Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 94-98, January 1978 Biochemistry
Ovalbumin: A secreted protein without a transient hydrophobic leader sequence ("signal hypothesis"/acetylation/NH2-terminal sequence/cell-free translation) RICHARD D. PALMITER, JEAN GAGNON, AND KENNETH A. WALSH Department of Biochemistry, University of Washington, Seattle, Washington 98195
Communicated by Hans Neurath, October 11, 1977
Edman degradation. In contrast to these secretory proteins, ovalbumin synthesized in this cell-free system is the same as the polypeptide synthesized and secreted in vivo. This paper establishes the sequence of primary translation product in
Ovalbumin mRNA was translated in a reticuABSTRACT locyte lysate. The primary translation product starts with methionine derived from Met-tRNAf. When the nascent polypep tide is about 20 residues long, this methionine is removed. The new NH2-terminal glycine is acetylated from acetyl-CoA when the polypeptide is 44 residues long. The se uence of 35 residues at the NH2 terminus of ovalbumin was determined by automated Edman degradation after a method was devised to prevent acetylation during protein synthesis in the reticulocyte lysate. This sequence is the same as that of secreted ovalbumin and does not resemble the transient "signal peptides" associated with most secretory proteins, including three other egg white proteins synthesized in the same cells as ovalbumin.
vitro.
MATERIALS A-ND METHODS Lysates. Rabbit reticulocyte lysates were prepared as before (5); for some experiments lysates were treated with staphylococcal nuclease (Worthington) (6) and passed through a Sephadex G-50 column to remove small molecules (1). Incubation conditions were essentially as before (1). For the preparations of unblocked ovalbumin, citrate synthase (Sigma Chemical Co., 10-25 units/ml) and oxaloacetate (1 mM) were included in the reaction mixture (7). mRNA and tRNA. For most studies, total hen polysomal RNA was used as a source of ovalbumin mRNA (mRNA,).' For studies requiring purified mRNA, the above RNA preparation was passed through oligo(dT)-cellulose twice and the mRNA fraction was sedimented twice on sucrose gradients (8). The peak mRNAO, activity fractions were >95% pure, judging from sodium dodecyl sulfate/acrylamide gel analysis of the total translation product. [a5S]Met-tRNA et and [35S]Met-tRNAmet were prepared from rabbit reticulocyte lysates and purified on BD-cellulose (1). Formyl-135S]Met-tRNA'et was prepared by incubating rabbit tRNA in an Escherichia coli extract with 0.1 mM leucovorin as a formyl donor (9). Sequence Determination. After incubation, radioactive ovalbumin was isolated by immunoprecipitation (1), dissolved in 300 ,ul of 10% HOAc,-and placed into the cup of a Beckman sequenator, model 890B. a-N-Formylovalbumin was deformylated by dissolving the immunoprecipitate and 1 mg of carrier ovalbumin in 300 Ail of 3 M HCl and incubating for 2 hr at 370 (10); after incubation it was placed directly into the sequenator cup. Ovalbumin-Synthesizing Polysomes. In vivo: oviduct tissue (1.2 g) from estrogen-treated chicks was incubated in 3 ml of Hanks' salt solution for 5 min with 200 ,Ci of [35S]methionine and 300 ,uCi of [3H]phenylalanine (11); the reaction was stopped by addition of cycloheximide to 10 ,M and quick cooling. Ribosomes were isolated by Mg2+ precipitation (12) and resuspended, and the ovalbumin-synthesizing polysomes from 68 A260 units were precipitated by indirect immunoprecipitation (13). In vitro: ovalbumin-synthesizing polysomes were isolated from the nuclease-treated reticulocyte lysate by incubating the lysate for 6 min with purified mRNA., and then stopping the reaction with 10 ,uM cycloheximide and 10 A260 units of carrier oviduct polysomes. The polysomes were centrifuged through a 0.5-1.0 M sucrose gradient for 200 min at 27,000 X
The mechanisms by which proteins pass through cellular membranes are poorly understood. Transmembrane transport of polypeptides is encountered in various situations, including the entrance of toxic peptides, such as diphtheria toxin, abrin, ricin, and some colicins into cells, the transfer of proteins from one cellular compartment to another, and the export of secretory proteins. In each case there are presumably specific membrane receptors that recognize the proteins to be transported and direct them through the membrane. In protein secretion, the initial events may involve the interaction of the nascent polypeptide with a hypothetical membrane receptor located in the endoplasmic reticulum. If this event is coupled with ribosome attachment to the membrane, then the energy of polypeptide elongation could provide the motive force necessary to drive the polypeptide through the membrane. Folding of the polypeptide on the other side of the membrane might effectively ensure unidirectional transport. Secreted proteins are typically synthesized as precursors containing 15-30 additional NH2-terminal residues. The current list includes over 20 examples, including immunoglobulin L and H chains, serum albumin, pancreatic enzymes, mellitin, and several polypeptide hormones, as well as a bacterial lipoprotein (1, 2). These sequences are unusually rich in hydrophobic residues (typically 85-95%) and are usually cleaved during membrane transport. They have been termed "signal peptides" because of their presumed recognition by membrane receptors (3). We are studying the synthesis and secretion of egg white proteins in the chicken oviduct. Lysozyme, ovomucoid, and conalbumin contain typical signal peptides of 18, 23, and 19 residues, respectively (1, 4). The precursor forms of these proteins were obtained by translating their respective mRNAs in a cell-free system derived from rabbit reticulocytes; the translation products were isolated by immunoprecipitation and their amino acid sequences were determined by automated 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. §1734 solely to indicate this fact.
Abbreviation: mRNA.,, ovalbumin mRNA. 94
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Palmiter et al.
Proc. Natl. Acad. Sci. USA 75 (1978) !.
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FIG. 1. Kinetics of leucine and acetate incorporation into immunoprecipitable ovalbumin. A regular lysate (4.2 ml) was incubated with oviduct polysomal RNA (950 ,ug) with [14C]leucine (7.5 jCi) and [3H]acetyl-CoA (40 uCi). At the indicated times aliquots were removed to measure radioactivity in completed ovalbumin molecules by immunoprecipitation (0). A 54-fold excess (650 MM) of unlabeled acetyl-CoA was added to part of the reaction mixture at 15 min (M); cycloheximide was added to another aliquot at 21 min (0).
The bottom half of the gradient, containing the polysomes, was layered over 15 ml of 1.0 M sucrose and centrifuged for 10 hr at 25,000 X g in the SW 27 rotor. The pellet was suspended in 2 mM EDTA and placed into the seg in an SW 27 rotor.
quenator.
