Download Sequences beyond the Cleavage Site Influence Signal Peptide

Vol. 263,No. 30,Issue of October 25, pp. 15791-15798, 1988
Printed in U.S A .
THEJOURNAL OF BIOLOGICAL
CHEMISTRY
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc
Sequences beyond the Cleavage Site Influence Signal Peptide
Function*
(Received for publication, April 29, 1988)
David W. AndrewsS, EvePerara, Cammie Lesser, and VishwanathR. Lingappa
From the Departments of Physiology and Medicine, University of California, Sun Francisco, California 94143-0444
The earliest events in protein secretion include tar- define the precise linear limits of a signal sequence. Moreover,
geting to and translocation across the endoplasmic
re- if cleavage is irrelevant for translocation per se it might be
ticulum membrane. To dissect the mechanism
by which possible that information beyond the signal cleavage site is
signal sequences mediate translocation ineukaryotes, important in directing chain translocation.
we are examining the behavior of fusion proteins and Examination of these issues became practical with the
deletion mutants incell-free systems. We demonstrate demonstration that cell-free expression of a plasmid conthat the protein domain being translocated can have
structed to encode a signal sequence at the amino terminus
profound impact on theefficiency of the translocation of a normally cytoplasmic protein, globin, results in transloprocess. Specifically, deletions in the mature prolactin cation of the encoded passenger protein to the ERlumen and
“passenger” domain, beyond thesignal cleavage site,
reduce the efficiency of signal function. The effect of normal cleavage of the signal (9). That study argued strongly
these deletionson signal function is observed whenthis that signal function is intrinsic to thesequence of amino acids
signal sequence is in its normal position, at the amino comprising the cleaved signal. While it did not address the
terminus, and when internalized by the addition
117of issue of the influence of sequences outside of the signal
amino acids of chimpanzee
a-globin. Alterations in the peptide, it did establish a system in which such questions
interaction ofthedeletionmutants
with the signal could be studied by using precise mutagenesis of both signal
and passenger coding regions.
recognition particle and with anothercomponentof
Recent work using similarly constructed fusion proteins
the translocation system, signal peptidase, were observed. Our results suggest that subtle changes in se- has reemphasized the enigma of selectivity and specificity of
quences beyond the signal cleavage site can alter the an obviously degenerate system for signal sequence recogniefficiency of co-translational translocation by affect- tion and function. A surprisingly large percentage of unseing various signal-receptor interactions.
lected genomic DNA fragments has been shown by Kaiser et
al. (10) to encode peptides with some signal-like activity in
yeast. Furthermore, Hurt and Schatz (11)have exposed the
The process by which a newly synthesized secretory protein potential that cytosolic proteins contain cryptic mitochonis efficiently targeted to the translocation machinery of the drial signal sequences by using gene fusion experiments.
endoplasmic reticulum (ER)’ is presumed to begin with the Nevertheless, cytosolic proteins do not, in fact, get transloemergence from the ribosome of a signal sequence. Using cell- cated across membranes while compartmentalized proteins
free translocation assays (1, 2) a subset of the protein com- are translocated, in general with high efficiency. While these
ponents involved in translocation has been identified (2-4). experiments further document the degeneracy of translocaTypically, signal sequences are cleaved from the protein dur- tion systems, the molecular basis for selective sequestration
ing or immediately after translocation to the ERlumen. For remains obscure.
The results presented here address directly the influence of
signal sequences in the form of amino-terminal extensions,
the site of cleavage has been presumed to define the carboxyl- protein domains outside the signal sequence on signal functerminal end of the signal. For this reason and because of the tion. We demonstrate that a series of progressive deletions
central role played by signal sequences in translocation, pre- beyond the signal cleavage site in preprolactin influence the
sequence signals have been studied in detail. At the amino interaction of these molecules with various components of the
acid level these sequences arenot similar, sharing only a ER translocation system. Our results also suggest how such a
common hydrophobic core, suggesting that receptors which recognition system, selective yet degenerate, may have
interact with signals recognizesome degenerate structural evolved. The implications of these findings for understanding
feature(s) (5). Furthermore, signal sequences are not neces- the phenomena elucidated by Kaiser et al. (10) and Hurt and
sarily cleaved to create the mature secreted molecule (6, 7), Schatz (11)are discussed.
