Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 94-98, January 1978 Biochemistry

Ovalbumin: A secreted 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

ABSTRACT mRNA was translated in a reticu- Edman degradation. In contrast to these secretory , locyte lysate. The primary translation product starts with me- ovalbumin synthesized in this cell-free system is the same as the thionine derived from Met-tRNAf. When the nascent polypep polypeptide synthesized and secreted in vivo. This paper es- tide is about 20 residues long, this methionine is removed. The new NH2-terminal glycine is acetylated from acetyl-CoA when tablishes the sequence of primary translation product in the polypeptide is 44 residues long. The se uence of 35 residues vitro. at the NH2 terminus of ovalbumin was determined by auto- mated Edman degradation after a method was devised to pre- MATERIALS A-ND METHODS vent acetylation during protein synthesis in the reticulocyte lysate. This sequence is the same as that of secreted ovalbumin Lysates. Rabbit reticulocyte lysates were prepared as before and does not resemble the transient "signal peptides" associated (5); for some experiments lysates were treated with staphylo- with most secretory proteins, including three other coccal nuclease (Worthington) (6) and passed through a Se- proteins synthesized in the same cells as ovalbumin. phadex G-50 column to remove small molecules (1). Incubation conditions were essentially as before (1). For the preparations The mechanisms by which proteins pass through cellular of unblocked ovalbumin, citrate synthase (Sigma Chemical Co., membranes are poorly understood. Transmembrane transport 10-25 units/ml) and oxaloacetate (1 mM) were included in the of polypeptides is encountered in various situations, including reaction mixture (7). the entrance of toxic peptides, such as diphtheria toxin, abrin, mRNA and tRNA. For most studies, total hen polysomal , and some colicins into cells, the transfer of proteins from RNA was used as a source of ovalbumin mRNA (mRNA,).' For one cellular compartment to another, and the export of secre- studies requiring purified mRNA, the above RNA preparation tory proteins. In each case there are presumably specific was passed through oligo(dT)-cellulose twice and the mRNA membrane receptors that recognize the proteins to be trans- fraction was sedimented twice on sucrose gradients (8). The ported and direct them through the membrane. In protein se- peak mRNAO, activity fractions were >95% pure, judging from cretion, the initial events may involve the interaction of the sodium dodecyl sulfate/acrylamide gel analysis of the total nascent polypeptide with a hypothetical membrane receptor translation product. [a5S]Met-tRNA et and [35S]Met-tRNAmet located in the endoplasmic reticulum. If this event is coupled were prepared from rabbit reticulocyte lysates and purified on with ribosome attachment to the membrane, then the energy BD-cellulose (1). Formyl-135S]Met-tRNA'et was prepared by of polypeptide elongation could provide the motive force incubating rabbit tRNA in an Escherichia coli extract with 0.1 necessary to drive the polypeptide through the membrane. mM leucovorin as a formyl donor (9). Folding of the polypeptide on the other side of the membrane Sequence Determination. After incubation, radioactive might effectively ensure unidirectional transport. ovalbumin was isolated by immunoprecipitation (1), dissolved Secreted proteins are typically synthesized as precursors in 300 ,ul of 10% HOAc,-and placed into the cup of a Beckman containing 15-30 additional NH2-terminal residues. The cur- sequenator, model 890B. a-N-Formylovalbumin was de- rent list includes over 20 examples, including immunoglobulin formylated by dissolving the immunoprecipitate and 1 mg of L and H chains, serum , pancreatic enzymes, mellitin, carrier ovalbumin in 300 Ail of 3 M HCl and incubating for 2 and several polypeptide hormones, as well as a bacterial lipo- hr at 370 (10); after incubation it was placed directly into the protein (1, 2). These sequences are unusually rich in hydro- sequenator cup. phobic residues (typically 85-95%) and are usually cleaved Ovalbumin-Synthesizing Polysomes. In vivo: oviduct tissue