.I. Mol. Riol.

(1984) 173, 243-251

How Signal Sequences Maintain Cleavage Specificity

GUNNAR VON HEIJNE

Research Group for Theoretical Biophysics

Department of Theoretical Physics
Royal Institute of Technology
S- IO0 44 &ockholm, Sweden

(Received 28 July 1983, and in revised form 26 October 19X.3)

The spe(ific*ity of’ the signal sequence cleavage reaction has been postulated to
rrsidr in a signal peptidase active site that can bind only to particular (i, i+Z)
pairs of amino acids. In this paper, we present further patterns of non-random
amino acid utilization in a region around in viva cleavage sites. and show that
thry can br interpreted in t,erms of selection acting to reduce the number of
potential rompeting sites in thr vicinity of the correct one.

1. Introduction
l’rot,eins destined for export are generally synthesized with a 15 to 25 amino acids
long, hydrophobic N-terminal extension that somehow initiates the export
process. This so-called signal sequence is removed from the protein once export is
under way through the action of an endoproteolytic “signal peptidase”. Usually.
the specificity of the cleavage reaction is very high, an observation that has been
hard to reconcile with the very limited degree of sequence homology found
amongst different signal sequences.
Tn a recent study (von Heijne, 1983), based on a collection of 78 eukaryotic
signal sequences, we showed that the region around the cleavage site shows strong
preferences for particular amino acids in particular positions. Small, neutral
rchsiducs abound in positions - 1 and - 3 (counting from the cleavage site between
positions - 1 and + 1) but are rare in -2. Conversely, aromatic, charged, and
large polar residues are absent from positions - 1 and - 3 (except Gln in - I), and
abundant in -2. Pro is absent from the region -3 to + 1 but quite common in
- -5. and Gly is found predominantly in positions - 4 and - 1. Upstream from
position -5 (eukaryotes) or -6 (prokaryotes). finally, hydrophobic residues
dominat,e st,rongly, forming a hydrophobic core in the middle part of the signal
sequences.
These observations led us to propose that an acceptable cleavage site must fulfil
a “(-3, -1) rule”, i.e. it must have either Ala, Ser, Gly, Cys, Thr or Gin in
position - 1, and must not have an aromatic (Phe, His, Tyr. Trp), charged (Asp.
(ilu, T,ys. Arg), or large polar (Asn, (iln) residue in position -3, as well as no Pro
2-M (;. voti HEI.JNE

in the region -3 to + 1. A similar suggest,ion has been made independently 1)~

Perlman & Halvorson (1983).
In this paper, we present further evidence in support of t,his model. and sho\t
that cells exploit’ the (-3, - 1) rule when designing cleavage sites. such that
cleavage ambiguities are avoided. In particular, we describe three different modes
for this ambiguity-reducing selection: (1) rrducaing the number of Ala residues
(and perhaps also Ser and Gly residues) in a region around the c~lravage site;
(2) putting “forbidden” residues (cf. above) in posit,ions such that a potential .‘- I
residue” does not give rise to an acceptable alternative cbleavagr site: and
(S) increasing the incidence of Ala residues, which may tit the signal peptidase
active sit,c better than any other residur. at the in c!iw cleavage sites. thus giving
these sites an edge over most compe’titors.