demonstrating that cleavage is not an obligate event in protein
MATERIALS ANDMETHODS
translocation. These and other examples of functional unGeneral Methods-Restriction endonucleases and Bal31exonuclecleaved signal sequences (8) raise the issue ofhow best to
* This work wassupported in part by National Institutes of Health
Grant GM 31626.The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore
be hereby marked “aduertisement” in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
$Recipient of a Medical Research Council of Canada Fellowship.
‘The abbreviations used are: ER, endoplasmic reticulum; SRP,
signal recognition particle(s); SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
ase were obtained from Boehringer Mannheim or from New England
BioLabs and were used according to the manufacturers’ instructions.
Placental RNase inhibitor was from Promega Biotec (Madison, WI).
Rabbit anti-human hemoglobin serum was from Cappel Laboratories
(Cochranville, PA) and rabbit anti-ovineprolactin from United States
Biochemical Corp. (Cleveland, OH). Proteinase K was from Merck
(Federal Republic of Germany); endoglycosidaseH was from DuPont
New England Nuclear; [35S]Methionine Translabel was from ICN
Biomedicals (Costa Mesa, CA); and TritonX-100 was from Boehringer Mannheim. Canine pancreatic microsomal membranes and SRP
15791
15792
Signal Peptide Function
were prepared as described (12). SRP receptor was measured from
electrophoretic nitrocellulose blots of membrane proteins separated
by SDS-PAGE. Blots were developed by incubation with lZ5I-labeled
antibody directed against purified receptor using a purified receptor
standard, both generous gifts of Dr. Shoji Tajima (National Cardiovascular Center Research Institute, Osaka, Japan).
Recombinant DNA Constructs-Construction of the plasmid encoding the parentmolecule (GsP) thatis composed of 117 residues of
chimpanzee a-globin, containing an eight amino acid glycosylation
site insertedat residue 19, fused to theamino terminusof the complete
coding region of bovine preprolactin has been described (13). Deletions were introduced in the DNA (pSPGsP) which encodes GsP by
Bal31 digestion from the PuuII site corresponding to amino acid 58
of the mature prolactin domain. After Bal31 digestion the linear
plasmid molecules werecut within the polylinker with PstI to remove
all prolactin sequences carboxyl-terminal to the deletion end point,
treated with mung bean nuclease, and recircularized with T4 DNA
ligase. An in-frame termination codon is provided by the XbaI site
whichfollows the PstI and SalI sites in the polylinker of pSP64.
Plasmids were selected by antibiotic resistance, screened by restriction endonuclease digestion, and sequenced by the dideoxy method
for double-stranded plasmids (14). The resulting plasmids encode the
prolactin signal sequence and varying lengths of mature prolactin a t
the carboxyl terminus of the globin coding region and were used for
construction of expression clones. BstEII-Sal1 fragments were purified from these molecules after the SalI site was blunted by mung
bean nuclease treatment. The fragments that encode part of the
globin domain, the signal sequence from prolactin, and mature prolactin to the deletion point were ligated into BstEII-PuuII sites of
pSPGsP. Corresponding mutants with amino-terminal signal sequences were constructed from these by simply removing the globin
coding region by digestion with NcoI followed by religation of the
plasmids.
Transcription-linked Translation-Transcription of SP6 plasmids
was as described previously (13). Aliquots of the transcription reaction mixture were used directly in the translation reactions at a final
concentration of 20%. Translation reactions of this kind have been
described for reticulocyte lysate (13) and wheat germ extract (12).
Proteins synthesized in uitro were labeled by [35S]methionineincluded
in the reaction and visualized by autoradiography after separationby
SDS-PAGE. Protein processing and translocation assays including
densitometry were as described (12).