during membrane transport. They have been termed "signal (1.2 g) from estrogen-treated chicks was incubated in 3 ml of peptides" because of their presumed recognition by membrane Hanks' salt solution for 5 min with 200 ,Ci of [35S]methionine receptors (3). and 300 ,uCi of [3H]phenylalanine (11); the reaction was stopped We are studying the synthesis and secretion of egg white by addition of cycloheximide to 10,M and quick cooling. Ri- proteins in the chicken oviduct. Lysozyme, , and bosomes were isolated by Mg2+ precipitation (12) and resus- contain typical signal peptides of 18, 23, and 19 pended, and the ovalbumin-synthesizing polysomes from 68 residues, respectively (1, 4). The precursor forms of these pro- A260 units were precipitated by indirect immunoprecipitation teins were obtained by translating their respective mRNAs in (13). In vitro: ovalbumin-synthesizing polysomes were isolated a cell-free system derived from rabbit reticulocytes; the from the nuclease-treated reticulocyte lysate by incubating translation products were isolated by immunoprecipitation and the lysate for 6 min with purified mRNA., and then stopping their sequences were determined by automated the reaction with 10 ,uM cycloheximide and 10 A260 units of carrier oviduct polysomes. The polysomes were centrifuged The costs of publication of this article were defrayed in part by the X payment of page charges. This article must therefore be hereby marked through a 0.5-1.0 M sucrose gradient for 200 min at 27,000 "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: mRNA.,, ovalbumin mRNA. 94 Downloaded by guest on October 2, 2021 Biochemistry: Palmiter et al. Proc. Natl. Acad. Sci. USA 75 (1978) 95

A !. A -1 E 400 'p I 602 C .; \b Cyclo- 2 i co 'Acetate 200 heximide

-J Acetyl-.X CoA , dues,respectively. 0.2tylA5lin fraction0 OTyr I 0 I- ~~~~~20- 020 30 5060 20 30 40 50 Fraction FIG. 2. Minimum size of nascent ovalbumin polypeptides con- taining [3H]acetate or [3H]tyrosine. (A) Ovalbumin nascent poly- peptides, 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 resi- dues, respectively. Acetyl-CoA elutes in fraction 55. (B) The logarithm 0 20 40 of polypeptide length is plotted against fraction number. The mini- Minutes mum size of acetate- and tyrosine- containing peptides was deter- FIG. 1. Kinetics of leucine and acetate incorporation into im- mined by subtracting 4.5 fractions from the extrapolation of the munoprecipitable ovalbumin. A regular lysate (4.2 ml) was incubated trailing edge of the radioactive profiles shown in (A). 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 cycloheximide immediately stops both [3H]acetate and ["'CI- to measure radioactivity in completed ovalbumin molecules by im- leucine incorporation into ovalbumin, suggesting that acetate munoprecipitation (0). A 54-fold excess (650 MM) of unlabeled ace- incorporation is tightly coupled with polypeptide elongation. tyl-CoA was added to part of the reaction mixture at 15 min (M); cy- In addition of a 54-fold excess of unlabeled acetyl-CoA cloheximide was added to another aliquot at 21 min (0). contrast, at 15 min of incubation has no effect on incorporation of [3H]acetate into immunoprecipitable ovalbumin for about 10 g in an 27 rotor. bottom half of the SW The gradient, containing mt, and has no effect on ["aClbeucine incorporation at any was over 15 ml of 1.0 M sucrose the polysomes, layered and time. These results are consistent with the incorporation of centrifuged for 10 hr at 25,000 X g in the SW 27 rotor. The [3H]acetate into the NH2 terminus of the nascent ovalbumin pellet was suspended in 2 mM EDTA and placed into the se- polypeptides within the first 2 min of ribosome transit along quenator. mRNAct. The effect of unlabeled is Determination of Nascent Ovalbumin acetyl-CoA delayed be- Size Polypeptides. cause only the polypeptides released from the ribosomes are A nuclease-treated lysate was incubated with 50 jCi of [3H]- immunoprecipitated under these conditions, and it takes 10 mi tyrosine or 50 ,uCi of [3H]acetyl-CoA for 7 min; then 1.6 ,ug of to chase all the 3H-labeled ovalbumin chains from the poly- purified mRNA., was added for 7 min. The reaction was somes. stopped with cycloheximide. Ribosomes