"export initiation domain" adjacent to the signal sequence is critical for ...

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 9751-9754, November 1991

Genetics

A 30-residue-long "export initiation domain" adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli (protein secretion/protein export/membrane protein)

HELENA ANDERSSON AND GUNNAR VON HEUNE Department of Molecular Biology, Karolinska Institute Center for Biotechnology, NOVUM, S-141 57 Huddinge, Sweden

Communicated by Jonathan Beckwith, July 29, 1991 (receivedfor review May 1, 1991)

ABSTRACT Signal sequences serve to target proteins to the secretory pathway in both prokaryotic and eukaryotic cells. However, although necessary, the presence of a signal sequence is not always sufficient to ensure efficient membrane translocation. One feature of the nascent chain that adversely affects secretion, at least in Escherichia coli, is the presence of positively charged amino acids immediately downstream of the signal sequence. We have exploited this sensitivity to positively charged residues to demonstrate the presence of a sharply delimited "export initiation domain" that comprises the signal sequence and its z30 downstream residues. A string of six consecutive lysines completely blocks translocation when placed inside this domain but not when placed only a few residues further away.

Signal sequences serve to target proteins for secretion in both prokaryotic and eukaryotic cells. Comparative sequence analysis as well as detailed mutational studies suggest a basic tripartite structure of the signal sequence with an N-terminal, positively charged tail (n region), a central hydrophobic region (h region), and a C-terminal cleavage region (c region), all of which are important for function (1). However, although necessary, the presence of a signal sequence is not always sufficient to ensure efficient secretion of the attached protein. Thus, stably folded or aggregated precursors cannot be translocated; such premature folding is normally prevented by chaperones that bind to nascent precursors and reduce their rate of folding (2-6). Another feature of the nascent chain that adversely affects secretion, at least in Escherichia coli, is the presence of positively charged amino acids immediately downstream of the signal sequence (7-15). In some cases, addition of as little as one extra arginine or lysine is sufficient to block secretion almost completely. Arginines and lysines, or even clusters of such residues, are found at similar overall frequencies in secreted and nonsecreted proteins (16), and there appears to be no intrinsic difficulty in moving internal chain segments containing many positively charged residues across the membrane (17). Rather, the effect of these residues would seem to be specific for the early steps of targeting and initiation of membrane translocation, although this has never been systematically analyzed. Here we show that a string of six consecutive lysine residues completely blocks or greatly reduces the rate of translocation of two related model proteins in E. coli when placed within 31 residues of an uncleaved signal sequence but not when placed at distances 38 residues or more from it. These results are consistent with a model in which the signal sequence and the following =30 residues form an "export initiation domain" that can only interact productively with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

the membrane or some factor of the secretory machinery if it does not include too many positively charged residues but in which the presence of such residues later in the chain poses few if any problems to the translocation machinery.

MATERIALS AND METHODS Enzymes and Chemicals. Trypsin, soybean trypsin inhibitor, chicken egg white lysozyme, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma. All enzymes were from Promega. Strains and Plasmids. Leader peptidase mutants were expressed from the pING1 plasmid (18) in E. coli strain MC1061

(19).

DNA Techniques. Site-specific mutagenesis was performed according to the method of Kunkel (20) as modified by Geisselsoder et al. (21). All mutants were confirmed by DNA sequencing of single-stranded M13 DNA using T7 DNA polymerase (Pharmacia). Cloning into the pING1 plasmid was performed as described (22). Assay of Membrane Topology. E. coli strains transformed with the pING1 vector carrying mutant leader peptidase (lep) genes under control of the arabinose promoter were grown at 370C in M9 minimal medium supplemented with ampicillin (100 ,ug/ml), 0.5% fructose, and all amino acids (50 ,Ag/ml each) except methionine. Overnight cultures were diluted 1:35 in 1 ml of fresh medium, shaken for 3.5 hr at 370C, induced with arabinose (0.2%) for 5 min, and labeled with [35S]methionine (150 ,Ci/ml; 1 Ci = 37 GBq). After 1 min, nonradioactive methionine was added (500 ,g/ml) and, after the indicated chase times, incubation was stopped by chilling on ice. Cells were spun at 15,000 rpm in an Eppendorf centrifuge for 1 min, resuspended in ice-cold buffer [40% (wt/vol) sucrose/33 mM Tris HC1, pH 8.0], and incubated with lysozyme (5 gg/ml) and 1 mM EDTA for 15 min on ice. Aliquots of the cell suspension were incubated 1 hr on ice, either with trypsin (0.75 mg/ml) or with trypsin (0.75 mg/ ml)/trypsin inhibitor (0.8 mg/ml)/PMSF (0.33 mg/ml). After addition of trypsin inhibitor and PMSF, samples were acid precipitated (trichloroacetic acid; final concentration, 1%o); resuspended in 10 mM Tris-HCI/2% SDS; immunoprecipitated (23) with antisera to Lep, OmpA, and AraB; washed; and analyzed by SDS/PAGE and fluorography. Radiosequencing. MC1061 transformed with the appropriate plasmid was grown overnight at 370C in the same medium as described above, diluted 1:35 into 0.5 ml of medium, shaken for 3.5 h at 370C, treated for 5 min with 0.2% arabinose, labeled for 10 mim with 150 pCi of [35S]methionine, acid and immunoprecipitated with Lep antiserum as described above, and purified by SDS/PAGE. The wet gel was exposed overnight, and the radioactive band was cut out, extracted with 0. 1% SDS by gentle shaking overnight at 370C, Abbreviation: PMSF, phenylmethylsulfonyl fluoride.