Translation Arrest Assays-Interaction of nascent preproteins with
signal recognition particle was assayed as signal recognition particle
(SRP)-mediated elongation arrest and subsequent release of this
arrest by detergent-solubilized membranes in wheat germ extract
translation reactions. To measure elongation arrest 30-50 nM SRP
was added to translation reactions which did not contain microsomal
membranes. Translation was allowed to proceed at 24 "C for 1h and
terminated by chilling on ice. This range of SRP concentration was
sufficient to give 50-90% inhibition of synthesis of wild type preprolactin (sP). After separation by SDS-PAGE, bands were visualized
by autoradiography and quantified by scanning densitometry. Percent
inhibition was defined as:
(
percent inhibition = 1 -
preprotein(+)
preprotein(-)
X
X
globin (-)
globin(+)
)
x 100
where globin was used as an internalcontrol for total synthesis and
the (+) or (-) indicates presence or absence of SRP, respectively.
When detergent-solubilized membranes are added to translation reactions, the apparent percent inhibition of synthesis by SRP is much
lower due to the arrestreleasing activity of the SRPreceptor (15,161
and signal processed by solubilized signal peptidase (17). Percent
inhibition for translation reactions including detergent-solubilized
membranes is calculated the same as without membranes, except that
preprotein synthesis must be corrected to include those molecules
processed by signal peptidase. In making this correction the loss of
one methionine in the signal sequence is included. Percent inhibition
release by solubilized membranes is calculated as:
percent release
)
percent inhibition(-mb) - percent inhibition (+mb)
x 100
percent inhibition(-mb)
=(
where globin was used as an internal control for total synthesis and
the (+mb) or (-mb) indicates presence or absence of solubilized
membranes, respectively.
Signal Cleauage-Removal of presequence signals by peptidase is
a convenient and reliable assay for translocation in cell-free translation reactions supplemented with intact microsomes. Such processing
can also be assayed independent of translocation by adding detergentsolubilized SRP-depleted membranes to thewheat germ extract translation reactions during or subsequent to translation (17). Percent
processing is calculated as:
percent processing =
processed protein
x 100
processed protein preprotein
+
where processed protein is corrected for the loss of one methionine
in the signal. To assay signal peptidase posttranslationally, cycloheximidewas added totranslations after 1 h a t 24 "C, solubilized
membranes were added, and incubation was continued for an additional hour.
RESULTS
To examine the possibility that sequences beyond the cleavage site influence signal sequence function, we constructed
the series of deletion mutants described in Fig. 1.The mutants
were derived from two parental molecules bearing signal sequences at either the amino terminus (sP) or at an internal
position (GsP). Authentic bovine preprolactin (sP) was chosen because it contains an amino-terminal cleaved signal
sequence. Moreover, this signal sequence has been thoroughly
studied with respect to function in uitro (18) and in vivo (19)
as well as for interaction with specific receptor components
(20, 21). We also chose the molecule, referred to here as GsP,
in which the same prolactin signal has been moved to an
internal location by fusing 110 amino acids of cy-globin, containing in addition an 8-residue glycosylation site, to the
amino terminus of preprolactin. Previous studies of this molecule have shown that theinternalized signal sequence interacts normally with SRP (12), mediates translocation of both
flanking protein domains, and is cleaved by signal peptidase
(13). Our approach was to determine the effect on prolactin
translocation of a series of progressive deletions introduced
in the coding region for mature prolactin in plasmids encoding
GsP and preprolactin.
The predicted amino acid sequences at thejunctions of the
particular deletion mutations generated and examined are
illustrated in Fig. 1. These constitute a family of deletion
mutants in which amino acid residues 22 to 58 (Gs+22p), 14
to 58 (Gs+l4p), 11 to 58 (Gs+llp), 1 to 58 (Gs+lp) and -7
to 58 (Gs-7p) of prolactin were removed from the parental
molecules. In addition to the deletion, the cloning strategy
employed resulted in a single amino acid substitution in four
of the five mutants. These include changing valine to glycine
for Gsf22p,leucine to glycine for Gs+14p,proline to glycine for
Gs+'p, cysteine to serine for G s - ~ ~while
, Gs+"p has no
substitution.