Size Determination of Nascent Ovalbumin Polypeptides. A nuclease-treated lysate was incubated with 50 jCi of [3H]tyrosine or 50 ,uCi of [3H]acetyl-CoA for 7 min; then 1.6 ,ug of purified mRNA., was added for 7 min. The reaction was stopped with cycloheximide. Ribosomes were isolated by ultracentrifugation, suspended in 8 M urea/0. 1 M NaOAc at pH 5.7, and applied to a DEAE-cellulose column (0.5 X 0.8 cm) equilibrated with the same buffer. After the column was washed with 0.1 M NaCI, the peptidyl-tRNA was eluted with 0.5 M NaCl in the same buffer. The polypeptide was cleaved from tRNA by addition of Na2CO3 to 0.3 M (pH 10.5) and incubation for 1 hr at 37°. The sample was then acidified, combined with marker CNBr fragments of whale myoglobin, and applied to a 0.9 X 32-cm column of Sephadex G-50 (fine) overlaid with a 14-cm column of G-25 (fine) and equilibrated with 8M urea/1.8 M HOAc. Fractions (450,ul) were collected and radioactivity was determined in Aquasol.
RESULTS Incorporation of Acetate into Ovalbumin from AcetylCoA. Preliminary attempts at Edman degradation of ovalbumin synthesized in the reticulocyte lysate were unsuccessful. We suspected that the molecules were blocked by acetylation, as they are in vivo, since reticulocyte lysates have a ribosomeassociated transacetylase (14). Fig. 1 shows that [3H]acetate and ["4C]leucine are incorporated into ovalbumin with similar kinetics when a rabbit reticulocyte is incubated with mRNAO,. There is a 10- to 12-min lag before either isotope is immunoprecipitated; this corresponds to the time required to synthesize a complete ovalbumin molecule under these assay conditions. Approximately 1.4 mol of acetate is incorporated from acetyl-CoA per mol of ovalbumin synthesized. Fig. 1 shows that inhibition of polypeptide elongation with 10 ,M
FIG. 2. Minimum size of nascent ovalbumin polypeptides containing [3H]acetate or [3H]tyrosine. (A) Ovalbumin nascent polypeptides, isolated as described in Materials and Methods, were combined with CNBr fragments of whale myoglobin (-.3 mg) and separated on a Sephadex column. Fragments a, b, and c (dotted lines) correspond to the internal standards containing 76, 55, and 22 residues, respectively. Acetyl-CoA elutes in fraction 55. (B) The logarithm of polypeptide length is plotted against fraction number. The minimum size of acetate- and tyrosine- containing peptides was determined by subtracting 4.5 fractions from the extrapolation of the trailing edge of the radioactive profiles shown in (A).
cycloheximide immediately stops both [3H]acetate and ["'CIleucine incorporation into ovalbumin, suggesting that acetate incorporation is tightly coupled with polypeptide elongation. In contrast, addition of a 54-fold excess of unlabeled acetyl-CoA at 15 min of incubation has no effect on incorporation of [3H]acetate into immunoprecipitable ovalbumin for about 10 mt, and has no effect on ["aClbeucine incorporation at any time. These results are consistent with the incorporation of [3H]acetate into the NH2 terminus of the nascent ovalbumin polypeptides within the first 2 min of ribosome transit along mRNAct. The effect of unlabeled acetyl-CoA is delayed because only the polypeptides released from the ribosomes are immunoprecipitated under these conditions, and it takes 10 mi to chase all the 3H-labeled ovalbumin chains from the polysomes.
Time of Acetylation. In order to determine more accurately when the ovalbumin nascent chains are acetylated, we measured the size of the smallest nascent polypeptide chains bearing [3H]acetate. Purified mRNA0v was translated in a nucleasetreated reticulocyte lysate for 7 min and the nascent ovalbumin polypeptides were isolated and separated on a Sephadex column with internal molecular weight standards (Fig. 2A). The trailing edge of the absorbance peaks extrapolate to a point on the abscissa 4.5 fractions to the right of the peak. This value was subtracted from the extrapolation of the [3H]acetate curve and plotted in Fig. 2B on the calibration curve. A minimum value of 44 residues for acetylated nascent chains was obtained. As a check on the methodology, [3H]tyrosine-labeled nascent chains were treated identically (Fig. 2A). In this case the minimum ovalbumin nascent chain bearing [3H]tyrosine is estimated to be 29 residues, a value in agreement with the location of the first tyrosine residue (Table 1). NH2-Terminal Sequence of Unblocked Ovalbumin. in order to determine the amino acid sequence of ovalbumin, we sought conditions that would prevent acetylation. We found that we could metabolize the endogenous pool of acetyl-CoA by adding citrate synthase and 1 mM oxaloacetate. Under these conditions the incorporation of [3H]acetate into ovalbumin can be reduced to less than 10% of controls (7). Using this metabolic trap, we were able to synthesize ovalbumin molecules whose amino acid sequence could be determined by Edman degradation. Ovalbumin was synthesized with each of the 20 radioactive amino acids in separate reactions. The sequence of these samples was then determined in-
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Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Amino acid sequence of the primary translation product from mRNAov*
1.5
Ac NH2-Met-Gly- Ser- Ile -Gly- Ala -Ala-Ser-Met-Glu- Phe- Cys-Phe-Asp- Val-Phe10 15 -1 +1 5 Lys-Glu-Leu- Lys- Val - His-His- Ala -Asn-Glu-Asn- Ile - Phe-Tyr-Cys20 25 30 Pro- Ile- X - Ile -Met- X - X -Leu35
1.0
* The primary translation product starts with Met (-1); the product isolated from the reticulocyte lysate after translation of mRNAo, in the presence of citrate synthase and oxaloacetate starts with Gly (+1); the product isolated from egg white starts with AcGly. X denotes an unidentified residue.
dividually, in pairs with different isotopes, or in combinations of up to 13 different amino acids that were subsequently separated by high-pressure liquid chromatography. Fig. 3 shows representative sequence data that establish the sequence of the first nine residues of ovalbumin. The entire NH2-terminal sequence derived by this procedure is shown in Table 1. Based on this information, we isolated and determined the sequence of CNBr peptides from unlabeled ovalbumin corresponding to residues 1-8 and 9-35. The sequence of these peptides is identical to that of ovalbumin synthesized in the reticulocyte lysate. Primary Translation Product from mRNAO,. Because all secreted proteins examined thus far have a hydrophobic leader sequence about 20 residues long that is cleaved in vivo, but not in vitro, we were surprised not to find a similar situation with ovalbumin. Perhaps the reticulocyte lysate contains a protease that cleaves a "signal peptide" from ovalbumin but not from other secretory proteins. Since the product synthesized in vitro begins with glycine, there must have been at least one proteolytic event (to remove initiator methionine) prior to our isolation of ovalbumin. Thus, it was essential to identify the sequence containing NH2-terminal methionine from initiator tRNA. We used two independent approaches to characterize this primary translation product. Both give the same result: the primary translation product is one residue longer than the unblocked in vitro product and that residue is methionine donated by Met-tRNAfet
The first approach relies on the fact that formyl-methionine 2
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FIG. 3. Representative sequence data for ovalbumin translated in reticulocyte lysates in the presence of citrate synthase and oxaloacetate. Hen polysomal RNA was translated in 1-2 ml of reaction mixtures with 100-200 ACi of the indicated amino acids. Glycine, alanine, and glutamic acid were part of a multiple label experiment and were separated by high-pressure liquid chromatography. [35S]Methionine and [3H]isoleucine were combined in one experiment; [3H]serine was part of a double-label experiment.
