75, 943948. 9. Sachs, H., Fawcett, P., Takabatake, Y. & Portanova, R. (1969) Recent Prog. Horm. Res. 25,447-491. 10. Walter, R., Audhya, T. K., Schlesinger, D. H., Shin, S., Saito, S. & Sachs, H. (1978) Endocrinology 100, 162-174. 11. Lauber, M., Carmier, M. & Cohen, P. (1979) FEBS Lett. 97, 343-347. 12. Gainer, H., Sarne, Y. & Brownstein, M. J. (1977) J. Cell Biol. 73, 366-380. 13. Brownstein, M. J. & Gainer, H. (1977) Proc. Nati. Acad. Sci. USA 74,4046-4049. 14. Brownstein, M. J., Robinson, A. G. & Gainer, H. (1977) Nature (London) 269, 259-261. 15. Fischer, E. A., Curd, J. G. & Chaiken, I. M. (1977) Immunochemistry 14, 595-602. 16. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 835-851. 17. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 852-862. 18. Chaiken, I. M. (1979) Anal. Biochem., in press. 19. Giudice, L. C., Waxdal, M. J. & Weintraub, B. D. (1979) Proc. Natl. Acad. Sci. USA 76, in press. 20. Shields, D. & Blobel, G. (1977) Proc. Natl. Acad. Sci. USA 74, 2059-2060. 21. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408-1412. 22. Roman, R., Brooker, J. D., Seal, S. N. & Marcus, A. (1976) Nature

(London) 260,359-360.

23. Dobberstein, B. & Blobel, G. (1977) Biochem. Biophys. Res. Commun. 74, 1675-1682. 24. Mans, R. J. & Novelli, G. D. (1971) Arch. Biochem. Biophys. 94, 48-53. 25. Kessler, S. W. (1976) J. Immunol. 117, 1482-1490. 26. Maizel, J. V. (1969) in Fundamental Techniques in Virology, eds. Hable, K. & Salzman, N. P. (Academic, New York), pp.

334-362.

27. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 28. Wuu, T.-C. & Crumm, S. E. (1976) Biochem. Biophys. Res. Commun. 68,634-639. 29. Roberts, B. E. & Paterson, B. M. (1973) Proc. Nati. Acad. Sci. USA

70,2330-2334.

30. McDowell, M. J., Joklik, W. K., Villa-Komaroff, L. & Lodish, H. F. (1972) Proc. Natl. Acad. Sci. USA 69,2649-2653. 31. Devillers-Thiery, A., Kindt, T., Scheele, G. & Blobel, G. (1975) Proc. Natl. Acad. Sci. USA 72,5016-5020. 32. Kruber, K. A. & Morris, M. (1979) Fed. Proc. Fed Am. Soc. Exp. Biol. 1130 (abstr. 4768). 33. Mains, R. E., Eipper, B. A. & Ling, N. (1977) Proc. Natl. Acad. Sci. USA 74,3014-3018. 34. Roberts, J. L. & Herbert, E. (1977) Proc. Natl. Acad. Sci. USA

74,4826-4830.

35. Roberts, J. L. & Herbert, E. (1977) Proc. Natl. Acad. Sci. USA

74,5300-5304.

36. Loh, Y. P. (1979) Proc. Natl. Acad. Sci. USA

76,796-800.