Our translocation assay system includes cell-free transcription of the constructed plasmids using SP6 RNA polymerase
followedby translation of the transcription products in a
wheat germ or a reticulocyte lysate protein synthesizing system. Translations arecarried out in the presence and absence
of canine pancreaticmicrosomal membranes. As a convenient
quantitative assay for signal function, we use percent processing calculated from the measured optical density of the
relevant bands on autoradiograms. Translocation of the passenger domains for all of the molecules wasconfirmed in both
reticulocyte lysate and wheat germ extract by protection of
the processed but not unprocessed moleculesfromexogenously added proteinase K, with protection abolished by
solubilization of the membrane with non-ionic detergent.
Deletions in the prolactin coding regions reduce, but do not
Signal Peptide Function
I
flobinpreprolactin
csp
cs+22p
Cr+ldp
Cr+llp
Cr+ Ip
cs-lp
-11
-18
I
15793
.I#
.I1
.SI
-119
THT-NNN~
K ~ ~ S ~ C ~ ~ ~ T M D S I C S S ~ K C ~ R ~ ~ ~ ~ ~ V ~ S : ~ ~ ~ G O C V V S ~ P V C P N C P C : C P ~ S ~ R ~ ~ ~ ~ R ~ " ~ ~ S H ~ I . . .
...D R I C C H I - . N N N ~
r
.~.QVSCCHl..~NNNC
-.PCNCPHT-.NNNT
...VSlCC~l...NNNC
.-~~LSTII~-NNN~
FIG.1. Amino acid sequencesurrounding the deletions introduced. The nomenclature for these molecules
is as follows: G, 117 residues of chimpanzee a-globin containing aglycosylation site; s, preprolactin signal sequence;
the number which follows s indicates the number of amino acids of prolactin remaining beyond the cleavage site
of the signal; P,full length mature prolactin; p , prolactin passenger, beginning a t amino acid 58 with the sequence
CHT andcontinuing to theend of prolactin a t residue 199 (openarrows).
46-
.-
80
L
m
30-
0
0
;60
C
2
k
40
I a.
12.
20
0
SP
0
40
r+22p
26
20
0
40
s+llp
26 20
0 2640
28
s+ l p
FIG.2. Translocation of mutants with amino-terminal signals at different membrane concentrations. Each clone is identified by name below the first lane of each series. SP6 polymerase
transcription products were translated in wheat germ extract. Translations (10 pl) were supplemented with 1pl of microsomal membranes
diluted to 40, 26, or 20 A m units/ml as indicated. Molecules were
fractionated by SDS-PAGE and visualized by autoradiography as
total products. The relative position of molecular mass markers is
indicated in kilodaltons.
abolish, translocation. Translocation assays for three of the
amino-terminal signal sequencebearing deletion mutants performed in the wheat germ cell-free system at three different
membrane concentrations are shown in Fig. 2. In thisexample
comparison of the intensities of the upper (non-translocated)
and lower (translocated) bands for the three deletion mutants
reveals that ateach membraneconcentration used the relative
number of processed molecules decreases as the deletion extends toward the signal cleavage site. The effect does not
depend on membrane concentration; however, it is most easily
visualized in this experiment when 1 p1of 26 A m units/ml
microsomes are added to a 10-pl translation reaction. Under
these conditions the lower band representing processed molecules decreasesin intensity markedly, whereasthere appears
to be some additional darkening of the upper band as one
goes from s+'$ to s+'p, respectively.
Densitometry of autoradiograms permits quantification of
the percent processing at all membrane concentrations used.