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FIG. 4. Edman degradation of ovalbumin synthesized with
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subjected to Edman degradation directly; (C and D) the other half was treated with 3 M HCl for 2 hr at 370 to remove the formyl group. The yield after acid treatment was about 40%.
is not cleaved from polypeptides by eukaryotic methionine aminopeptidase (15). Oviduct mRNA was translated (30 min) in the presence of f[-5S]Met-tRNAM'et and [3H]alanine. Ovalbumin was isolated by immunoprecipitation and its sequence was determined with and without acidic deformylation. Without deformylation, all the ovalbumin molecules were blocked; there was no evidence of MS or 3H in any of the first eight cycles of the Edman degradation (Fig. 4, 'Ai and B). However, after 2 hr of treatment of the immunoprecipitate in 3 M HC1 at 370, Edman degration yielded [-5S]methionine in position 1 and a doublet of [3H]alanine at positions 6 and 7 (Fig. 4 C and D). The characteristic alanine doublet is displaced one cycle compared to the sequence obtained by preventing acetylation (Fig. 3), as would be expected if the ovalbumin molecules were one residue longer. The second approach for detecting the primary translation product takes advantage of the early acetylation of nascent ovalbumin molecules. Purified mRNAO, was translated in a mRNA-dependent lysate for a brief period (6 min; about 1/2 of a ribosome transit time); the ovalbumin-synthesizing polysomes generated in this time contained three to six ribosomes and were separated from the bulk of the monoribosomes and any released peptides by ultracentrifugation. The polysome pellet was then subjected to Edman degradation. Since most of the ovalbumin molecules are acetylated, only the sequence of those chains shorter than 44 residues long can be determined. We expected two sequences: one beginning with methionine and one with glycine. The displacement of these two sequences during Edman degradation indicates the number of residues between initiator metbionine and the new NH2-terminal glycine while their abundance indicates the lengths of the nascent chains when methionine is cleaved and acetylation takes place. When mRNAO, was translated in the presence of [s5S]methionine, we noted radioactive peaks at cycles 1, 8, and 9. Just prior to acetylation, ovalbumin should have methionine at position 8 that would be accessible to Edman degradation. The peaks at positions 1 and 9 were related to each other by a reasonable step-wise yield and might represent an ovalbumin molecule extended at the NH2 terminus by one methionine residue. In order to ascertain the source of the methionine in positions 1, 8, and 9 of ovalbumin, we labeled ovalbumin nascent chains separately with methionine from [&iS]Met-tRNAfeM or [-`SIMet-tRNAMet. Fig. 5 upper shows that the methionine in position 1 is derived from Met-tRNArmet, whereas those at positions 8 and 9 are from Met-tRNAet. These experiments also
Biochemistry:
Palmiter et al.
Met-tRNAf 2000
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 5. Edman degradation of ovalbumin nascent chains synthesized in lysates supplemented with [35S]Met-tRNAfet, [35S]Met-tRNAMet, [3H]isoleucine, or [3H]glutamate. Nuclease- and Sephadex G-50-treated lysates (250 IAl) were incubated for 6 min with purified mRNAov and the indicated labeled amino acid or acyl-tRNA. Ovalbumin-synthesizing polysomes were isolated as described in Materials and Methods and subjected to Edman degradation.
included -3H]isoleucine, which was detected at positions 3 and 4 in equal abundance (Fig. 5), whereas the normal lysate product has isoleucine only in position 3 (Fig. 3). In another experiment we labeled ovalbumin with [3H]leucine, [3H]tyrosine, and [3H]glutamate. Fig. 5 shows glutamate residues at positions 9 and 10 (rather than 9 only, Fig. 3), again with nearly equal abundance. There were no discernible peaks with either leucine or tyrosine. Primary Ovalbumin Translation Product In Vivo. In order to ascertain whether the translation of ovalbumin follows the same pathway in vivo, we labeled oviduct explants with [a5S]methionine and [3H]phenylalanine for 5 min, then isolated the ovalbumin-synthesizing polysomes by immunoprecipitation. This procedure accomplished the same goal as the previous method, but in this case the polysomes were purified after translation whereas in the previous method the mRNA was purified before translation. Fig. 6 shows the pattern of '5S and 3H released during successive cycles of Edman degradation. Methionine residues were released in cycles 1, 8, and 9. This pattern of methionine release and the relative abundance of methionine in cycles 8 and 9 is the same as that observed with ovalbumin synthesized in vitro. The pattern of [3H]phenylalanine release (Fig. 6B) is consistent with a mixture of nonacetylated ovalbumin molecules with phenylalanine residues at positions 10, 12, and 15 (Table 1) and the primary translation product (with its extra methionine) with phenylalanine residues at positions 11, 13, and 16. The high background in these experiments is due to the fact that only 10% of the nascent chains are accessible to sequencing, the others being acetylated. DISCUSSION All the available evidence suggests that the ovalbumin polypeptide synthesized in the reticulocyte lysate is the same length as the molecule synthesized in intact tubular gland cells. When analyzed on sodium dodecyl sulfate/acrylamide gels, the in vitro translation product actually migrates slightly faster than ovalbumin synthesized in vivo (16); this disparity is undoubtedly due to the lack of glycosylation in vitro, since ovalbumin synthesized in vio in the presence of tunicamycin, an inhibitor of glycosylation, also migrates slightly faster than controls (17). Moreover, comparison of peptide maps of ovalbumin synthe-
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FIG. 6. Edman degradation of ovalbumin nascent chains labeled in vivo. Explants of oviduct tissue were labeled for 5 min and ovalbumin-synthesizing polysomes were isolated by immunoprecipitation. The recovery of (A) methionine and (B) phenylalanine during Edman degradation is shown. (Inset) A confirming experiment with [35S]methionine.
sized in vivo and in vitro reveals no extra peptides from the in vitro product (16). Our sequence of the NH2 terminus of ovalbumin (Fig. 3 and Table 1) is identical to that of ovalbumin isolated from egg white (J. Gagnon, R. Palmiter, and K. Walsh, unpublished data). The residues in positions 3 and 4 are reversed compared to the sequence reported by Narita and Ishii (18). We have confirmed our alignment on four occasions, and thus feel confident that it is correct. According to the "signal hypothesis," the first step in secretion involves the recognition of the hydrophobic leader sequence of the nascent polypeptide by membrane receptors. This recognition leads to membrane transport, which is followed by proteolytic removal of the "signal peptide" (3). At first we thought that the ovalbumin could have a typical hydrophobic leader sequence that is recognized by a proteolytic enzyme in the cell-free system and hence is cleaved in vitro as well as in vivo. However, we performed two different types of experiments to find the primary translation product in vitro, and in each case concluded that the product is only one residue longer than that of the secreted protein; the extra residue is methionine and it is derived from Met-tRNAf et. The same primary translation product was deduced with nascent ovalbumin chains obtained from intact cells. Thus, it is unlikely that the lack of a typical "signal peptide" is due to incorrect initiation in vitro. The finding that ovalbumin does not have a transient, hydrophobic leader sequence raises important questions about the mechanism of ovalbumin secretion, as well as the function of such sequences in other secretory proteins, including other egg white proteins secreted from the same cells as ovalbumin. There are several variations on the secretory pathway mentioned above that might be invoked to explain ovalbumin secretion. The simplest idea is that the NH2-terminal sequence of ovalbumin functions as a "signal": it may interact with membrane receptor in a manner analogous to other "signal peptides", but then not be cleaved during membrane transport. The NH2terminal sequence of ovalbumin shows a weak resemblance to the other hydrophobic "signal peptides"; 11 of the first 15 residues of ovalbumin are hydrophobic and the NH2 terminus
98
Biochemistry: Palmiter et al.