2. Materials and Methods

Twenty l)rokaryotic and 6.5 eukaryotic signal sequences are included iI1 this studs.
I’nless stated otherwise, the original references art: cited by Michaelis & Beckwith (1982)
(prokaryotir sequences) or von Heijne (1983) (eukaryotic sequences).
Prokaryotir sequences: phage Ml3 major and minor coat proteins. alkaline phosphatase,
maltose-binding protein, leucine-specific-binding protein. leucine/isoleucine/valine-binding
protein. histidine-binding protein. lysine/arginine/ornithinr-binding, protein. amp(’
p-lactamase. TEM p-lactamase, arabinose-binding protein, lam13 prot,em. ompA protein.
ompF protein, heat-labile toxin A and K subunits. r-amylase (Palva et al.. 1982). omp(’
Ijrotein (Mizuno et al., 1983). [IhoE: Ijrotein (C)l-erbeekr rt al., 1983). and Ilrotein ;1 (Liifdahl
et al.. 1983).
Eukaryotic sequences: rat whey phosphoprotein. human serum albumin. rat r-l acid
glycoprotein. mouse thgrotropin c( and ,6 subunits ((iurr et 01.. 1983). hagfish. angler&h and
human insulin. mouse embryonic \‘n immunoglobulin. rat rc immunoglobulin. mouse
H-chain immunoglobulin. mouse i, immunoglobulin. rabbit YH immunoglobulin (Bernstein
d al., 1982). caiman VH immunoplobulin (Litman rl al., 1983). human E-chain
immunoglobulin (Kenten et al., 1982),- ovine fl- and ti-casein. ovine x- and /Clactalbumin.
rabbit a-lactalbumin, cockerel VI,l)T,-II, bee melitt,in. rat tactin, human placental lactogen.
human choriogonadotropin g and fi subunits. rabbit uteroglobin, rat growt,h hormone.
bovine parathyroid hormone. human fibroblast interferon. human immune interferon. rat
relaxin. chicken tropoelastin K. chicken ovomucoid, chicken Igsozyme. chicken
conalbumin. human a- 1 an6trypsin. rat prostat’ic binding proteins Cl and C2. rat
apolipoprotein Al, adenovirus glycoprotein. vesicular stomatitis virus glyroprotein. rabies
virus (ERA and WS) glycoproteins (Yelverton rt al., 1983). human influenzaiYirtoria!
haemagglutinin. canine trvpsiongen 2 + 3. mouse liver amylase. rat carboxy-
peptidase A, Torpedo caZ$~~&n acetglcholine receptor CX.,6. 7 and 6 subunits (Soda et al..
1983), maize zein protein 222.1 (Marks & Larkins. 1982), human HLA-DR r- and PI-chains
(Lee et al., 1982; Long it al., 1983). mouse (‘3 complement (Wiebauer ef al., 1982), rat
pancreatic Rh’ase (MacDonald it al.. 1982), mouse opiomrlanocortin (L:hler & Herbert.
1983), rat pituitary glycoprotein hormone a subunit (Godine rt al., 1982). rat somat’ostatin
(Goodman rt al., 1983), human anti-thrombin ITT (Chandra et al., 1983). rat Thy-l
(Moriuchi et al., 1983). bovine AvpNpII hormone (Land et al., 1983), yeast invertase
(I’erlman h Halvorson. 1983) and hamster glucagon (Bell et al.. 1983).
The 2 samples have been analysed in terms of amino acid composition at each position
(counting either from the cleavage site or from the C terminus of t’he hydrophobic core. in
which case a subscript C is used. e.g. +2,). and have been screened for potential cleavage
sites other than the one used in viao, using computer programs written in FORTRAN.
The observed amino acid distributions have been compared with expected values
 (!I,EAvA(;E OF Sl(:NAI, SEQlIltNCES 245

c~nlculatrtl for binomial distributions with mean amino acid frequencies as given by Levitt
(1978) for rukaryotic proteins. and by Lehninger (1970) for Eschrrichia coli proteins. In
positions -3 and - 1, only acceptable residues have been included in the expected
distributions (with the same relative frequencies as in t,he tabulations of Leant and
I,?hllirlgtT).