The results of an experiment similar to that shown in Fig. 2
but employing all of the prolactin deletion mutants are depicted in Fig. 3. The trend illustrated in Fig. 2 extends over
the range of mutants analyzed with s+*$ translocated close
to wild type efficiency and further deletions resulting in a
gradual decreasein translocation efficiency. Protease protection assays confirmed that all of the molecules translocated
were processed by signal peptidase as no fulllength protected
molecules were observed,
data not shown. Efficient processing
was observed even for S - ~ Pwhich lacks the original cleavage
r+22p
r+l4p
s+llp
r+lp
r-7p
Signal
FIG.3. Quantification of translocation efficiency for mutants with amino-terminal signals. Percent translocation is de-
fined as the ratio of processed molecules to the total (unprocessed +
processed molecules) when translation is performed in wheat germ
extract in the presence of three different concentrations of membranes. The names of each deletion mutant are indicated below the
A m units; hatched bar, 2.6 X loT3
histograms. Solid bar, 4.0 X
Am units; stippled bar, 2.0 X
Am units microsomes. The results
of one experiment are shown; nd, not done.
site entirely, indicating that another cryptic signalpeptidase
site is present in this mutant. This cryptic site is presumed
to be near the deletion junction as the migration position of
the mature portion, -7p, is consistent withcleavage at a
consensus sequence observednear this point.
The same deletions in the prolactin domain of GsP also
reduced translocation efficiency. However, translocation of
molecules with internal signal sequences in the wheat germ
cell-freesystemis of such low efficiency that the values
obtained for the deletion mutants rapidly fell to zero (data
not shown). In order to increase total translocation such that
meaningful values could
be recorded forthe deletion mutants
with internal signals, we used the reticulocyte lysate cell-free
systemsupplementedwithsalt-washedmicrosomalmembranes. Washing membraneswithhigh
concentrations of
potassium salts greatly reduces the nonspecific inhibition of
protein synthesis observedwhenmicrosomes are added to
translation reactions, which limits the amount of membranes
usefullyadded to cell-free translations. This processalso
extracts endogenousSRP bound to themicrosomes. By allowinglarger additions of microsomes, salt washing permits
higher overall translocation to be achieved. In addition to
providing SRP to replace that lost in salt washing, use of
reticulocyte lysate results in increased targeting efficiency
over the wheat germ system, whichalso results in higher
overall translocation. We used this inherently more efficient
translocation system to examine the effect of the deletions on
translocation efficiency forboth amino-terminal and internal
signal sequences.
Signal Peptide Function
15794
As expected the percent translocation in reticulocyte lysate
far exceeds that obtained withwheat germ extract. Nevertheless, when the amino-terminal signal containing deletion mutants characterized in Figs. 2 and 3 are translocated in reticulocyte lysate the trendobserved in wheat germ persists. The
translocation propertiesof several of these molecules in reticulocyte lysate are illustrated in Fig. 4.Wild type preprolactin,
Fig. 4, lane I, in the presence of microsomes is efficiently
processed to prolactin, lane 3, and becomes protected from
exogenously added proteinase K, compare lanes 2 and 4;
however, sensitivity to the protease is restored when the
membrane is solubilized with detergent, &ne 5. Protection
from exogenous protease is observed only for processed molecules. Complete protease protection assays are presented in
Fig. 4 for s+*%, lanes 6-9;s+'p, lanes 10-13; and s-~P,lanes
14-17.The results of a control experiment showing the lack
of translocation activity for the passenger domain, p, alone is
presentedin lanes 18 and 19. Processing of the deletion
mutants decreases as described above for the wheat germ
system, compare the upper and lower bands in lanes 3, 7,11,
and 15.The results of quantification of translocation observed
in two independent experiments under these conditions are
displayed in Fig. 5.Although in reticulocyte lysate the percent
translocation is higher for all of the molecules examined, the
decline in efficiency due to the deletions in the coding region
of prolactin is reflected by the decline in processing from
100% to justover 40%.