is blocked. However, when a longer, more typical sequence of 20 residues is considered, only 13 are hydrophobic and the hypothesis of an uncleaved signal peptide seems less likely. An intriguing possibility is that the transacetylase is itself the receptor. If this enzyme is associated with the endoplasmic reticulum in the oviduct, then in the process of acetylating the NH2 terminus of ovalbumin it may direct the nascent chain into a membrane secretory channel. If this is the case, then the hydrophobicity of the NH2-terminal sequence may be of secondary importance. There are several more remote possibilities that may be considered. One is that a hydrophobic "signal sequence" is located elsewhere within the molecule, rather than at the NH2 terminus. Alternatively, a "signal peptide" might be translated as a separate polypeptide from mRNAO,. This can tentatively be ruled out by our sequence data from the entire nascent-chain population derived from purified mRNA0v (Fig. 5). We did not observe a second sequence of specific activity comparable to that of ovalbumin, but we have not tried all 20 amino acids, and hence cannot definitively rule out this possibility. Another suggestion is that ovalbumin nascent chains may have some hydrophobic group covalently attached near the NH2 terminus (e.g., to a serine or cysteine residue) that could serve the function of a "signal" and subsequently be removed. This idea has not been adequately tested, but there is a precedent in the case of penicillinase, which has a fatty acid transiently attached at the NH2 terminus (19). In any event, it is clear that proteolysis of a "signal sequence" is not essential for membrane transport or secretion of ovalbumin. The timing of methionine aminopeptidase action and acetylation of ovalbumin can be deduced from the data presented here. By determining the sequence of all the ovalbumin polypeptides that are not blocked, the yield of radioactivity in position n+1 ranges from 75-100% of a yield in position n both in vitro (Fig. 5) and in vivo (Fig. 6). The radioactivity in position n+1 represents those nascent polypeptides that still retain the NH2-terminal methionine while that in position n represents those from which methionine has been cleaved. Thus, assuming that the rate of elongation is constant, the number of residues incorporated before methionine is removed is 75-100% of the number incorporated between that event and acetylation. The size of the smallest nascent ovalbumin polypeptides bearing acetate is 44 residues (Fig. 2). With this information we calculate that initiator methionine is removed when ovalbumin nascent chain is 19-22 residues long, and acetylation occurs when the peptide is 22-26 residues longer. Initiator methionine is removed from hemoglobin when these polypeptides are 15-20 residues long (20), suggesting that the timing of this event may be related to accessibility of the methionine aminopeptidase to the nascent chain. The rate of polypeptide elongation under our in vitro conditions is about 0.65 residues/sec; thus, acetylation takes place 68 sec after initiation. The rate of polypeptide elongation on mRNAO, in vivo is about 5 residues/sec (21). Hence, in vivo, the NH2-terminal methionine is cleaved 4 sec after initiation and acetylation of the new
Proc. Nati. Acad. Sci. USA 75 (1978)
NH2-terminal glycine takes place 4 sec later. This conclusion is contrary to a recent report suggesting that methionine is cleaved 1-2 min after ovalbumin synthesis in vivo (22). We conclude that there are at least two distinct mechanisms of protein secretion in the chick oviduct. Under optimal hormonal stimulation, ovalbumin is synthesized and secreted at a rate close to 106 molecules/min per tubular gland cell (21). Thus, if there are specific membrane receptors for secretion of different polypeptides, then the oviduct should be a rich source of those involved in ovalbumin secretion, as well as those thought to recognize the "signal peptides" on lysozyme, ovo-
mucoid, and conalbumin. We thank Linda Strimple for secretarial assistance, and Stephen Thibodeau for help in developing some of the methods. This work was funded by grants from the National Institutes of Health: HD-09172, GM-15731, and RR-05432. J.G. is supported by the Medical Research Council of Canada. R.D.P. is an investigator of the Howard Hughes Medical Institute. 1. Palmiter, R. D., Gagnon, J., Ericsson, L. H. & Walsh, K. A. (1977)
J. Biol. Chem. 252,6386-6393. 2. Inouye, S., Wang, S., Sekizawa, J., Halegoua, S. & Inouye, M. (1977) Proc. Natl. Acad. Sci. USA 74, 1004-1008. 3. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 852-862. 4. Palmiter, R. D., Thibodeau, S. N., Gagnon, J. & Walsh, K. A. (1978) in Proceedings of the 11th FEBS Meeting, Regulatory Proteolytic Enzymes and Their Inhibitors, eds. Magnussen, S., Otteson, M., Foltmann, B., Dano, K. & Neurath, H. (Pergamon Press, Oxford), in press. 5. Palmiter, R. D. (1973) J. Biol. Chem. 248,2095-2106. 6. Pehlam, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, 247-256. 7. Palmiter, R. D. (1978) J. Biol. Chem., in press. 8. Haines, M. E., Carey, N. H. & Palmiter, R. D. (1974) Eur. J. Biochem. 43, 549-560. 9. Stanley, W. M., Jr. (1972) Anal. Biochem. 48,202-216. 10. Elson, N. A., Brewer, H. B. & Anderson, W. F. (1974) J. Biol. Chem. 249, 5227-5235. 11. Palmiter, R. D., Palacios, R. & Schimke, R. T. (1972) J. Biol.
Chem. 247,3296-3304. 12. Palmiter, R. D. (1974) Biochemistry 13,3606--3615. 13. Shapiro, D. J., Taylor, J. M., McKnight, G. S., Palacios, R., Gonzalez, C., Kiely, M. L. & Schimke, R. T. (1974) J. Biol. Chem. 249, 3665-3671. 14. Traugh, J. A. & Sharp, S. B. (1977) J. Biol. Chem. 252, 37383744. 15. Housman, D., Jacobs-Lorena, M., Raj Bhandary, U. L. & Lodish, H. F. (1970) Nature 227,913-918. 16. Rhoads, R. E., McKnight, G. S. & Schimke, R. T. (1971) J. Biol.
Chem. 246,7407-7410. 17. Struck, D. K. & Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 18. Narita, K. & Ishii, J. (1962).J. Biochem. 52,367-373. 19. Yamamoto, S. & Lampen, J. 0. (1976) Proc. Natl. Acad, Sci. USA 73, 1457-1461. 20. Jackson R. & Hunter, T. (1970) Nature 227,672-676. 21. Palmiter, R. D. (1975) Cell 4, 189-197. 22. Prasad, C. & Peterkofsky, A. (1976) Arch. Biochem. Biophys. 175,
730-736.