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Proc. Natl. Acad ScL USA 88 (1991)

Genetics: Andersson and von Heijne 6K(9O

A

6K(74

A

wildtype

\14k

m~

6K(1 5)

_

_

_

_

0m

6K(27)

6K(38) 6K(54)

-

m

-

6K(74)

-

-

trypsin FIG. 1. (A) Wild-type Lep. (B) Lep derivative used in this study. In B, four lysines have been added to the N terminus of the H1 transmembrane region (residues 4-22), and residues 30-52 in P1 as well as transmembrane region H2 (residues 62-76) have been deleted, converting H1 into an uncleaved signal sequence (25). The positions of the lysine strings in the mutants discussed in the text are indicated by arrows.

chase (min)

0

5

10

B 4KA30-52,A62-76

-_

6K(1 4)

Applied Biosystems 470A protein

6K(21)

__

Quantitation. With the help of autoradiograms, 4-mmdiameter circles were cut from the gels at the center of each band. Gel pieces were dissolved in 150 Al of water and 450 ,ul of gel solubilizer (Protosol, DuPont), and dpm were obtained by counting in 2 ml of liquid scintillation fluid (Emulsifier Safe, Packard). The background in each lane was estimated by assaying a band cut out from an empty region between the Lep and AraB bands and was subtracted from all other counts. To correct for cell lysis, the fraction of trypsinresistant Lep at each time point was normalized to the fraction of trypsin-resistant AraB (a cytoplasmic marker).

6K(31 )

_

and sequenced

on an

sequencer.

6K(43) 6K(54) 6K(70)

-

6K(90)

trypsin

chase (min) RESULTS To systematically study the effects on translocation of positively charged residues placed at different distances from a signal sequence, we have analyzed mutants derived from the well-characterized inner membrane protein leader peptidase (Lep) (24) and from a previously characterized Lep derivative in which the N-terminal hydrophobic region of the wild-type protein (Hi) has been turned into an uncleaved signal sequence (25) (Fig. 1). Both proteins require SecA for assembly (ref. 26; unpublished data) and thus utilize the normal secretory machinery. The periplasmic P2 domain is rapidly translocated across the inner membrane in wild-type Lep as judged by a standard trypsin accessibility assay (25) (Fig. 2A), but only slowly in the derivative (Fig. 2B), thus allowing us to observe the effects of positively charged residues in two different contexts. This is important, since it has been shown that an increase in the kinetic efficiency of a signal sequence can to some extent overcome the block imposed by one or a few positively charged residues, although the difference becomes minimal when the number of charged residues is increased (15). In an attempt to saturate the positive charge effect we thus inserted a string of six consecutive lysines 15, 27, 38, 54, or 74 residues from the end of H2 in wild-type Lep [i.e., after residues 91, 103, 114, 130, and 150; designated 6K(15), etc.] and 14, 21, 31, 43, 54, 70, or 90 residues from the end of H1 in the Lep derivative (i.e., after residues 59, 81, 91, 103, 114, 130, and 150 as numbered in the wild-type sequence) and assayed their effects on translocation. As shown in the pulse-chase experiments of Fig. 2, translocation of the P2 domain in wild-type Lep is essentially complete within a 1-min pulse. However, no translocation is observed with the 6K(15) construct, even after a 10-min

-

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1.0

C

0.8

0.6

t 0

0.4 z

4-

0.2

0.0 0

20

40 60 distance

80

100

FIG. 2. A string of six lysine residues blocks translocation when present 27 residues- or less downstream of the uncleaved signal sequence but not when placed 31 or more residues away. (A) Six-lysine insertions in wild-type Lep. (B) Six-lysine insertions in a Lep derivative where H1 functions as the translocation signal. (C) Fraction of molecules translocated during a 5-min chase as a function of the number of residues between the Hi or H2 regions and the six-lysine insertion.

chase. On the other hand, the P2 domain in mutants 6K(38), 6K(54), and 6K(74) translocates with wild-type kinetics. The 6K(27) mutant is markedly unstable, and no more than -30%1o translocates during the 10-min chase. In the case of the Lep derivative where H1 functions as a translocation signal, translocation of the parent is slow, with a half-time of =2 min.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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20 D 1

2

3

4

5

6

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7

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cycle

FIG. 3. Radiosequencing of [35S]methionine-labeled 6K(14) (stippled bars) and 6K(90) (hatched bars) proteins. The total activity of the 6K(90) sample applied to the sequencer was approximately half that of the 6K(14) sample.