3. Results and Discussion

If the postulated (-3. - 1) model is correct, then position + 1 is unique. It is
under selective pressure as part of the mature protein, it is close enough to the
cleavage sit,e to interfere with correct processing. but it is. at the same time. the
only residue in the mature chain that can be prevent’ed from competing in the
cleavage reaction by sequence modifications outside the mature chain: namely. by
putting a “forbidden” residue in position -2. Thus. it, is free to vary in response
to selection acting exclusively on the post-export functions of the mature protein,
whereas position:, +2 onwards will be selected both for post-export function and
for reducing cleavage ambiguities.
This uniqueness of position + 1 is readily apparent in Tables 1 and 2. ala.
which is by far t)he most preferred residue in position - 1, is quite common in
position + 1 (especially in prokaryotes) where, in 15 out of 16 cases, it is made
inoffensive by a forbidden residue in -2 (Table 2). In contrast, Ala is markedly
reduced in incidence in positions -2 and $2 to +5. Ser and Gly, the other t,wo
good - 1 residues. are more evenly distribut,ed (but note t,hat Gly is absent from
position - 3). with what is at best a very slight reduction in + 1 to + .5. Cys. Thr
and Gin. finally, show no obvious new patterns beyond the ( -3, - 1) rule. except
t hst they are very rare in position - 1 (data not shown). In prokaryotes. Cys. Thi
and (:ln have never been observed in this position.
Tnterestingly. both the number of alanines and the number of acceptable
cleavage sites increase quite dramatically at position -3, indicating that
positions upstream from -2 do not normally get tested by the cleavage enzyme.
All other residues are distribut,ed according to the patterns described earlier
(van Heijne. 1!483): in addition, charged and large polar residues seem to be
enriched in positions + 1 to +5 (there are 158 such residues in this region.
significantly more than the expected number of 121 (P > 0.95)).
Even when these new patterns of select,ion are taken into account,, however, a
relatively large number of acceptable, potential cleavage sites are still found in
the vicinity of the in viva sites. Some of these ambiguities may in fact represent
true alternatives. in the sense that they may be utilized in viva, generating
N-terminal heterogeneity in the mature proteins (see below), but most of them are
probably not used to any appreciable extent.
The strong preponderance of Ala in positions - 1 and -3 is perhaps indicat,ive
of a scale of substrate preferences at the signal peptidase active site. If so, t,hen
the final outcome of the cleavage reaction would be the result of competition
between the potential sites, with the order of preferences being Ala > Gly.
Ser > (‘ys. Thr > Gin ( > Asn !, see below) for position - 1, and Ala > Val > (1ys,
Ser. Thr > Leu. Tle > Gly for position -3. If this is correct. Gly. Ser. Cys, Thr
and C:ln n-ould not be expected to be under much negative selection near the
-r
+

TI

Y
 CLEAVAGE OF SIGNAL SEQUEXCES “47
1 MMAAGPRTSLLLAFALLCLPWTQVVG-A*FPAMSLSGLF

2 MNSQVSARKACTLLLLMMSNLLFCQN-VQT"LPVCSGGDCQ

3 MKLAITLALVTLALLCSPASA'G-ICPRFAXVI

4 MILCSYWHVGLVLLLFSCCCLVLG*S"EHETRLVAN

5 MENVRRMALGLVFMMALALSGVG-A*S-VMEDTLLSV

6 MGNIHFVYLLISCLYYSGCS-G*VNEEERLIND

7 NGLEKSLFLFSLLJLVLGWVQPSLG*G-ESSRDK

8 MLLQAFLFLLAGFAAKISA*S-MXXXXXXXX

9 MRYMILGLLALAAVCS-A'A-KKVEFKEPA

FIG. 1. Signal sequences with known (nos 1 and 2) or possible alternative cleavage sites in positions
-2 and + 1. Sequence 1, bovine growth hormone; 2, rat preprolactin; 3, rabbit uteroglobin:
4, acetylcholine receptor a subunit; 5, acetylcholine receptor b subunit; 6. acetylcholine receptor 6
subunit; 7, pancreatic RNase; 8, yeast invertase; 9, adenovirus glycoprotein. References are cited b?
van Heijne (1983) or in Materials and Methods. * The normal (or predominant) cleavage site;
*, acceptable alternative sites. Amino acids are given in the one-letter code; i.e. A, Ala; C, Cys; D, Asp;
E, Glu; F. Phe; G, Gly; H, His; I, Ile; K, Lys; I,, Leu; M, Met; N. Asn; P, Pro; Q, Gin; R, Arg; S. Ser:
T, Thr: V. Val: W, Trp; Y, Tyr; X, unknown.

cleavage site, as indeed they are not (Table l), since they do not compete well
with Ala for position - 1.
Still, there are examples of ambiguous cleavage to be found in the literature.
Bovine growth hormone is perhaps the best example, where 65% of the cleavage
takes place at an Ala residue, the remaining 35% having a neighbouring
acceptable Gly residue in position - 1, Figure 1 (Lingappa et al., 1977). Human
interferon, cloned into yeast cells, is also cleaved at more than one acceptable site
(Hitzeman et al., 1983). Finally, rat preprolactin is miscleaved when a more bulky
Thr analogue is incorporated at the normal cleavage site, cleavage now taking
place three residues upstream at an Asn residue (the nearest acceptable site.