Ovalbumin: A secreted protein without a transient hydrophobic leader sequence ("signal hypothesis"/acetylation/NH2-terminal sequence/cell-free translation) RICHARD D. PALMITER, JEAN GAGNON, AND KENNETH A. WALSH Department of Biochemistry, University of Washington, Seattle, Washington 98195
Communicated by Hans Neurath, October 11, 1977
Edman degradation. In contrast to these secretory proteins, ovalbumin synthesized in this cell-free system is the same as the polypeptide synthesized and secreted in vivo. This paper establishes the sequence of primary translation product in
Ovalbumin mRNA was translated in a reticuABSTRACT locyte lysate. The primary translation product starts with methionine derived from Met-tRNAf. When the nascent polypep tide is about 20 residues long, this methionine is removed. The new NH2-terminal glycine is acetylated from acetyl-CoA when the polypeptide is 44 residues long. The se uence of 35 residues at the NH2 terminus of ovalbumin was determined by automated Edman degradation after a method was devised to prevent acetylation during protein synthesis in the reticulocyte lysate. This sequence is the same as that of secreted ovalbumin and does not resemble the transient "signal peptides" associated with most secretory proteins, including three other egg white proteins synthesized in the same cells as ovalbumin.
vitro.
MATERIALS A-ND METHODS Lysates. Rabbit reticulocyte lysates were prepared as before (5); for some experiments lysates were treated with staphylococcal nuclease (Worthington) (6) and passed through a Sephadex G-50 column to remove small molecules (1). Incubation conditions were essentially as before (1). For the preparations of unblocked ovalbumin, citrate synthase (Sigma Chemical Co., 10-25 units/ml) and oxaloacetate (1 mM) were included in the reaction mixture (7). mRNA and tRNA. For most studies, total hen polysomal RNA was used as a source of ovalbumin mRNA (mRNA,).' For studies requiring purified mRNA, the above RNA preparation was passed through oligo(dT)-cellulose twice and the mRNA fraction was sedimented twice on sucrose gradients (8). The peak mRNAO, activity fractions were >95% pure, judging from sodium dodecyl sulfate/acrylamide gel analysis of the total translation product. [a5S]Met-tRNA et and [35S]Met-tRNAmet were prepared from rabbit reticulocyte lysates and purified on BD-cellulose (1). Formyl-135S]Met-tRNA'et was prepared by incubating rabbit tRNA in an Escherichia coli extract with 0.1 mM leucovorin as a formyl donor (9). Sequence Determination. After incubation, radioactive ovalbumin was isolated by immunoprecipitation (1), dissolved in 300 ,ul of 10% HOAc,-and placed into the cup of a Beckman sequenator, model 890B. a-N-Formylovalbumin was deformylated by dissolving the immunoprecipitate and 1 mg of carrier ovalbumin in 300 Ail of 3 M HCl and incubating for 2 hr at 370 (10); after incubation it was placed directly into the sequenator cup. Ovalbumin-Synthesizing Polysomes. In vivo: oviduct tissue (1.2 g) from estrogen-treated chicks was incubated in 3 ml of Hanks' salt solution for 5 min with 200 ,Ci of [35S]methionine and 300 ,uCi of [3H]phenylalanine (11); the reaction was stopped by addition of cycloheximide to 10 ,M and quick cooling. Ribosomes were isolated by Mg2+ precipitation (12) and resuspended, and the ovalbumin-synthesizing polysomes from 68 A260 units were precipitated by indirect immunoprecipitation (13). In vitro: ovalbumin-synthesizing polysomes were isolated from the nuclease-treated reticulocyte lysate by incubating the lysate for 6 min with purified mRNA., and then stopping the reaction with 10 ,uM cycloheximide and 10 A260 units of carrier oviduct polysomes. The polysomes were centrifuged through a 0.5-1.0 M sucrose gradient for 200 min at 27,000 X
The mechanisms by which proteins pass through cellular membranes are poorly understood. Transmembrane transport of polypeptides is encountered in various situations, including the entrance of toxic peptides, such as diphtheria toxin, abrin, ricin, and some colicins into cells, the transfer of proteins from one cellular compartment to another, and the export of secretory proteins. In each case there are presumably specific membrane receptors that recognize the proteins to be transported and direct them through the membrane. In protein secretion, the initial events may involve the interaction of the nascent polypeptide with a hypothetical membrane receptor located in the endoplasmic reticulum. If this event is coupled with ribosome attachment to the membrane, then the energy of polypeptide elongation could provide the motive force necessary to drive the polypeptide through the membrane. Folding of the polypeptide on the other side of the membrane might effectively ensure unidirectional transport. Secreted proteins are typically synthesized as precursors containing 15-30 additional NH2-terminal residues. The current list includes over 20 examples, including immunoglobulin L and H chains, serum albumin, pancreatic enzymes, mellitin, and several polypeptide hormones, as well as a bacterial lipoprotein (1, 2). These sequences are unusually rich in hydrophobic residues (typically 85-95%) and are usually cleaved during membrane transport. They have been termed "signal peptides" because of their presumed recognition by membrane receptors (3). We are studying the synthesis and secretion of egg white proteins in the chicken oviduct. Lysozyme, ovomucoid, and conalbumin contain typical signal peptides of 18, 23, and 19 residues, respectively (1, 4). The precursor forms of these proteins were obtained by translating their respective mRNAs in a cell-free system derived from rabbit reticulocytes; the translation products were isolated by immunoprecipitation and their amino acid sequences were determined by automated 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. §1734 solely to indicate this fact.
Abbreviation: mRNA.,, ovalbumin mRNA. 94
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Proc. Natl. Acad. Sci. USA 75 (1978) !.
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FIG. 1. Kinetics of leucine and acetate incorporation into immunoprecipitable ovalbumin. A regular lysate (4.2 ml) was incubated with oviduct polysomal RNA (950 ,ug) with [14C]leucine (7.5 jCi) and [3H]acetyl-CoA (40 uCi). At the indicated times aliquots were removed to measure radioactivity in completed ovalbumin molecules by immunoprecipitation (0). A 54-fold excess (650 MM) of unlabeled acetyl-CoA was added to part of the reaction mixture at 15 min (M); cycloheximide was added to another aliquot at 21 min (0).
The bottom half of the gradient, containing the polysomes, was layered over 15 ml of 1.0 M sucrose and centrifuged for 10 hr at 25,000 X g in the SW 27 rotor. The pellet was suspended in 2 mM EDTA and placed into the seg in an SW 27 rotor.
quenator.
Size Determination of Nascent Ovalbumin Polypeptides. A nuclease-treated lysate was incubated with 50 jCi of [3H]tyrosine or 50 ,uCi of [3H]acetyl-CoA for 7 min; then 1.6 ,ug of purified mRNA., was added for 7 min. The reaction was stopped with cycloheximide. Ribosomes were isolated by ultracentrifugation, suspended in 8 M urea/0. 1 M NaOAc at pH 5.7, and applied to a DEAE-cellulose column (0.5 X 0.8 cm) equilibrated with the same buffer. After the column was washed with 0.1 M NaCI, the peptidyl-tRNA was eluted with 0.5 M NaCl in the same buffer. The polypeptide was cleaved from tRNA by addition of Na2CO3 to 0.3 M (pH 10.5) and incubation for 1 hr at 37°. The sample was then acidified, combined with marker CNBr fragments of whale myoglobin, and applied to a 0.9 X 32-cm column of Sephadex G-50 (fine) overlaid with a 14-cm column of G-25 (fine) and equilibrated with 8M urea/1.8 M HOAc. Fractions (450,ul) were collected and radioactivity was determined in Aquasol.