The same deletions in the prolactin domain of GsP reduce
translocation efficiency markedly as shown in Fig. 6.Protease
protection assays are presented in this figure for wild type
GsP, lanes 1-5, and Gs+14p,lanes 6-10.Comparing the translocation of prolactin as determined by signal cleavage in lanes
3 and 8 illustrates the considerable decrease in efficiency due
to the deletion which commences 14 amino acids beyond the
cleavage site. Translocation is not observed when a molecule
containing the two coding regions but no signal sequence (Gp)
is assayed, Fig. 6 lanes 11-14.Quantification of translocation
efficiency for deletions carboxyl-terminal to thecleavage site
of the now internalized signal sequence of prolactin reveals a
dramatic decrease in translocation efficiency as shown in Fig.
7. As described above for the amino-terminal signal bearing
molecules, the magnitude of the effect increases as the deletion end point nears the signal. This similarity suggests that
the reduction in translocation due to deletions in the domain
following the signal is a phenomenon independent from the
reduction in translocation efficiency observed due tothe
addition of a protein domain in front of the signal sequence
(Fig. 4, lane 3, and Fig. 6, lane 3, also Ref. 13).
Extending the deletions in the GsP parent moleculeby
truncating the plasmid PsPGs+lp at the deletion end point
results in afusion protein encoding 117 residues of chimpanzee a-globin, including a glycosylation site, followed by the
signal sequence from prolactin and one aminoacid of mature
prolactin at the extreme carboxyl terminus of the molecule.
That such molecules score as translocation positive in our
assay system, albeit with very low efficiency as shown in Fig.
8, confirms our earlier suggestion that theability to mediate
translocation is intrinsic to thissignal sequence (9). Notethat
the small number of molecules which become glycosylated
when translated in the presence of membranes, lanes 3 and 5
arrows, are well protected from protease, lanes 4 and 6.Endoglycosidase H sensitivity was used to confirm our identification of this band (data notshown).
Although the prolactin signal sequence is able to direct the
translocation of all of the mutants, the marked decrease in
efficiency associated with the deletions suggests one or more
steps in the process are significantly impaired. To examine
the mechanism underlying the reduction intranslocation
efficiency, we assayed the interaction of the nascent polypeptide chains with two components of the translocation apparatus, SRP and signal peptidase. For these assays we employed the wheat germ translation system because it does not
contain endogenous SRP. The results of these experiments
for the deletion mutants with amino-terminal signal sequences are summarized in Fig. 9.
Translation of preprolactin in the presence of exogenously
added SRP, without membranes, in the wheat germ system
results in an inhibition of synthesis due to direct interaction
of the signal sequence with SRP (22). To quantify this interaction we used a subsaturating concentration of SRP which
results in apartial inhibition of synthesis, as theseconditions
should be the most sensitive to perturbation. In Fig. 9, panel
A , 30 nM SRP is demonstrated to be subsaturating, resulting
in 65% inhibition of synthesis of the wild type molecule, sP,
whereas at 40 nM SRP thisvalue increased to 95%. As is also
shown in Fig. 9, panel A , the translation arrest induced by
either 30 or 40 nM SRP is indistinguishable from that displayed by wild type for each of the deletion mutants.
Interaction of the SRP arrested translation complex with
the SRP receptor releases arrest concomitant with initiation
FIG.4. Localization of translocated protein domains by proteolysis of translation products. Each
clone is identified by name above the first
lane of each series. SP6 polymerase transcriptionproducts
were translatedin
reticulocyte lysate inthe presence or
absence of salt-extracted microsomal
membranes and subjected to posttranslational proteolysis with proteinase
K. Molecules were fractionated by SDSPAGE and visualized by autoradiography as total products. Designations are
as follows: Mb, dog pancreas microsomes; Pk, post-translational digestion
with proteinase K; Det, 0.1% Triton X100. The relative position of molecular
mass markers is indicatedin kilodaltons.