were isolated by ul- Time of Acetylation. In order to determine more accurately in 8 M 1 M at tracentrifugation, suspended urea/0. NaOAc pH when the ovalbumin nascent chains are acetylated, we mea- 5.7, and applied to a DEAE-cellulose column (0.5 X 0.8 cm) sured the size of the smallest nascent polypeptide chains bearing equilibrated with the same buffer. After the column was [3H]acetate. Purified mRNA0v was translated in a nuclease- washed with 0.1 M NaCI, the peptidyl-tRNA was eluted with treated reticulocyte lysate for 7 min and the nascent ovalbumin 0.5 M NaCl in the same buffer. The polypeptide was cleaved polypeptides were isolated and separated on a Sephadex column from tRNA by addition of Na2CO3 to 0.3 M (pH 10.5) and in- with internal molecular weight standards (Fig. 2A). The trailing cubation for 1 hr at 37°. The sample was then acidified, com- of the absorbance to a on the bined with marker CNBr of whale and edge peaks extrapolate point ab- fragments myoglobin, scissa 4.5 fractions to the right of the peak. This value was to a 0.9 X 32-cm column of G-50 ov- applied Sephadex (fine) subtracted from the extrapolation of the [3H]acetate curve and with a 14-cm column of G-25 and with erlaid (fine) equilibrated plotted in Fig. 2B on the calibration curve. A minimum value 8M M HOAc. Fractions were collected and urea/1.8 (450,ul) of 44 residues for acetylated nascent chains was obtained. As radioactivity was determined in Aquasol. a check on the methodology, [3H]tyrosine-labeled nascent chains were treated identically (Fig. 2A). In this case the min- RESULTS imum ovalbumin nascent chain bearing [3H]tyrosine is esti- Incorporation of Acetate into Ovalbumin from Acetyl- mated to be 29 residues, a value in agreement with the location CoA. Preliminary attempts at Edman degradation of ovalbu- of the first tyrosine residue (Table 1). min synthesized in the reticulocyte lysate were unsuccessful. NH2-Terminal Sequence of Unblocked Ovalbumin. in We suspected that the molecules were blocked by acetylation, order to determine the amino acid sequence of ovalbumin, we as they are in vivo, since reticulocyte lysates have a ribosome- sought conditions that would prevent acetylation. We found associated transacetylase (14). Fig. 1 shows that [3H]acetate and that we could metabolize the endogenous pool of acetyl-CoA ["4C]leucine are incorporated into ovalbumin with similar ki- by adding citrate synthase and 1 mM oxaloacetate. Under these netics when a rabbit reticulocyte is incubated with mRNAO,. conditions the incorporation of [3H]acetate into ovalbumin can There is a 10- to 12-min lag before either isotope is immu- be reduced to less than 10% of controls (7). noprecipitated; this corresponds to the time required to syn- Using this metabolic trap, we were able to synthesize oval- thesize a complete ovalbumin molecule under these assay bumin molecules whose amino acid sequence could be deter- conditions. Approximately 1.4 mol of acetate is incorporated mined by Edman degradation. Ovalbumin was synthesized from acetyl-CoA per mol of ovalbumin synthesized. Fig. 1 with each of the 20 radioactive amino acids in separate reac- shows that inhibition of polypeptide elongation with 10,M tions. The sequence of these samples was then determined in- Downloaded by guest on October 2, 2021 96 Biochemistry: Palmiter et al. Proc. Natl. Acad. Sci. USA 75 (1978)

Table 1. Amino acid sequence of the primary translation product 1.5 from mRNAov* Ac 1.0 NH2-Met-Gly- Ser- Ile -Gly- Ala -Ala-Ser-Met-Glu- Phe- Cys-Phe-Asp- Val-Phe- -1 +1 5 10 15 00.5 Lys-Glu-Leu- Lys- Val - His-His- Ala -Asn-Glu-Asn- Ile -Phe-Tyr-Cys- 0 20 25 30 0 Pro- Ile- X - Ile -Met- X - X -Leu- x 1.5 35 a.E 0 * The primary translation product starts with Met (-1); the product 1.0 isolated from the reticulocyte lysate after translation of mRNAo, in the presence of citrate synthase and oxaloacetate starts with Gly 0.5 (+1); the product isolated from egg white starts with AcGly. X de- notes an unidentified residue. 