RESULTS Incorporation of Acetate into Ovalbumin from AcetylCoA. Preliminary attempts at Edman degradation of ovalbumin synthesized in the reticulocyte lysate were unsuccessful. We suspected that the molecules were blocked by acetylation, as they are in vivo, since reticulocyte lysates have a ribosomeassociated transacetylase (14). Fig. 1 shows that [3H]acetate and ["4C]leucine are incorporated into ovalbumin with similar kinetics when a rabbit reticulocyte is incubated with mRNAO,. There is a 10- to 12-min lag before either isotope is immunoprecipitated; this corresponds to the time required to synthesize a complete ovalbumin molecule under these assay conditions. Approximately 1.4 mol of acetate is incorporated from acetyl-CoA per mol of ovalbumin synthesized. Fig. 1 shows that inhibition of polypeptide elongation with 10 ,M
FIG. 2. Minimum size of nascent ovalbumin polypeptides containing [3H]acetate or [3H]tyrosine. (A) Ovalbumin nascent polypeptides, isolated as described in Materials and Methods, were combined with CNBr fragments of whale myoglobin (-.3 mg) and separated on a Sephadex column. Fragments a, b, and c (dotted lines) correspond to the internal standards containing 76, 55, and 22 residues, respectively. Acetyl-CoA elutes in fraction 55. (B) The logarithm of polypeptide length is plotted against fraction number. The minimum size of acetate- and tyrosine- containing peptides was determined by subtracting 4.5 fractions from the extrapolation of the trailing edge of the radioactive profiles shown in (A).
cycloheximide immediately stops both [3H]acetate and ["'CIleucine incorporation into ovalbumin, suggesting that acetate incorporation is tightly coupled with polypeptide elongation. In contrast, addition of a 54-fold excess of unlabeled acetyl-CoA at 15 min of incubation has no effect on incorporation of [3H]acetate into immunoprecipitable ovalbumin for about 10 mt, and has no effect on ["aClbeucine incorporation at any time. These results are consistent with the incorporation of [3H]acetate into the NH2 terminus of the nascent ovalbumin polypeptides within the first 2 min of ribosome transit along mRNAct. The effect of unlabeled acetyl-CoA is delayed because only the polypeptides released from the ribosomes are immunoprecipitated under these conditions, and it takes 10 mi to chase all the 3H-labeled ovalbumin chains from the polysomes.
Time of Acetylation. In order to determine more accurately when the ovalbumin nascent chains are acetylated, we measured the size of the smallest nascent polypeptide chains bearing [3H]acetate. Purified mRNA0v was translated in a nucleasetreated reticulocyte lysate for 7 min and the nascent ovalbumin polypeptides were isolated and separated on a Sephadex column with internal molecular weight standards (Fig. 2A). The trailing edge of the absorbance peaks extrapolate to a point on the abscissa 4.5 fractions to the right of the peak. This value was subtracted from the extrapolation of the [3H]acetate curve and plotted in Fig. 2B on the calibration curve. A minimum value of 44 residues for acetylated nascent chains was obtained. As a check on the methodology, [3H]tyrosine-labeled nascent chains were treated identically (Fig. 2A). In this case the minimum ovalbumin nascent chain bearing [3H]tyrosine is estimated to be 29 residues, a value in agreement with the location of the first tyrosine residue (Table 1). NH2-Terminal Sequence of Unblocked Ovalbumin. in order to determine the amino acid sequence of ovalbumin, we sought conditions that would prevent acetylation. We found that we could metabolize the endogenous pool of acetyl-CoA by adding citrate synthase and 1 mM oxaloacetate. Under these conditions the incorporation of [3H]acetate into ovalbumin can be reduced to less than 10% of controls (7). Using this metabolic trap, we were able to synthesize ovalbumin molecules whose amino acid sequence could be determined by Edman degradation. Ovalbumin was synthesized with each of the 20 radioactive amino acids in separate reactions. The sequence of these samples was then determined in-
96
Biochemistry: Palmiter et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Amino acid sequence of the primary translation product from mRNAov*
1.5
Ac NH2-Met-Gly- Ser- Ile -Gly- Ala -Ala-Ser-Met-Glu- Phe- Cys-Phe-Asp- Val-Phe10 15 -1 +1 5 Lys-Glu-Leu- Lys- Val - His-His- Ala -Asn-Glu-Asn- Ile - Phe-Tyr-Cys20 25 30 Pro- Ile- X - Ile -Met- X - X -Leu35
1.0
* The primary translation product starts with Met (-1); the product isolated from the reticulocyte lysate after translation of mRNAo, in the presence of citrate synthase and oxaloacetate starts with Gly (+1); the product isolated from egg white starts with AcGly. X denotes an unidentified residue.
dividually, in pairs with different isotopes, or in combinations of up to 13 different amino acids that were subsequently separated by high-pressure liquid chromatography. Fig. 3 shows representative sequence data that establish the sequence of the first nine residues of ovalbumin. The entire NH2-terminal sequence derived by this procedure is shown in Table 1. Based on this information, we isolated and determined the sequence of CNBr peptides from unlabeled ovalbumin corresponding to residues 1-8 and 9-35. The sequence of these peptides is identical to that of ovalbumin synthesized in the reticulocyte lysate. Primary Translation Product from mRNAO,. Because all secreted proteins examined thus far have a hydrophobic leader sequence about 20 residues long that is cleaved in vivo, but not in vitro, we were surprised not to find a similar situation with ovalbumin. Perhaps the reticulocyte lysate contains a protease that cleaves a "signal peptide" from ovalbumin but not from other secretory proteins. Since the product synthesized in vitro begins with glycine, there must have been at least one proteolytic event (to remove initiator methionine) prior to our isolation of ovalbumin. Thus, it was essential to identify the sequence containing NH2-terminal methionine from initiator tRNA. We used two independent approaches to characterize this primary translation product. Both give the same result: the primary translation product is one residue longer than the unblocked in vitro product and that residue is methionine donated by Met-tRNAfet
The first approach relies on the fact that formyl-methionine 2
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FIG. 3. Representative sequence data for ovalbumin translated in reticulocyte lysates in the presence of citrate synthase and oxaloacetate. Hen polysomal RNA was translated in 1-2 ml of reaction mixtures with 100-200 ACi of the indicated amino acids. Glycine, alanine, and glutamic acid were part of a multiple label experiment and were separated by high-pressure liquid chromatography. [35S]Methionine and [3H]isoleucine were combined in one experiment; [3H]serine was part of a double-label experiment.
