Structural Requirement at the Cleavage Site for Efficient Processing

THE JOURNAL OF BIOLOGICAL CHEMISTRV 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 24, Issue of August 25, pp. 10970-10975,1986 Printed in U.S.A.

Structural Requirement at the Cleavage Site for Efficient Processing of the Lipoprotein Secretory Precursor of Escherichia coli*

(Received for publication, March 25, 1986)

Sumiko Inouye, Guy Duffaud, and Masayori Inouye From the Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11 794

A phenotypically silent mutation in the signal peptide of the Escherichia coli outer membrane prolipoprotein was combined with other mutations in the mature lipoprotein structure. Under conditions where the indi- vidual mutations permit normal lipoprotein secretion, the prolipoprotein with both mutations was unable to be normally modified or processed. These results dem- onstrate that a given signal peptide is fully functional only if it is structurally compatible with the protein to be secreted. This structural compatibility between the signal peptide and the secretory protein is considered to be dependent on the secondary structure formed at or near the signal peptide cleavage site.

Most secretory proteins are synthesized first as precursors containing an extra amino-terminal extension or signal peptide (Watson, 1984). Prokaryotic signal peptides have several major structural homologies which include: ( a ) 1-5 basic amino acid residues in the amino-terminal region, ( b ) a hydrophobic region consisting of approximately 15 amino acid residues directly following the positively charged amino-terminal region, (c ) in most signal peptides, a proline or glycine residue located within the hydrophobic domain, ( d ) serine and/or threonine residue(s) located close to the carboxyl- terminal end of the hydrophobic region, and ( e ) an alanine or glycine residue at the carboxyl-terminal end (cleavage site) (Inouye et al., 1984).

The signal peptide is considered to play two functional roles for the secretion of a protein across the membrane. The first role is to direct the amino-terminal end of the secretory protein from the cytoplasm to the outside of the membrane, and the second role is to serve as substrate for the signal peptidase, an enzyme which removes the signal peptide during translocation of the protein across the membrane. These functions are likely related to the unique structural features of the signal peptide described above. A systematic approach to elucidate the structural and functional relationships of the signal peptide has been undertaken using oligonucleotide- directed site-specific mutagenesis to alter various regions of the signal peptide of the Escherichia coli prolipoprotein. We have studied the importance of the number of basic amino acid residues at the amino terminus of the precursor protein and the effect of their substitution by acidic amino acids (Inouye et al., 1982; Vlasuk et al., 1983). The hydrophobic region of the signal peptide has been studied for its total length as well as for the particular disposition of glycine,

* This work was supported by Grant GM19043 from the National Institute of General Medical Sciences and Grant NP387L from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

proline, serine, and threonine residues within it (Inouye et al., 1984; Vlasuk et al., 1984; Pollit et al., 1985). Finally, the specificity of the amino acid residues at the cleavage site has also been investigated (Inouye et al., 1983, a and b; Pollit et al., 1986).

The present work concentrates on the structural requirements surrounding the cleavage site of the prolipoprotein signal peptide. We have engineered a mutant lipoprotein by oligonucleotide-directed site-specific mutagenesis, in which Ser-Asn at positions 3 and 4 in the mature lipoprotein has been changed to Ile-Ile, likely altering the secondary structure of the cleavage site. Like the wild type prolipoprotein, the prolipoprotein carrying this mutational alteration was able to be secreted across the cytoplasmic membrane at 30 “C. How- ever, when the Ile-Ile substitution mutation was combined with a silent mutation in the signal peptide in which the glycine residue at the cleavage site was replaced with an alanine residue (A20), further altering the cleavage site structure, the signal peptide was no longer cleaved at 30 “C. Since the Ile-Ile substitution increases the hydrophobicity at the cleavage site, we have engineered an additional set of mutants by replacing the Ile a t position 4 with a lysine residue. The new Ile-Lys mutation was fully functional at 30 “C, but be- came completely defective at 30 “C when combined with the silent A20 mutation. Thus, we propose that a given signal peptide must be structurally compatible with the protein to be secreted in order for the signal peptide to be fully functional, and that this structural compatibility allows the formation of the proper secondary structure at or near the signal peptide cleavage site.