0 2 4 6 8 2 4 6 8 Sequenator cycle isotopes, or in combinations dividually, in pairs with different FIG. 4. Edman degradation of ovalbumin synthesized with of up to 13 different amino acids that were subsequently sep- f[35S]Met-tRNAM`et and [3H]alanine. (A and B) Half the sample was arated by high-pressure liquid chromatography. Fig. 3 shows subjected to Edman degradation directly; (C and D) the other half representative sequence data that establish the sequence of the was treated with 3 M HCl for 2 hr at 370 to remove the formyl group. first nine residues of ovalbumin. The entire NH2-terminal se- The yield after acid treatment was about 40%. quence derived by this procedure is shown in Table 1. Based on this information, we isolated and determined the sequence is not cleaved from polypeptides by eukaryotic methionine of CNBr peptides from unlabeled ovalbumin corresponding aminopeptidase (15). Oviduct mRNA was translated (30 min) to residues 1-8 and 9-35. The sequence of these peptides is in the presence of f[-5S]Met-tRNAM'et and [3H]alanine. Oval- identical to that of ovalbumin synthesized in the reticulocyte bumin was isolated by immunoprecipitation and its sequence lysate. was determined with and without acidic deformylation. Primary Translation Product from mRNAO,. Because all Without deformylation, all the ovalbumin molecules were secreted proteins examined thus far have a hydrophobic leader blocked; there was no evidence of MS or 3H in any of the first sequence about 20 residues long that is cleaved in vivo, but not eight cycles of the Edman degradation (Fig. 4, 'Ai and B). in vitro, we were surprised not to find a similar situation with However, after 2 hr of treatment of the immunoprecipitate in ovalbumin. Perhaps the reticulocyte lysate contains a protease 3 M HC1 at 370, Edman degration yielded [-5S]methionine in that cleaves a "" from ovalbumin but not from position 1 and a doublet of [3H]alanine at positions 6 and 7 (Fig. other secretory proteins. Since the product synthesized in vitro 4 C and D). The characteristic alanine doublet is displaced one begins with glycine, there must have been at least one proteo- cycle compared to the sequence obtained by preventing acet- lytic event (to remove initiator methionine) prior to our isolation ylation (Fig. 3), as would be expected if the ovalbumin mole- of ovalbumin. Thus, it was essential to identify the sequence cules were one residue longer. containing NH2-terminal methionine from initiator tRNA. We The second approach for detecting the primary translation used two independent approaches to characterize this primary product takes advantage of the early acetylation of nascent translation product. Both give the same result: the primary ovalbumin molecules. Purified mRNAO, was translated in a translation product is one residue longer than the unblocked mRNA-dependent lysate for a brief period (6 min; about 1/2 of in vitro product and that residue is methionine donated by a ribosome transit time); the ovalbumin-synthesizing polysomes Met-tRNAfet generated in this time contained three to six ribosomes and were The first approach relies on the fact that formyl-methionine separated from the bulk of the monoribosomes and any released peptides by ultracentrifugation. The polysome pellet was then 2 subjected to Edman degradation. Since most of the ovalbumin Glcne 8'Aane 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 20- Methionine glycine. The displacement of these two sequences during E 2 Glutmmip indicates the number of residues between o_ acid Edman degradation 10 initiator metbionine and the new NH2-terminal glycine while X their abundance indicates the lengths of the nascent chains a.E o h~ 'Io when methionine is cleaved and acetylation takes place. When mRNAO, was translated in the presence of [s5S]me- thionine, 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 2 4 6 8 10 246 810 peaks at positions 1 and 9 were related to each other by a rea- Sequenator cycle sonable step-wise yield and might represent an ovalbumin FIG. 3. Representative sequence data for ovalbumin translated molecule extended at the NH2 terminus by one methionine in reticulocyte lysates in the presence of citrate synthase and oxa- residue. In order to ascertain the source of the methionine in loacetate. Hen polysomal RNA was translated in 1-2 ml of reaction positions 1, 8, and 9 of ovalbumin, we labeled ovalbumin nas- mixtures with 100-200 ACi of the indicated amino acids. Glycine, al- with methionine from anine, and glutamic acid were part of a multiple label experiment and cent chains separately [&iS]Met-tRNAfeM were separated by high-pressure liquid chromatography. [35S]Me- or [-`SIMet-tRNAMet. Fig. 5 upper shows that the methionine thionine and [3H]isoleucine were combined in one experiment; in position 1 is derived from Met-tRNArmet, whereas those at [3H]serine was part of a double-label experiment. positions 8 and 9 are from Met-tRNAet. These experiments also Downloaded by guest on October 2, 2021 Biochemistry: Palmiter et al. Proc. Natl. Acad. Sci. USA 75 (1978) 97