TABLE 2
Number of forbidden and acceptable sites (forbidden : acceptable)
with Ala, Ser and Gly at the potential cleavage site

Position -5 -4 -3 -2 fl +2 +3 +4 +5

1:3 I:3 3:s 0 :0 10 : 0 0 :0 0 :0 0 :0 0 :0

2:5 4:3 12:6 2 :0 5: 1 1 1 1:0 I:1 0 :0
3:s 5:6 15.14 2:o I5:I 1 I I 0 I-1 0 :0

1 :o 0 :3 1:2 1 0 0 :0 0 0 0 :0 0 :0 0 :0
4:5 2 :0 7:" 3.2 2.3 14 3 1 0 :0 1:4
5:5 P:3 8:4 42 2-3 14 3:l 0 :0 1:4

0 :2 0: 1 0:O 0 :0 0 :0 0.1 0 :2 0:O 2 :0

0:5 4:12 0 :0 2:l 2:2 1.2 0 :0 212 4:l
cl:7 4: 13 0:O 2:l 212 1 3 0 :2 2:2 6: I

See the legend to Table 1 for abbreviations

 “1X (:. I-ON HEI.INI1:
presuming that Asn can. if forced, tit into the - 1 site at the signal peptidase)
(Hortin & Boime, 1981).
These observations suggest that’ some degree of miscleavage may be a not t,()o
uncommon phenomenon. In Figure I. WV give a c:ouple of examples of sequences
chosen from our data-base with acceptable sites next to the irr r+o site where
miscleavage might occur.
In addition to the (-3, - 1) rule, the position of the hydrophobic core of t,hr
signal sequence may caonceivably influence the relative strengt,h of csomprt,ing
sites. The (’ terminus of the hydrophobic segment is fairly well correlated with the
position of the in r?iz~ cleavage site. usually falling in position -6 (eukar-yot,es) or
-7 (prokaryotes) (T’erlman & Halvorson, 1983: van Heijne. 1983). Thus. an
acceptable site at position +5, or + 6, may have an increased cleavage
probabilit,y, further reducing thr ambiguity.
This correlation between the cleavage site and the position of t,he (’ terrninus of
the hydrophobic core, coupled with thfa observation that ambiguit.y-reducing
selection seems to be of much reduced intensity upst’ream from position -2, also
suggests that the tirst t,hree or four residues following the core C terminus are poor
substrates for the signal peptidasc>. If so. then the hydrophobic (‘or?. besides its
role in the initiation of the export process. also defines a “window” for cleavage:
this window would ext,end from residue +1, or $5, t,o residue + 10, or + 11, (thr
strongest ambiguity-reducing selection sterns t.o atfect the seven residues -2 to
+5 counting from the cleavage site, cf. above), i.e. the cleavage peptidase would
have to find an acceptable site in this region. or else no cleavage would take place.
Tn fact, only three out, of 39 signal sequemes analysed by Perlman K: Halvorson
(198.3) have their cleavage sites outside a + 4, t’o + 10, window.
Our working hypothesis, then, is that: (1) cleavage of the signal sequence is
influenced b-y the position of the (’ t’erminus of the hydrophobic core, which
defines an approximately seven-residues-wide window for processing in the region
$4, to + IO,: and (2) acceptable c*leavagt~ sites inside this window compete for
access to thr signal pept’idase active site, the outc*ornr depending on the “goodness
of tit“ of the various sites (cf. scales of preferrncsc> abovr). An acceptable site at or
nrxt. to positjions +.i, (eukaryotrs) or + 6, (prokaryotes) may have an extra edge
when competing for t,he signal pcptidase.
In addition to the statistical observations presented here. further evidence in
favour of this model comes from a few c:leavage-tlefiuirnt rnut’ants t’ha,t have been
isolated in various laboratories.
Two caasrs where the (-3. - I ) rulr is violated have been found so far. First. an
cx-amylase/fi-lactamase hybrid protein nhrrr t,he fusionjoint is such that no
acceptable cleavage sit.e remains does not get proc~sed. but is nevert,heless
rxported (Palva it al.. 1982; and personal cbommunication: see Fig. 2). Second. in a
b-lac:t,amase mutant where residues - 3 to -6 have been altered to a strrt,ch of
hydrophobic residues with a Phr (forbidden) in posit8ion -3, the protein is
cxport,ed but the signal sequence is not &aved (Koshland et al.. 1982). Two
additional mutants. where residue --3 is left t,htA same as in t’he wild-type (Val. an
acceptable residue in - 3), but, where a Pro in position -4 is changed into either a
Ser or a I,eu are also rxported. but csleavrd slowly and only to a limited extent
 CLEAVAGE OF SIGNAL SEQUENC’ES 210