EXPERIMENTAL PROCEDURES’

RESULTS

Design of Mutations at the Signal Peptide Cleavage Region- We have previously shown (Vlasuk et al., 1984) that there is a very high probability for forming a &turn structure at the signal peptide cleavage site of prolipoprotein when its secondary structure is analyzed by the rules developed by Chou and Fasman (1978) (see Fig. 1A). In order to determine if this predicted @-turn structure is essential for cleavage of the signal peptide, we proceeded to alter the primary structure of the prolipoprotein SO as to reduce the predicted probabilities of the P-turn structure at the cleavage site to various levels.

Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. 3-6, and additional references) are presented in miniprint. Miniprint is easy read with the aid of a standard magni- fying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-960, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

10970

Compatibility between the Signal Peptide and the Secretory Protein 10971

FIG. 1. Predicted secondary structures of wild type and mutant prolipoprotein signal peptides. Second- ary structures were predicted for wild type ( A ) , mutant A20 ( B ) , mutant I23124 (C) , mutant A20123124 ( D ) , mutant I23K24 ( E ) , and mutant A20123K24 ( F ) signal peptides according to Chou and Fasman (1978). Amino acid sequence re- fers to the prolipoprotein signal peptide sequence with the NH2-terminal methionine residue being number 1. The solid lines represent the probabilities of 01-

helical structure, and the dotted lines represent the probabilities of @turn structure. The arrow designates the signal peptide cleavage site. A part of the amino acid sequence around the cleavage site is shown in each case. The bored amino acid residues are those that have been changed from the wild type sequence.

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When the glycine residue a t position 20 (the carboxyl terminus of the prolipoprotein signal peptide) was changed to alanine (A20 mutation, previously designated C1 mutant (In- ouye et ai., 1983b)), no effect was detected on either the cleavage of the signal peptide or the secretion of the lipoprotein. Analysis of the secondary structure of the A20 prolipoprotein, shown in Fig. 1B, shows a slight reduction in P-turn probability and a concomitant increase in a-helical structure on the amino-terminal side of the cleavage site. Apparently, these structural alterations have no effect on the function of the signal peptide.

To further reduce the P-turn probability, we chose to change the primary structure of the prolipoprotein in the region of the mature lipoprotein, while maintaining the same signal peptide structure. This was achieved by individually changing both the serine and the asparagine residues a t positions 23 and 24 of prolipoprotein, respectively (corresponding to positions 3 and 4 of the mature lipoprotein, respectively) to

AMINO ACID SEQUENCE

isoleucine residues. This mutation, designated 123124, significantly reduces the predicted 0-turn structure at the cleavage site as shown in Fig. 1C. An even greater reduction of P-turn structure can be achieved by combining the A20 and I23124 mutations creating A20123124. Fig. 1D shows that the 0-turn probability at the cleavage site is very low for this mutant. However, in this set of mutations, 2 polar residues (Ser-Asn) are substituted with 2 hydrophobic residues (Ile-Ile). We can also predict a similar change of structure at the cleavage site using polar amino acid residues. This is achieved replacing the isoleucine residue at position 24 of mutant I23124 with a lysine residue. Fig. 1, E and F, shows that the lysine substitution causes the same structural alteration at the cleavage site as the isoleucine substitution (compare Fig. 1, E with C and F with D ) .