Met-tRNAf Met-tRNAm 2000 1600-

1000 300 '6 6 0 X 2 4 6108 E 0 0 Ee4 e~8 X soieucine 150 Glutamic acid U 30 20 2 10 200 34 0 6B 100- 6

0 0 0 24 68 10 0 24 6 810 Sequenator cycle FIG. 5. Edman degradation of ovalbumin nascent chains syn- U thesized in lysates supplemented with [35S]Met-tRNAfet, 0 [35S]Met-tRNAMet, [3H]isoleucine, or [3H]glutamate. Nuclease- and 0 2 4 6 8 10 12 14 16 18 Sephadex G-50-treated lysates (250 IAl) were incubated for 6 min with Sequenator 'yI purified mRNAov and the indicated labeled amino acid or acyl-tRNA. FIG. 6. Edman degradation of ovalbumin nascent chains labeled Ovalbumin-synthesizing polysomes were isolated as described in in vivo. Explants of oviduct tissue were labeled for 5 min and oval- Materials and Methods and subjected to Edman degradation. bumin-synthesizing polysomes were isolated by immunoprecipitation. The recovery of (A) methionine and (B) phenylalanine during Edman included -3H]isoleucine, which was detected at positions 3 and degradation is shown. (Inset) A confirming experiment with [35S]- 4 in equal abundance (Fig. 5), whereas the normal lysate methionine. product has isoleucine only in position 3 (Fig. 3). In another sized in vivo and in vitro reveals no extra peptides from the in experiment we labeled ovalbumin with [3H]leucine, [3H]ty- vitro product (16). Our sequence of the NH2 terminus of rosine, and [3H]glutamate. Fig. 5 shows glutamate residues at ovalbumin (Fig. 3 and Table 1) is identical to that of ovalbumin positions 9 and 10 (rather than 9 only, Fig. 3), again with nearly isolated from egg white (J. Gagnon, R. Palmiter, and K. Walsh, equal abundance. There were no discernible peaks with either unpublished data). The residues in positions 3 and 4 are reversed leucine or tyrosine. compared to the sequence reported by Narita and Ishii (18). We Primary Ovalbumin Translation Product In Vivo. In order have confirmed our alignment on four occasions, and thus feel to ascertain whether the translation of ovalbumin follows the confident that it is correct. same pathway in vivo, we labeled oviduct explants with According to the "signal hypothesis," the first step in secretion [a5S]methionine and [3H]phenylalanine for 5 min, then isolated involves the recognition of the hydrophobic leader sequence the ovalbumin-synthesizing polysomes by immunoprecipita- of the nascent polypeptide by membrane receptors. This rec- tion. This procedure accomplished the same goal as the previous ognition leads to membrane transport, which is followed by method, but in this case the polysomes were purified after proteolytic removal of the "signal peptide" (3). At first we translation whereas in the previous method the mRNA was thought that the ovalbumin could have a typical hydrophobic purified before translation. Fig. 6 shows the pattern of '5S and leader sequence that is recognized by a proteolytic enzyme in 3H released during successive cycles of Edman degradation. the cell-free system and hence is cleaved in vitro as well as in Methionine residues were released in cycles 1, 8, and 9. This vivo. However, we performed two different types of experi- pattern of methionine release and the relative abundance of ments to find the primary translation product in vitro, and in methionine in cycles 8 and 9 is the same as that observed with each case concluded that the product is only one residue longer ovalbumin synthesized in vitro. The pattern of [3H]phenylal- than that of the secreted protein; the extra residue is methionine anine release (Fig. 6B) is consistent with a mixture of non- and it is derived from Met-tRNAf et. The