45,
I
15'
Mb
F k - +
Del-
1
2
+ + + - + + + - + + + - + ++ ++ +- ++
-- +- +.+ --. - -+ -+ +- -- +- -+ +- -" _
t - 3
4
5
6
7
8
9
10
11 12 13
14 15 16 17
18 19
Signal Peptide Function
120
15795
1
I
6o
S+NP
s+22p
SP
s+lp
s-7p
CSP
Gs+22p
C s +Cl 4s p+Cl sl p+ l p
FIG.5. Quantification of translocation efficiency for mutants with amino-terminal signals in reticulocyte lysate. Percent translocation is defined in Fig. 3. The names of each deletion
mutant are indicated below the histograms. Salt-washed microsomes
were added as in Fig. 7 . Stippled bar and hatched bar represent two
independent experiments.
cs-7p
Signal
Signal
FIG. 7. Quantification of translocation efficiency for mutants with internal signals in reticulocyte lysate. Percent translocation is defined in Fig. 3. The names of each deletion mutant are
indicated below the histograms. Sufficient salt-washed microsomes
were added to translocate 50% of GsP molecules. The results of one
experiment are shown.
CS-7
B
CCP
SP
Cs+l4p
151
Mb -
-.
Det 1
2
Pk-
+
-I
3
t
+
4
FIG.6. Localization of translocated domains for mutants
with internal signals. Each clone is identified by name aboue the
first laneof each series. SP6 polymerase transcription productswere
translated in reticulocyte lysate in thepresence or absence of microsomal membranes and
subjected to post-translational proteolysis with
proteinase K as described (17). Molecules were fractionated by SDSPAGE and visualized by autoradiography as total products. Designations are as in Fig. 4. The relativeposition of molecular mass
markers is indicated inkilodaltons.
MbPk+
1
- + + + +
- - c - +
2
3
4
5
6
FIG.8. Localization of translocated protein domains bypro-
teolysis of translation products. Each clone is identified by name
aboue the first lane of each series. SP6 polymerase transcription
products were translated in reticulocyte lysate in the presence or
absence of microsomal membranes and subjected to post-translational proteolysis with proteinase K. Molecules were fractionated by
SDS-PAGE andvisualized by autoradiography following immunoprecipitation with antiserum to human a-globin (Cappel Laboratories);
of translocation (15).Thus, interaction with the SRP receptor
lanes 5 and 6 are the same as lanes 3 and 4 only 3 times longer
is the next assayable step in the sequence of events leading exposure. Arrows indicate the glycosylated protease protected prodto translocation. T o measure release of SRP-mediated inhi- ucts. Designations are as inFig. 4. The relative position of molecular
bition of synthesis for these deletion mutants, we supple- mass markers is indicated in kilodaltons.
mented wheat germ translations with intact or detergentsolubilized microsomes. Addition of detergent-solubilized
membranes permits arrestrelease to be monitored independent from translocation. S R P was added to these reactionsat
40 nM in order to maximize the range of values possible for
percent release of inhibition of synthesis.
As expected the deletion mutants were translocated with
reduced efficiency compared withsP, when S R P depleted, but
otherwise intact microsomes were added to the translation
reactions. Moreover, the pattern of decreasing translocation
efficiency as the deletion end point nears the cleavage site
was, with one exception, the same as reportedabove, see Fig.
9, panel B, open bars. Translocation decreased from 80% for
the wild type molecule SP to 48% for s-'p. Surprisingly, the
translocation of s+'%, 41%, was the lowest obtained for any
of the mutants under these conditions. This molecule was
consistently translocatedwith lower efficiency in this system
even when the concentrations of SRP and microsomes were
varied, Fig. 9, panel B, hatched bars. This effect, seen only
whentranslocationdependson
added exogenous purified
SRP, suggests the lower efficiency observed for s+'% may
result from aberrant SRPdirected targeting.