Expression of the Mutant lpp Genes-The lpp genes used in this study are cloned in an expression cloning vector, PIN-11, under the control of both the lpp promoter and the lac pro-

10972 Compatibility between the Signal Peptide and the Secretory Protein

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

a- -*c b-

d a -b

FIG. 2. Immunoprecipitates from the pulse-labeled membrane fractions of wild type (pYM141) and mutants A20, 123124, and A20123124. E , co(i dA221 (lpp-) harboring pYM141 (wild type) or mutant plasmids were pulse-labeled with [""Sjmethio- nine for 15 s at various temperatures after a 20-min induction of the lpp genes with IPTG. The membrane fraction was prepared, soluhi- lized, and treated with anti-lipoprotein serum as described under "Experimental Procedures." The immunoprecipitates were then ana- lvzed bv SDS-polyacrylamide gel electrophoresis using the phosphate buffer svstem (Inouye et al., 1982). Lanes I-.3, wild type lpp; lanes 4- 6.1pp-AZO; lanrs 7-9,lpp-I23124; lanes 10-12,lpp-A20123124. Samples prepared from the cultures grown at 30 "C were applied to lanes I , 4, 7, and IO; at 37 "C, to lanes 2, 5 , 8, and 11; a t 42 "C, to lanes 3, 6, 9, and 12. Rand a represents unmodified and lipid-modified prolipoproteins. and band 6 the fully modified mature lipoprotein.

moter-operator (Nakamura and Inouye, 1982). The expression of the cloned lpp gene can then be induced in E. coli SB221 Ipp-lF' lacP by adding isopropyl-e-D-thiogalactopyranoside (IPTG'). a lac inducer to the culture medium.

E. coli carrying the plasmid bearing lpp-123124 (I23124 plasmid) were capable of forming colonies on L-broth plates containing 2 mM IPTG at 30 "C, but not at 37 "C or 42 "C. On the ot,her hand, E. coli carrying the plasmid bearing lpp- 123K24 (I23K24 plasmid) were capable of forming colonies at 30 "C and also at 37 "C, but not at 42 "C, whereas E. coli carrying either plasmid lpp-A20123124 (A20123124 plasmid) or lpp-A20123K24 (A20123K24 plasmid) were sensitive to IPTG at all temperatures tested. It has been shown that the accumulation of prolipoprotein in the membrane resulted in growth inhibition (Inouye et al., 1983, a and b, 1984; Vlasuk et al., 1984). Therefore, these results suggest that the I23124 prolipoprotein is normally processed a t 30 "C, but not, a t 37 "C or 42 "C, while the I23K24 prolipoprotein can be processed a t :E "C. However, processing ofeit.her A20123124 or A20123K24 would be defective at any temperature tested.

In order to examine lipoprotein production in I23124 and A20123124 mutants, cells carrying the mutant plasmids were pulse-labeled with ['"'Slmethionine for 15 s after a 20 min induction of the lpp genes with IPTG. Membrane fractions were then prepared, solubilized with SDS, and treated with anti-lipoprotein serum. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis using the phosphate buffer system (Inouye et al., 1982) to facilitate separation of the mature lipoprotein (band b in Fig. 2) from both the unmodified and the lipid-modified prolipoproteins, which migrate to a higher position (band a in Fig. 2) (Inouye ct al., 1982). As can be seen in Fig. 2, both the wild type lpp (lancs 1-3) and lpp-A20 genes (lanes 4-6) produce mainly the mature lipoprotein at all temperatures tested. In the case of the I23124 prolipoprotein, the mature lipoprotein was produced at 30 "C (band b in lane 7, Fig. 2; also see band b in Fig. 3R for longer labeling). However, the rate of processing appears to be slower, resulting in the accumulation of the prolipoprotein (band a in lane 7 , Fig. 2). At higher temperatures (37 "C in lane 8 and 42 "C in lane 9 ) , the mature ~~ ~ ~~~~~

'The abbreviations used are: IPTG, isopropyl-1-8-D-thiogalacto- -~

pyranoside: SDS, sodium dodecyl sulfate.

lipoprotein was barely detectable. In contrast, the A20123124 mutation blocks the production of the mature lipoprotein from the prolipoprotein not only at 37 "C (lane 1 1 ) and 42 "C (lane 12) , hut also at 30 "C (lane I O ) . The cells' inability to modify and process prolipoprotein seems to coincide with the observed IPTG sensitivity of the cells carrying the mutant lpp gene as described ahove. There was little accumulation of prolipoprotein in the soluble fraction in all cases (data not shown).