The 6K(14) and 6K(21) constructs are not translocated at all, after a 10-min chase. Translocation in mutants 6K(43), 6K(54), 6K(70), and 6K(90) is as fast or even faster than in the parent, whereas mutant 6K(31) has an intermediary phenotype with little detectable translocation (-10%) after a 1-min pulse and partial translocation (-70%) after a 10-min chase. Although the reason for the faster translocation of the more C-terminal 6K mutants relative to the parent is not known, it may be caused by, e.g., an effect on the folding kinetics of the nascent protein (27). To obtain further insight into the orientation relative to the membrane of the translocated and nontranslocated mutants, we took advantage of the fact that the initiator fMet is retained on the periplasmically exposed N terminus of wildtype Lep and other Lep mutants with the wild-type orientation but is removed, presumably by the cytoplasmic deformylase and methionyl-aminopeptidase enzymes (28, 29), in Lep constructs that have an inverted orientation with the N terminus facing the cytoplasm (30). Since the N-terminal ., [35S]methiosequence of Lep (31) is fMet-Ala-Asn-Met nine-labeled protein will either be refractory to radiosequencing if the fMet is still present, or it will show a peak of activity in cycle 3 if the fMet is removed. When mutants 6K(14) and 6K(90) (H1 translocation signal) were subjected to radiosequencing, both gave a clear peak in cycle 3 (Fig. 3), suggesting that the N terminus is exposed to the cytoplasm, either stably or at least long enough to allow deformylation and methionine removal to take place, regardless of whether the P2 domain is translocated or not. Mutants 6K(15) and 6K(74) (H2 translocation signal) gave no sequence (data not shown), suggesting that H1 is inserted in its wild-type orientation (periplasmic N terminus) in both mutants. Thus, the sixlysine insertions appear only to affect the translocation of the P2 domain while leaving the part of the molecule upstream of the signal sequence in its normal location relative to the membrane. even

.

.

DISCUSSION The results presented above demonstrate the presence of a critical export initiation domain encompassing the signal sequence and an additional, well-defined downstream region. In the wild-type protein, the lysine stretch essentially blocks translocation when present 27 residues downstream of the H2 translocation signal, but it has no effect when placed at a distance of 38 residues or more. Similarly, when H1 is used as the translocation signal, the lysine-stretch prevents translocation from a distance of 21 residues but not when it is 42

9753

or more residues distant from H1. Interestingly, a construct where the distance is 31 residues is largely translocated but with markedly slower kinetics. Thus, although the H1 and H2 translocation signals and most of their downstream regions have completely unrelated sequences and support translocation with markedly different kinetics, the lengths of the critical domains defined by the lysine insertions are remarkably similar. When placed within =30 residues of the translocation signals the lysine stretch severely compromises an early step(s) of the translocation process; when placed only a few more residues further away, it has no observable detrimental effects. We note that although it is formally possible that the major effect of positively charged residues on translocation is indirect and may result from, e.g., different folding kinetics of the different constructs, the close correlation between the secretion phenotype and the distance of the six-lysine stretch from the translocation signal makes such alternative interpretations less likely. The existence of a sharply delimited export initiation domain extending -30 residues from the signal sequence is particularly interesting in light of the recent demonstration that a small N-terminal domain of SecA-bound pro-OmpA protein inserts sufficiently far across inverted E. coli inner membrane vesicles to allow cleavage of the signal sequence but not to allow the formation of detectable proteaseprotected fragments when incubation is carried out in the presence of a nonhydrolyzable ATP analogue (32). According to the loop or helical hairpin models of protein secretion (33-36), the signal sequence would initially form the N-terminal half of a loop inserting into the membrane with its Nand C-terminal ends both on the cytoplasmic side; the C-terminal half of this loop would consist of residues immediately adjacent to the signal sequence. We suggest that this postulated loop corresponds to the export initiation domain defined here and that its insertion into the membrane, triggered by the binding of ATP to the SecA protein complexed with the precursor and the membrane-embedded SecY/E protein (32), constitutes an early, critical step in protein translocation in E. coli. Antisera were gifts from Dr. Andreas Kuhn (Basel) (AraB) and from Dr. Ulf Henning (Tubingen) (OmpA). Purified Lep used to produce Lep antiserum in rabbits was a gift from Dr. Bill Wickner (University of California, Los Angeles). Oligonucleotide synthesis and radiosequencing were done by Zekiye Cansu and Anne Peters at the Karolinska Institute Center for Biotechnology; IngMarie Nilsson provided expert technical assistance. This work was supported by grants from the Swedish Natural Sciences Research Council and the Swedish National Board for Technical Development to G.v.H. We would also like to thank Dr. Jon Beckwith (Harvard Medical School) for useful suggestions.

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