10 MIQKAKRTVSFRLVLMCTLLFVSLPITKTSA*VNGTLMQYFEWYTP
lb QACPPETLVKVKDAEDPLCA

20 MSIQHFRVALIPFFAAFCLPVFA*HPETLVKVKDAEDQ
2b AFLF
2c s
2d L

30 MKKSLVLKASVAVATLVPMLSFA*AEGDDPAKAAFDSL
3b I.
3c Y

PIG 2. (‘Iravage-drficient mutant signal sequences. la. h, cc-amSlase/8-lactamase h~hrid protrin:

21 (1. p-lactamase; Xa (2, phage Ml3 coat protein (see the text). In all cases, entry “a” IS the normal.
l,lravage-c’omprtent sequence (*).

(Prop, to SW), or not at all (Pro-, to Leu). In these two cases, the (-3, - 1) rule
is not violated. Exchanging Pro-, for a more hydrophobic residue, however, has
t,he effect of extending the hydrophobic core towards the mature protein, and t’hr
window for cleavage defined by the core C terminus now encompasses not’ the
original cleavage site but part of the mature chain, where no potential site exists
(see Fig. 2).
A similar case has been described for the phage Ml3 coat protein, where an Asp
to 1,~ or Asp to Tyr (i.e. charged t*o hydrophobic) replacement in position +2 in
the mature protein slows down cleavage (Boeke et al., 1980). Again, cleavage
might be impaired in these mutants as the result of an erroneous window rather
than as t’he result of a direct effect on the signal peptidase active site.
Tn this discussion, we have deliberately left out cleavage mutants obtained for
the Iii. cnli lipoprotein (Inouye et al., 1983), since it is known that this and similar
lipoproteins. as well as a number of membrane-bound penicillinases (Nielsen B
I,ampen. 1982), are processed by a special signal peptidase t)hat does not cleave
other export~etl bacterial proteins (Tokunaga et al.. 1982).

4. Conclusion
The (-3, - 1) signal peptidase recognition site suggested in an earlier paper
(von Heijne, 1983), and also by Perlman & Halvorson (1983), is well suit’ed t)o
explain both results from studies on cleavage-deficient mutants proteins, as well
as the patterns of amino acid selection around the cleavage site presented here. In
particular, we suggest that the very low incidence of Ala in positions - 2 and + 2
to +5, and the observation that 15 out of 16 atanine residues found in position
+ 1 do not make acceptable cleavage sites according to the (-3. - 1) rule, show
t,hat a region approximately between positions - 2 and + 5 is subject to selection
aimed at reducing cleavage sit’e ambiguity. A st’retch of about seven residues
would thus be accessible to the signal peptidase active site. its position would be
determined by the position of the (1 terminus of the hydrophobic core (the
cleavage window would start 4 to 5 residues downstream from the core (’
t,erminus). and cleavage would take place at the “strongest”’ site allowed by the
( - 3, - 1) rule inside the cleavage window. In some cases, more than one site of
comparable st’rength may be used in ~ivo, giving rise to N-terminal heterogeneity
among the mature chains. A first attempt at formulat’ing a quantitative
2.50 G. VON HEIJNE

prediction scheme along these lines has been published (von Heijne, 1983).
Finally, the very low incidence of Cly in position -3 is noteworthy in the
context of a (-3, - 1) recognition site.

This work was supported by a grant from the Swedish Xatural Sciences Research
Council.

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Edited by 8. Brenner

Note added in proof: We have recently made an observation further underlining the
special status of Ala residues near the cleavage site (von Heijne & Flinta, unpublished
results): Ala when in position + I, is remarkably often followed in position + 2 by either
Pro (9 out of 20 cases found in our current data base of 130 eukaryotic and 29 prokaryotic
signal sequences) or Glu (6 out of 20 cases). Other residues in position + 1; such as Gly or
Ser. do not seem t)o correlate with the kind of residue found in position 12.