Pulse-Chase Experiments-The results shown in Fig. 2 indicate that the signal peptide of I23124 prolipoprotein is functional at least at 30 "C to produce mature lipoprotein, whereas that ofthe A20123124 prolipoprotein is defective even a t :30 "C. In order to examine the rate at which mutant. prolipoproteins are processed, pulse-chase experiments were performed. Cells harboring the I23124 plasmid or the A20123124 plasmid were pulse-labeled with [""Slmethionine for 30 s at both 30 "C and 42 "C after a 20-min induction with IPTG. This labeling was followed with a chase with nonra- dioactive methionine for 2 min and 15 min. The immunoprecipitates were then analyzed by SDS-polyacrylamide gel electrophoresis using both the Tris buffer and the phosphate buffer systems (Fig. 3). In the case of the lpp-123124 gene, the lipid-modified prolipoprotein (band a in lanes 1-3, Fig. 3 A ) , and both the modified and unmodified prolipoprotein (band a in lanes 1-3, Fig. 3B) appear to be chased into the mature lipoprotein during a 15-min chase. Degradation of the precursor during the chase is unlikely, since these molecules appear to be very stable as judged from the experiments at 42 "C (lanes 4-6, Fig. 3, A and B ) . At 42 "C, the I23124 prolipoprotein was quite stable; band a in Fig. 3B (unmodified plus modified prolipoproteins) remained almost constant during the 15-min chase. Since there is no band b in lane 4 of Fig. 3B (the mature lipoprotein), it is concluded that band b in lane 4 of Fig. 3A consists of only the unmodified prolipoprotein, which mi- grates a little faster than the mature lipoprotein (band b in lane 3, Fig. 3 A ) in the Tris buffer system. In the 30-s pulse experiment, a small amount of the modified prolipoprotein was produced as judged from band a in lane 4 of Fig. 3A, which gradually increased in intensity during the chase. After the 15-min chase period, a small amount of the mature lipoprotein can be observed as well (band b in lane 6 , Fig. 3B) . Thus, it appears that the processing of I23124 prolipoprotein is temperature-sensitive. At 42 "C, lipid modification as well as signal-peptide cleavage was inhibited; in particular, the lipid modification step was most severely inhibited.

In contrast to the I23124 prolipoprotein, the processing of the A20123124 prolipoprotein to the mature lipoprotein could not be observed either at 42 "C (lanes 10-12 in Fig. 3 ) or at 30 "C (lanes 7-9 in Fig. 3). Band b was not detected even after a 15-min chase at these temperatures (lanes 9 and 12 in Fig. 3B). It was not immediately clear if the A20123124 prolipoprotein that was accumulated was lipid-modified or not, since it migrated t,o an intermediat.e position (band c) in the Tris buffer system (lanes 7-12, Fig. 3 A ) between band a (the modified prolipoprotein) and band b (the unmodified prolipoprotein plus the mature 1ipoprot.ein). Therefore, in order to further characterize the A20123124 prolipoprotein, the cells were labeled with ["Hlglycerol for 30 min a t 42 "C after a 20- min induction with IPTG. The immunoprecipitates were then analyzed by SDS-polyacrylamide gel electrophoresis using the phosphate buffer system. In the case of the I23124 prolipoprotein, both band a and band b were labeled with ["Hlglycerol (lane 2, Fig. 4), consistent with the result obtained with ['"SI methionine shown on lane 6 of Fig. 3R. On the other hand, band a was not labeled a t all in the case of the A20123124

Compatibility between the Signal Peptide and the Secretory Protein 10973

prolipoprotein, demonstrating that the band c protein (7-12, Fig. 3A) is the unmodified prolipoprotein. Thus, it can be concluded that the lipid modification reaction was completely defective in the A20123124 mutant. It should be noted that the prolipoprotein accumulated in the membrane fraction was very stable since little change in its amount was observed during the chase (Fig. 3).