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FIG. 4. Edman degradation of ovalbumin synthesized with
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subjected to Edman degradation directly; (C and D) the other half was treated with 3 M HCl for 2 hr at 370 to remove the formyl group. The yield after acid treatment was about 40%.
is not cleaved from polypeptides by eukaryotic methionine aminopeptidase (15). Oviduct mRNA was translated (30 min) in the presence of f[-5S]Met-tRNAM'et and [3H]alanine. Ovalbumin was isolated by immunoprecipitation and its sequence was determined with and without acidic deformylation. Without deformylation, all the ovalbumin molecules were blocked; there was no evidence of MS or 3H in any of the first eight cycles of the Edman degradation (Fig. 4, 'Ai and B). However, after 2 hr of treatment of the immunoprecipitate in 3 M HC1 at 370, Edman degration yielded [-5S]methionine in position 1 and a doublet of [3H]alanine at positions 6 and 7 (Fig. 4 C and D). The characteristic alanine doublet is displaced one cycle compared to the sequence obtained by preventing acetylation (Fig. 3), as would be expected if the ovalbumin molecules were one residue longer. The second approach for detecting the primary translation product takes advantage of the early acetylation of nascent ovalbumin molecules. Purified mRNAO, was translated in a mRNA-dependent lysate for a brief period (6 min; about 1/2 of a ribosome transit time); the ovalbumin-synthesizing polysomes generated in this time contained three to six ribosomes and were separated from the bulk of the monoribosomes and any released peptides by ultracentrifugation. The polysome pellet was then subjected to Edman degradation. Since most of the ovalbumin molecules are acetylated, only the sequence of those chains shorter than 44 residues long can be determined. We expected two sequences: one beginning with methionine and one with glycine. The displacement of these two sequences during Edman degradation indicates the number of residues between initiator metbionine and the new NH2-terminal glycine while their abundance indicates the lengths of the nascent chains when methionine is cleaved and acetylation takes place. When mRNAO, was translated in the presence of [s5S]methionine, we noted radioactive peaks at cycles 1, 8, and 9. Just prior to acetylation, ovalbumin should have methionine at position 8 that would be accessible to Edman degradation. The peaks at positions 1 and 9 were related to each other by a reasonable step-wise yield and might represent an ovalbumin molecule extended at the NH2 terminus by one methionine residue. In order to ascertain the source of the methionine in positions 1, 8, and 9 of ovalbumin, we labeled ovalbumin nascent chains separately with methionine from [&iS]Met-tRNAfeM or [-`SIMet-tRNAMet. Fig. 5 upper shows that the methionine in position 1 is derived from Met-tRNArmet, whereas those at positions 8 and 9 are from Met-tRNAet. These experiments also
Biochemistry:
Palmiter et al.
Met-tRNAf 2000
Proc. Natl. Acad. Sci. USA 75 (1978)
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300
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Sequenator cycle
FIG. 5. Edman degradation of ovalbumin nascent chains synthesized in lysates supplemented with [35S]Met-tRNAfet, [35S]Met-tRNAMet, [3H]isoleucine, or [3H]glutamate. Nuclease- and Sephadex G-50-treated lysates (250 IAl) were incubated for 6 min with purified mRNAov and the indicated labeled amino acid or acyl-tRNA. Ovalbumin-synthesizing polysomes were isolated as described in Materials and Methods and subjected to Edman degradation.
included -3H]isoleucine, which was detected at positions 3 and 4 in equal abundance (Fig. 5), whereas the normal lysate product has isoleucine only in position 3 (Fig. 3). In another experiment we labeled ovalbumin with [3H]leucine, [3H]tyrosine, and [3H]glutamate. Fig. 5 shows glutamate residues at positions 9 and 10 (rather than 9 only, Fig. 3), again with nearly equal abundance. There were no discernible peaks with either leucine or tyrosine. Primary Ovalbumin Translation Product In Vivo. In order to ascertain whether the translation of ovalbumin follows the same pathway in vivo, we labeled oviduct explants with [a5S]methionine and [3H]phenylalanine for 5 min, then isolated the ovalbumin-synthesizing polysomes by immunoprecipitation. This procedure accomplished the same goal as the previous method, but in this case the polysomes were purified after translation whereas in the previous method the mRNA was purified before translation. Fig. 6 shows the pattern of '5S and 3H released during successive cycles of Edman degradation. Methionine residues were released in cycles 1, 8, and 9. This pattern of methionine release and the relative abundance of methionine in cycles 8 and 9 is the same as that observed with ovalbumin synthesized in vitro. The pattern of [3H]phenylalanine release (Fig. 6B) is consistent with a mixture of nonacetylated ovalbumin molecules with phenylalanine residues at positions 10, 12, and 15 (Table 1) and the primary translation product (with its extra methionine) with phenylalanine residues at positions 11, 13, and 16. The high background in these experiments is due to the fact that only 10% of the nascent chains are accessible to sequencing, the others being acetylated. DISCUSSION All the available evidence suggests that the ovalbumin polypeptide synthesized in the reticulocyte lysate is the same length as the molecule synthesized in intact tubular gland cells. When analyzed on sodium dodecyl sulfate/acrylamide gels, the in vitro translation product actually migrates slightly faster than ovalbumin synthesized in vivo (16); this disparity is undoubtedly due to the lack of glycosylation in vitro, since ovalbumin synthesized in vio in the presence of tunicamycin, an inhibitor of glycosylation, also migrates slightly faster than controls (17). Moreover, comparison of peptide maps of ovalbumin synthe-
U
0
0 2 4 6 8 10 12 14 16 18 Sequenator 'yI
FIG. 6. Edman degradation of ovalbumin nascent chains labeled in vivo. Explants of oviduct tissue were labeled for 5 min and ovalbumin-synthesizing polysomes were isolated by immunoprecipitation. The recovery of (A) methionine and (B) phenylalanine during Edman degradation is shown. (Inset) A confirming experiment with [35S]methionine.
sized in vivo and in vitro reveals no extra peptides from the in vitro product (16). Our sequence of the NH2 terminus of ovalbumin (Fig. 3 and Table 1) is identical to that of ovalbumin isolated from egg white (J. Gagnon, R. Palmiter, and K. Walsh, unpublished data). The residues in positions 3 and 4 are reversed compared to the sequence reported by Narita and Ishii (18). We have confirmed our alignment on four occasions, and thus feel confident that it is correct. According to the "signal hypothesis," the first step in secretion involves the recognition of the hydrophobic leader sequence of the nascent polypeptide by membrane receptors. This recognition leads to membrane transport, which is followed by proteolytic removal of the "signal peptide" (3). At first we thought that the ovalbumin could have a typical hydrophobic leader sequence that is recognized by a proteolytic enzyme in the cell-free system and hence is cleaved in vitro as well as in vivo. However, we performed two different types of experiments to find the primary translation product in vitro, and in each case concluded that the product is only one residue longer than that of the secreted protein; the extra residue is methionine and it is derived from Met-tRNAf et. The same primary translation product was deduced with nascent ovalbumin chains obtained from intact cells. Thus, it is unlikely that the lack of a typical "signal peptide" is due to incorrect initiation in vitro. The finding that ovalbumin does not have a transient, hydrophobic leader sequence raises important questions about the mechanism of ovalbumin secretion, as well as the function of such sequences in other secretory proteins, including other egg white proteins secreted from the same cells as ovalbumin. There are several variations on the secretory pathway mentioned above that might be invoked to explain ovalbumin secretion. The simplest idea is that the NH2-terminal sequence of ovalbumin functions as a "signal": it may interact with membrane receptor in a manner analogous to other "signal peptides", but then not be cleaved during membrane transport. The NH2terminal sequence of ovalbumin shows a weak resemblance to the other hydrophobic "signal peptides"; 11 of the first 15 residues of ovalbumin are hydrophobic and the NH2 terminus
98
Biochemistry: Palmiter et al.