same primary trans- acetylated ovalbumin molecules with phenylalanine residues lation product was deduced with nascent ovalbumin chains at positions 10, 12, and 15 (Table 1) and the primary translation obtained from intact cells. Thus, it is unlikely that the lack of product (with its extra methionine) with phenylalanine residues a typical "signal peptide" is due to incorrect initiation in at positions 11, 13, and 16. The high background in these ex- vitro. periments is due to the fact that only 10% of the nascent chains The finding that ovalbumin does not have a transient, hy- are accessible to sequencing, the others being acetylated. drophobic leader sequence raises important questions about the mechanism of ovalbumin secretion, as well as the function of DISCUSSION such sequences in other secretory proteins, including other egg All the available evidence suggests that the ovalbumin poly- white proteins secreted from the same cells as ovalbumin. There peptide synthesized in the reticulocyte lysate is the same length are several variations on the secretory pathway mentioned as the molecule synthesized in intact tubular gland cells. When above that might be invoked to explain ovalbumin secretion. analyzed on sodium dodecyl sulfate/acrylamide gels, the in The simplest idea is that the NH2-terminal sequence of oval- vitro translation product actually migrates slightly faster than bumin functions as a "signal": it may interact with membrane ovalbumin synthesized in vivo (16); this disparity is undoubt- receptor in a manner analogous to other "signal peptides", but edly due to the lack of in vitro, since ovalbumin then not be cleaved during membrane transport. The NH2- synthesized in vio in the presence of tunicamycin, an inhibitor terminal sequence of ovalbumin shows a weak resemblance to of glycosylation, also migrates slightly faster than controls (17). the other hydrophobic "signal peptides"; 11 of the first 15 Moreover, comparison of peptide maps of ovalbumin synthe- residues of ovalbumin are hydrophobic and the NH2 terminus Downloaded by guest on October 2, 2021 98 Biochemistry: Palmiter et al. Proc. Nati. Acad. Sci. USA 75 (1978) is blocked. However, when a longer, more typical sequence of NH2-terminal glycine takes place 4 sec later. This conclusion 20 residues is considered, only 13 are hydrophobic and the is contrary to a recent report suggesting that methionine is hypothesis of an uncleaved signal peptide seems less likely. An cleaved 1-2 min after ovalbumin synthesis in vivo (22). intriguing possibility is that the transacetylase is itself the re- We conclude that there are at least two distinct mechanisms ceptor. If this enzyme is associated with the endoplasmic re- of protein secretion in the chick oviduct. Under optimal hor- ticulum in the oviduct, then in the process of acetylating the monal stimulation, ovalbumin is synthesized and secreted at NH2 terminus of ovalbumin it may direct the nascent chain into a rate close to 106 molecules/min per tubular gland cell (21). a membrane secretory channel. If this is the case, then the hy- Thus, if there are specific membrane receptors for secretion of drophobicity of the NH2-terminal sequence may be of secon- different polypeptides, then the oviduct should be a rich source dary importance. of those involved in ovalbumin secretion, as well as those There are several more remote possibilities that may be thought to recognize the "signal peptides" on lysozyme, ovo- considered. One is that a hydrophobic "signal sequence" is lo- mucoid, and conalbumin. cated elsewhere within the molecule, rather than at the NH2 We thank Linda Strimple for secretarial assistance, and Stephen terminus. Alternatively, a "signal peptide" might be translated Thibodeau for help in developing some of the methods. This work was as a separate polypeptide from mRNAO,. This can tentatively funded by grants from the National Institutes of Health: HD-09172, be ruled out by our sequence data from the entire nascent-chain GM-15731, and RR-05432. J.G. is supported by the Medical Research population derived from purified mRNA0v (Fig. 5). We did not Council of Canada. R.D.P. is an investigator of the Howard Hughes observe a second sequence of specific activity comparable to Medical Institute. that of ovalbumin, but we have not tried all 20 amino acids, and hence cannot definitively rule out this possibility. Another 1. Palmiter, R. D., Gagnon, J., Ericsson, L. H. & Walsh, K. A. (1977) suggestion is that ovalbumin nascent chains may have some J. Biol. Chem. 252,6386-6393. hydrophobic group covalently attached near the NH2 terminus 2. Inouye, S., Wang, S., Sekizawa, J., Halegoua, S. & Inouye, M. (1977) Proc. Natl. Acad. Sci. USA 74, 1004-1008. (e.g., to a serine or cysteine residue) that could serve the function 3. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 852-862. of a "signal" and subsequently be removed. This idea has not 4. Palmiter, R. D., Thibodeau, S. N., Gagnon, J. & Walsh, K. A. been adequately tested, but there is a precedent in the case of (1978) in Proceedings of the 11th FEBS Meeting, Regulatory penicillinase, which has a fatty acid transiently attached at the Proteolytic Enzymes and Their Inhibitors, eds. Magnussen, S., NH2 terminus (19). In any event, it is clear that proteolysis of Otteson, M., Foltmann, B., Dano, K. & Neurath, H. (Pergamon a "signal sequence" is not essential for membrane transport or Press, Oxford), in press. secretion of ovalbumin. 5. Palmiter, R. D. (1973) J. Biol. Chem. 248,2095-2106. The timing of methionine aminopeptidase action and acet- 6. Pehlam, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, ylation of ovalbumin can be deduced from the data presented 247-256. 7. Palmiter, R. D. (1978) J. Biol. Chem., in press. here. By determining the sequence of all the ovalbumin poly- 8. Haines, M. E., Carey, N. H. & Palmiter, R. D. (1974) Eur. J. peptides that are not blocked, the yield of radioactivity in po- Biochem. 43, 549-560. sition n+1 ranges from 75-100% of a yield in position n both 9. Stanley, W. M., Jr. (1972) Anal. Biochem. 48,202-216. in vitro (Fig. 5) and in vivo (Fig. 6). The radioactivity in posi- 10. Elson, N. A., Brewer, H. B. & Anderson, W. F. (1974) J. Biol. tion n+1 represents those nascent polypeptides that still retain Chem. 249, 5227-5235. the NH2-terminal methionine while that in position n repre- 11. Palmiter, R. D., Palacios, R. & Schimke, R. T. (1972) J. Biol. sents those from which methionine has been cleaved. Thus, Chem. 247,3296-3304. assuming that the rate of elongation is constant, the number of 12. Palmiter, R. D. (1974) Biochemistry 13,3606--3615. residues incorporated before methionine is removed is 75-100% 13. Shapiro, D. J., Taylor, J. M., McKnight, G. S., Palacios, R., Gon- event zalez, C., Kiely, M. L. & Schimke, R. T. (1974) J. Biol. Chem. 249, of the number incorporated between that and acetylation. 3665-3671. The size of the smallest nascent ovalbumin polypeptides bearing 14. Traugh, J. A. & Sharp, S. B. (1977) J. Biol. Chem. 252, 3738- acetate is 44 residues (Fig. 2). With this information we cal- 3744. culate that initiator methionine is removed when ovalbumin 15. Housman, D., Jacobs-Lorena, M., Raj Bhandary, U. L. & Lodish, nascent chain is 19-22 residues long, and acetylation occurs H. F. (1970) Nature 227,913-918. when the peptide is 22-26 residues longer. Initiator methionine 16. Rhoads, R. E., McKnight, G. S. & Schimke, R. T. (1971) J. Biol. is removed from hemoglobin when these polypeptides are Chem. 246,7407-7410. 15-20 residues long (20), suggesting that the timing of this event 17. Struck, D. K. & Lennarz, W. J. (1977) J. Biol. Chem. 252, may be related to accessibility of the methionine aminopep- 1007-1013. tidase to the nascent chain. The rate of polypeptide elongation 18. Narita, K. & Ishii, J. (1962).J. Biochem. 52,367-373. 19. Yamamoto, S. & Lampen, J. 0. (1976) Proc. Natl. Acad, Sci. USA under our in vitro conditions is about 0.65 residues/sec; thus, 73, 1457-1461. acetylation takes place 68 sec after initiation. The rate of 20. Jackson R. & Hunter, T. (1970) Nature 227,672-676. polypeptide elongation on mRNAO, in vivo is about 5 resi- 21. Palmiter, R. D. (1975) Cell 4, 189-197. dues/sec (21). Hence, in vivo, the NH2-terminal methionine 22. Prasad, C. & Peterkofsky, A. (1976) Arch. Biochem. Biophys. 175, is cleaved 4 sec after initiation and acetylation of the new 730-736. Downloaded by guest on October 2, 2021