Effect of the Polarity of Amino Acid Residues Located at the Cleavage Site-As pointed out earlier, the I23124 mutants alter the structure of the cleavage site of the prolipoprotein and likely render this region more hydrophobic. In order to study a possible effect of this elevated hydrophobicity at the cleavage site area, the I23K24 mutations have been designed.

E. coli carrying the 123K24 and A20123K24 plasmids were pulse-labeled with [35S]methionine for 30 s after a 20-min induction of the lpp gene with IPTG. The production of lipoprotein and precursor was analyzed as described above. Fig. 5 shows the result of the pulse experiment at 30 "C, 37 "C, and 42 "C. Contrary to 123124, the 123K24 product is fully processed at 30 "C (lane 4, band 6 , Fig. 5), almost fully processed at 37 "C, but to a lesser extent at 42 "C (lanes 5 and 6, bands a and 6, respectively, Fig. 5, (compare with lanes I, 2, and 3 of Fig. 5). However, this partial reversion to the wild type behavior, following substitution of isoleucine with lysine, is not observed at all for the A20123K24 mutation, which remains totally unprocessed (band a, lanes 7-9, Fig. 5). Thus, as in the case of the I23124 mutation, the I23K24 mutation, which by itself is not defective at least at 30 "C, becomes completely defective when it is combined with the silent mutation AZO.

We further examined the effect of the I23K24 mutation on the rate of processing by a pulse-chase experiment at 30 "C and 42 "C. Cells harboring the I23K24 plasmid or the A20123K24 plasmid were pulse-labeled with [35S]methionine for 30 s at both 30 "C and 42 "C after a 20-min induction with IPTG. This labeling was followed by a chase with nonradioac- tive methionine for 2 min. The immunoprecipitates were then analyzed by SDS-polyacrylamide gel in the phosphate buffer system (Fig. 6). It can be seen that at 30 "C, the lpp-123K24 gene product was immediately processed (lanes 1 and 2, band 6, Fig. 6). However, at 42 "C, a significant amount of precursor prolipoprotein was observed after the pulse and was slowly chased to the mature lipoprotein (lanes 3 and 4, bands a and 6, Fig. 6). As is the case of the A20123124 plasmid, the product of the lpp-A20123K24 gene was unprocessed not only at 42 "C but also at 30 "C (lanes 5-8, band a, Fig. 6). As in the case of the A20123124 mutant, the lipid modification step was severely impaired for the A20123K24 mutant (data not shown).

DISCUSSION

The present work clearly demonstrates that a phenotypically silent mutation in the signal peptide (A20 mutation which substitutes glycine 20 at the cleavage site to alanine) causes a visible deleterious effect on the functions of the signal peptide when the mutation is combined with another mutation, 123124, in the protein structure. At 30 "C, the I23124 prolipoprotein was capable of being processed to produce the mature lipoprotein. Although accumulation of the prolipoprotein was observed in an early chase (band a in lane 2, Fig. 3B), it was almost completely chased into the mature lipoprotein during a 15-min chase (lane 3, Fig. 3 B ) . Since the bands at position a in the Tris buffer system (the lipid-modified prolipoprotein; lanes 1 and 2, Fig. 3A) were very faint in contrast to bands at position a in the phosphate buffer system (the lipid-modified plus the unmodified prolipoprotein; lanes 1 and 2, Fig 3B), it can be concluded that band a in lanes 1

and 2 of Fig. 3B mainly consists of the unmodified prolipoprotein. Thus, the I23124 mutation appears to have an effect mainly on the lipid modification reaction at 30 "C. However, at 42 "C, not only the lipid modification but also the cleavage of the signal peptide were severely inhibited (lanes 4-6, Fig. 3, A and B).