is blocked. However, when a longer, more typical sequence of 20 residues is considered, only 13 are hydrophobic and the hypothesis of an uncleaved signal peptide seems less likely. An intriguing possibility is that the transacetylase is itself the receptor. If this enzyme is associated with the endoplasmic reticulum in the oviduct, then in the process of acetylating the NH2 terminus of ovalbumin it may direct the nascent chain into a membrane secretory channel. If this is the case, then the hydrophobicity of the NH2-terminal sequence may be of secondary importance. There are several more remote possibilities that may be considered. One is that a hydrophobic "signal sequence" is located elsewhere within the molecule, rather than at the NH2 terminus. Alternatively, a "signal peptide" might be translated as a separate polypeptide from mRNAO,. This can tentatively be ruled out by our sequence data from the entire nascent-chain population derived from purified mRNA0v (Fig. 5). We did not observe a second sequence of specific activity comparable to that of ovalbumin, but we have not tried all 20 amino acids, and hence cannot definitively rule out this possibility. Another suggestion is that ovalbumin nascent chains may have some hydrophobic group covalently attached near the NH2 terminus (e.g., to a serine or cysteine residue) that could serve the function of a "signal" and subsequently be removed. This idea has not been adequately tested, but there is a precedent in the case of penicillinase, which has a fatty acid transiently attached at the NH2 terminus (19). In any event, it is clear that proteolysis of a "signal sequence" is not essential for membrane transport or secretion of ovalbumin. The timing of methionine aminopeptidase action and acetylation of ovalbumin can be deduced from the data presented here. By determining the sequence of all the ovalbumin polypeptides that are not blocked, the yield of radioactivity in position n+1 ranges from 75-100% of a yield in position n both in vitro (Fig. 5) and in vivo (Fig. 6). The radioactivity in position n+1 represents those nascent polypeptides that still retain the NH2-terminal methionine while that in position n represents those from which methionine has been cleaved. Thus, assuming that the rate of elongation is constant, the number of residues incorporated before methionine is removed is 75-100% of the number incorporated between that event and acetylation. The size of the smallest nascent ovalbumin polypeptides bearing acetate is 44 residues (Fig. 2). With this information we calculate that initiator methionine is removed when ovalbumin nascent chain is 19-22 residues long, and acetylation occurs when the peptide is 22-26 residues longer. Initiator methionine is removed from hemoglobin when these polypeptides are 15-20 residues long (20), suggesting that the timing of this event may be related to accessibility of the methionine aminopeptidase to the nascent chain. The rate of polypeptide elongation under our in vitro conditions is about 0.65 residues/sec; thus, acetylation takes place 68 sec after initiation. The rate of polypeptide elongation on mRNAO, in vivo is about 5 residues/sec (21). Hence, in vivo, the NH2-terminal methionine is cleaved 4 sec after initiation and acetylation of the new
Proc. Nati. Acad. Sci. USA 75 (1978)
NH2-terminal glycine takes place 4 sec later. This conclusion is contrary to a recent report suggesting that methionine is cleaved 1-2 min after ovalbumin synthesis in vivo (22). We conclude that there are at least two distinct mechanisms of protein secretion in the chick oviduct. Under optimal hormonal stimulation, ovalbumin is synthesized and secreted at a rate close to 106 molecules/min per tubular gland cell (21). Thus, if there are specific membrane receptors for secretion of different polypeptides, then the oviduct should be a rich source of those involved in ovalbumin secretion, as well as those thought to recognize the "signal peptides" on lysozyme, ovo-
mucoid, and conalbumin. We thank Linda Strimple for secretarial assistance, and Stephen Thibodeau for help in developing some of the methods. This work was funded by grants from the National Institutes of Health: HD-09172, GM-15731, and RR-05432. J.G. is supported by the Medical Research Council of Canada. R.D.P. is an investigator of the Howard Hughes Medical Institute. 1. Palmiter, R. D., Gagnon, J., Ericsson, L. H. & Walsh, K. A. (1977)
J. Biol. Chem. 252,6386-6393. 2. Inouye, S., Wang, S., Sekizawa, J., Halegoua, S. & Inouye, M. (1977) Proc. Natl. Acad. Sci. USA 74, 1004-1008. 3. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 852-862. 4. Palmiter, R. D., Thibodeau, S. N., Gagnon, J. & Walsh, K. A. (1978) in Proceedings of the 11th FEBS Meeting, Regulatory Proteolytic Enzymes and Their Inhibitors, eds. Magnussen, S., Otteson, M., Foltmann, B., Dano, K. & Neurath, H. (Pergamon Press, Oxford), in press. 5. Palmiter, R. D. (1973) J. Biol. Chem. 248,2095-2106. 6. Pehlam, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, 247-256. 7. Palmiter, R. D. (1978) J. Biol. Chem., in press. 8. Haines, M. E., Carey, N. H. & Palmiter, R. D. (1974) Eur. J. Biochem. 43, 549-560. 9. Stanley, W. M., Jr. (1972) Anal. Biochem. 48,202-216. 10. Elson, N. A., Brewer, H. B. & Anderson, W. F. (1974) J. Biol. Chem. 249, 5227-5235. 11. Palmiter, R. D., Palacios, R. & Schimke, R. T. (1972) J. Biol.
Chem. 247,3296-3304. 12. Palmiter, R. D. (1974) Biochemistry 13,3606--3615. 13. Shapiro, D. J., Taylor, J. M., McKnight, G. S., Palacios, R., Gonzalez, C., Kiely, M. L. & Schimke, R. T. (1974) J. Biol. Chem. 249, 3665-3671. 14. Traugh, J. A. & Sharp, S. B. (1977) J. Biol. Chem. 252, 37383744. 15. Housman, D., Jacobs-Lorena, M., Raj Bhandary, U. L. & Lodish, H. F. (1970) Nature 227,913-918. 16. Rhoads, R. E., McKnight, G. S. & Schimke, R. T. (1971) J. Biol.
Chem. 246,7407-7410. 17. Struck, D. K. & Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 18. Narita, K. & Ishii, J. (1962).J. Biochem. 52,367-373. 19. Yamamoto, S. & Lampen, J. 0. (1976) Proc. Natl. Acad, Sci. USA 73, 1457-1461. 20. Jackson R. & Hunter, T. (1970) Nature 227,672-676. 21. Palmiter, R. D. (1975) Cell 4, 189-197. 22. Prasad, C. & Peterkofsky, A. (1976) Arch. Biochem. Biophys. 175,
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