When the A20 silent mutation was combined with the I23124 mutation, the lipid modification was completely blocked both at 30 "C and at 42 "C. These results can be correlated with the predicted secondary structure at the signal peptide cleavage region. As shown in Fig. lA, a P-turn structure is predicted at the cleavage site of the wild type prolipoprotein and the A20 mutation has little effect on the &turn structure (Fig. 1B). On the other hand, the I23124 mutation significantly reduces the predicted 6-turn structure. This reduction in the 6-turn probability at the cleavage site of I23124 prolipoprotein may well explain temperature-sensitive processing of the mutant prolipoprotein. The combination of the A20 and the I23124 mutations causes a dramatic reduction of the predicted 0-turn structure (Fig. 1D). Thus, it appears reasonable to propose that the 6-turn structure at the cleavage site is essential for the processing of the prolipoprotein signal peptide by signal peptidase I1 (Hussain et al., 1980). This result is in accord with the loop model (Inouye et al., 1977, 1979; Inouye and Halegoua, 1980). In this model, the insertion of the hydrophobic signal peptide in the membrane bilayer results in the formation of a loop. This would in turn expose the cleavage site on the periplasmic side of the inner membrane for cleavage by the signal peptidase. It is possible to assume that the formation of such a loop would create structural requirements, particularly at the cleavage site (for a review, see Duffaud et al., 1985).

The substitution of 2 polar residues (Ser-Asn) at positions 23 and 24 by 2 highly hydrophobic residues (Ile-Ile) might also be a reason for the deficient processing of the mutants. We investigated this possibility by designing a new mutant arising from the substitution of the isoleucine now at position 24 with a lysine residue. These new mutants (plasmids I23K24 and A20123K24) show the same structural pattern around the cleavage site as the mutants described above (compare Fig. 1, C and D with E and 3'). The choice of lysine allows the conservation of the introduced structural alteration while replacing a hydrophobic residue with a hydrophilic residue. As a result, I23K24 mutant was more efficiently processed at all temperatures than mutant I23124 (compare lanes 1-3 and 4-6 in Fig. 5). However, at 42 "C, some precursor was accumulated (see lane 6, Fig. 5), which was identified as unmodified prolipoprotein (data not shown). These results indicate that hydrophobicity at the cleavage site region may have some effect on the processing of the prolipoprotein. However, the A20123K24 mutation was as deficient as the A20123124 mutation, not only at 42 "C but also at 30 "C (lanes 7-9, Fig. 5 , and lanes 5-8, Fig. 6). Thus, it seems that the essential factor for impaired processing in all these mutants is the structural alteration at the cleavage site region, namely the reduction of 6-turn structure.

It should be noted that the signal peptide has two major functions. The first is to facilitate translocation of a protein across the membrane, and the second is to serve as substrate for a signal peptidase, which cleaves the signal peptide upon translocation of the protein across the membrane. The first function is the most essential role of the signal peptide, and the second function is not essential for the first. Prolipopro- teins have been shown to be translocated across the cytoplasmic membrane and assembled into the outer membrane without the signal peptide being cleaved (Lin et al., 1978;

10974 Compatibility between the Signal Peptide and the Secretory Protein

Inouye pf al., 1983, a and b). The /3-turn structure at the cleavage site is a proposed requirement only for t.he cleavage event, not for protein translocation across the membrane, since the mutant prolipoproteins described in this report were also found in the outer membrane fraction without the signal peptide being cleaved (result not shown).

The present work clearly demonstrates that a given signal peptide can be fully functional only when its structure is compatible with the structure of the protein to which the signal peptide is attached. The compatibility between the signal peptide and t,he secretory protein can be achieved by altering the primary structure of either the signal peptide (for example, from A20123124 to 123124) or the protein (for example, from A20123K24 to A20). In this regard, it is interest- ing to note that some mutational alterations in the mature protein sequence (not in the signal peptide) have been shown to affect the functions of signal peptides that are processed by a different signal peptidase (Russel and Model, 1981; Bankaitis et a/., 1984).

Acknordedgments-We wish to thank Girija Ramakrishnan and Dr. Charles Lunn for critical reading of this manuscript.

REFERENCES

Rankaitis, V. A., Rasmussen, B. A., and Bassford, P. J. (1984) Cell 37,243-252

Chou, P. Y., and Fasman, G. D. (1978) Annu. Reu. Riochem. 47,251- "76

Duffaud, G. D., Lehnhardt, S. K., March, P. E., and Inouye, M. (1985) in Membrane Protein Biosynthesis and Turnover (Knauf, P. A., and Cook, ,J. S., eds) pp. 65-104, Academic Press, New York

Supplementary material to: "Structural WQuirement at the cleavnpe 5ite

for efficient processing o f the lipoprotein secretory precursor of

Escherichia coli". ~-

hv

Sumiko lnouvr. Guv Duffnud, and Hasagari lnouve

Ilepartment of Riochemistry

State Universitv of h'ew York at Stony Rrook

Gtonv Rrook, K'ew York 11794

- EXPERI?TNTAL PROCEDURES

Cella were g r o w as previously described (Inouye & s., 1987). Plasmid pY\!111 (Rakamura and Inouve, l q U 2 ) and pIN-III-~-A20 - (pC1: lnouve 1.. designation is given for each mutation based on two rules: (1) numbers always 19R3) were used f o r site-specific mutagenesis experiments. A svstematic

hv the single-letter code of the new amino acid followed hv the position at refer to the wild tvpe amino acid positions, and ( 2 ) substitution is indicated

which the substitution takes p l a c e (Duffaud et 51.. 19%) Oligon, ic leo t ide-d i rec t rd Site-specific Mutagenesis

plasmid DNA as template acrording to norinaga 5 &. (19U4). DNA sequencing Site-specific mornyenesis was carried out with use of double-stranded

v a s carried out accordinp to tlaxam and Gilhert (1980) o r Sangcr et al. ( 1 9 7 7 ) . Two o1ipodeoxyrih"nueleotides were synthesized hy a System % c x s v n 1450 automated DNA Synthesizer: an IR-mer, TCCTCCATCATCGCTMA. and 19-mer GCTCCATCMACCTAAMT. W r e s s i m of the Mutant Lipoprotein Cenes

Pulse and pulse-chase enpcriments were carried out as described previously ( lnooye et a l . , 1982) . The labeling experiment with [ 3 H ] g l y c e r o l was performed a s d e c r z e d (Inoxtve et g . , 1 9 8 3 a ) . Preparation of membrane fractions, immunoprecipitation using rahhit anti-lipoprotein serum, and SnS-polyacrglanide gel electrophoresis were perlormed as described (Inouye

"

- al.. 1982).

KFSL'LTS ~

WTANTS CORSTR1:CTlON The mutdnls used in this studv were constructed in two Steps. An

18-ncr o l i g o d e o x v r i h o n l l c l e o t i d e , TGCTTCATCAXCCCTAAA (Cys-Ser-Ile-IIe-Ala-I.ys;

of the wild-tape llNA S ~ Q U ~ ~ C E at these positions, respectivelv with T) was the first and second underlined nucleotides were designed to replace G and ,\

employed for site-specific ml~tnqenesis. This oli@onucleotide was used to construct both the 123124 and A?01?11?4 mutants from the wild-tvpe & gene and the ipp-n?o ~ e n e . respectively. Subsequently, a 19-mer oligodeexy

2 1 26

Hussain, M., Ichihara, S., and Mizushima, S. (1980) J . Biol. Chem. 255, 0707-3712

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Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3438- 3441

Inouye, S., Franceschini, T., Sato, M., Itakura, K., and Inouye, M. (1983a) EMBO J . 2,87-91

Inouye, S., Hsu, C. S., Itakura, K., and Inouye, M. (1983b) Science

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Pollit, S., Inouye, S., and Inouye, M. (1986) J. Biol. Chem. 261,

Russel, M., and Model, P. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,

Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983)

Vlasuk, G. P., Inouye, S., and Inouye, M. (1984) J. Biol. Chem. 259,

Watson, M. E. E. (1984) Nucleic Acids Res. 12, 5145-5164 Additional references are found on p. 10975.

221, 59-61

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1,289-299

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6195-6200

A 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

6 1 - 2 - 3 4 5 6 7 8 9 10 11 12

Figure 3 . Immunoprecipitates from a Pulse-chase Experiment at 30' and 42'C Using

Mutants 123124 and A20123724.

Figure 3 except that the labeling time was 30 seconds instead of 15 seconds. Cells harboring the mutant plasmids were pulse-labeled as described in

After the pulse, cells were chased with "on-radioactive methionine for 2 and 15 min. The immunoprecipitates were analyzed by SDS-polyacrylamide g e l electrophoresis using the Tris buffer system ( A ) a s well as the phosphate-huffer svstem ( 8 ) Inouve e t X . , 1982). Lanes I-b, mutRnt 1 2 3 1 2 4 : lane 7-12. mutant A20121124. s n i p l e ~ f ~ m experiments at 30'C were applied to lanes 1-3 and 7-9 and those at 42'C were applied to lanes 4 - 6 and 10-12. Samples from the 30 seconds pulse experiments were applied to lanes 1 , 4 , 7 , and 10; from the 2 min-chase experiments to lanes 2,5,R, and 11; from the 1 5 mi" chase experiments to lanes 3 , 6 , 9 , and I?. Rand a in ( A ) represents the

plus the mature lipoprotein. The unmodified A?(lI23IZ4 prolipoprotein minrated lipid-modiiied prolipoprotein, and hand b in ( A ) the unmodified prolipoprotein

are the same as described for Figure ? . to the band c position in ( A ) as described in the text. Rands a and b in ( R )

Compatibility between the Signal Peptide and the Secretory Protein

1 2 3 4

Figure 4 . lmmunoprccipitates Irom Cells l a b e l e d with j3H]Clvcerol. c 3 l i s harboring the 123124 plasmid or the A20127124 plasmid were labeled

with [ Hlglyrerol for 30 mi" a t 4?'C a f t e r 3 ?O-mi" induction of the Ipp genes with IPTG. The proteins immunoprecipitated with enti-lipoprotein serup~ were

phosphate-huffer system ( I n a u y e e t al., 1982). Lane 2, immunoprecipitates then analyzed by SDS-polvacrylamide gel electrophoresis using the

and 4 [ S]methianine-lah~Ied samples from mutant I23124 and A20123724, irom m"taT5 1 2 3 1 2 4 ; lane 3, immunoprezpitatcs from mutant A20123124. Lanes 1

respectively. The same sample, applied to lane h o f Figure 3. was applied t o l ane 1, and the same sample that was applied to lane 12 of Figure 3 was applied to l a n e L .

1 2 3 4 5 6 7 8 9

10975

1 2 3 4 5 6 7 8

Figure h .

Mutants 123K24 and A?OI?3K24. immunoprecipitates From a Pulse-chase Experiment at 30' and 4?'C using

Cells harboring the mutant plasmids were pulse-labeled as descrihed in Figure I except that the labeling time was 30 seconds instead of 15 seconds. After the pulse, cells were chased with non-radioactive methionine for I min. Thc immunoprecipitates were analyzed by SllS-polyacrylamide gel electrophoresis using the phosphace-buffer system (Inouye &., 1982). Lanes 1 - 4 , mutant 123K24; lanes 5-8, mutant A20123K24. Samples from experiments at 3OoC were applied t o lanes 1, 2 , 5 and 6 and those at 41'C were applied to lanes 3, L , 7 , and 8. Samples from the 30 S P C pulse experiments were applied to lanes 1. 3, 5 and 7 ; from the 7 min chase experiments to lanes ? , L , h and 8. Bands a and h are the same a s described in Figure 5.

REFERENCES

?laxam, A . M . , and Gilhert, 14. (1480). Xethods Enrymol. 6 5 , 4 9 9 - 5 5 9 . Sanger , F., Nicklen, S . , and Coulson, A.R. (1977). Proc. Natl. Acad. S c i .

U.S.A. 7 4 , 5463-5167.

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Structural Requirement at the Cleavage Site for Efficient Processing