T o measure release of S R P arrest, independent of translo-
Signal Peptide Function
SP
loo
S+22p
S+14p
S+llp
S+lp
S-7p
T
cation, wheat germ extract translation reactions both with
and without SRP were supplemented with salt-washed detergent-solubilized microsomes (15). To optimize the sensitivity
of this assay, solubilized membranes were added such that the
final concentration of SRP receptor, approximately 1.7 nM,
resulted in a partial release, 65%, of the SRP induced inhibition of synthesis. In contrast to the relatively uniform inhibition of synthesis described above for the deletion mutants,
Fig. 9, panel A, the extentto which this SRP-mediated arrest
could be overcome by solubilized membranes varied dramatically, Fig. 9,panel C. Release of inhibition was much less than
wild type for the deletion mutants s+14pand s+llp,whereas it
wasmore than 85% complete, greater than wild type, for
mutants s+'Zp and s-~P.
Differences between the deletion mutants were also observed when co-translational accessibility to signal peptidase
was assayed in wheat germ translation reactions devoid of
SRP, but supplemented with detergent-solubilized SRP-depleted microsomes, Fig. 9, panel D. In the two experiments
shown the sensitivity of sc2$ was the same as wild type sP,
while s+14pand s+"p were much more sensitive and s+'p and
S-~Pwere much less sensitive to cleavage. To confirm signal
peptidase was acting co-translationally the assays were also
performed post-translationally. For sP, sensitivity to peptidase was approximately 2-fold greater when detergent-solubilized membranes were added co-translationally, resulting in
52% rather than 27% of molecules being cleaved. Some but
not all of the deletion mutants were also more susceptible to
processing when peptidase was presentco-translationally,
data not shown. These alterations in signal peptidase sensitivity, dependent only on the time of enzyme addition, constitute prima facia evidence for processing occurring during
protein synthesis in this co-translational
solubilizedpeptidase
assay.
80
DISCUSSION
60
40
20
0
SP
loo
S+22p
S+14p
S + l Sl p+ Sl p- 7 p
The results of our translocation assays demonstrate that
the precise sequence of a secreted protein domain can influence the ability of the signal peptide to direct its translocation
across the ER membrane. The sequences that compose the
prolactin passenger domain are normally translocated with
high efficiencyas demonstratedby the parentalconstructions
sP and GsP. Therefore, our finding of diminished passenger
translocation for the deletion mutants would appear to reflect
a secondary effect of protein folding which either masks the
signal directly or generates a substrateless easily transported.
A similar finding, demonstrated in prokaryotes, suggests that
this influence of protein domains beyond the cleavage site
may be a general phenomenon. In that study deletions near
the amino terminus of the "passenger" domain of a secreted
fusion protein appear to abolish signal function (23). Furthermore, in prokaryotes effects on protein translocation due
to alterations in the passenger domain are not limited to the
sequences immediately following the signal. A single amino
acid substitution 183 amino acids from the site of cleavage
has been shown to completely abolish translocation of the
I
40
20
SRP, 40 nM; microsomes at 1.7 nM SRP receptor; hatched bars, SRP
40 nM; microsomes a t 1.3 nM SRP receptor. Panel C, percent release
SP
S+22p
S+14p
s + l lsp+ lSp- 7 p
FIG. 9. Interaction of deletion mutants with SRP and signal
peptidase. The deletion mutants are identified by name below the
panels. Panel A , X, inhibition of translation in wheat germ extract
reactions supplemented with 30 nM SRP; open boxes, 40 nM SRP;
Panel B, translocation in wheat germ translation reactions supplemented with SRP and intact SRP-depleted microsomes. Open bars,
of inhibition of synthesis. Wheat germ translation reactions were
supplemented with SRP, 40 nM, and detergent-solubilized microsomes as 1.7 nM SRP receptor. Averages for two separate experiments
are shown. Error barsrepresent the average range of values obtained.
Panel D, percent processing by signal peptidase. Reactions were
supplemented with detergent-solubilized membranes at the onset of
translation at 1.7 nM SRP receptor. Averages for two independent
experiments are shown. Error bars indicate the complete range of
values obtained for each mutant.
Signal Peptide Function
maltose binding protein in Escherichia coli (24). Alterations
distal to the cleavage site are more likely to affect translocation inprokaryotes because it is believed to occur after protein
synthesis is complete. In eukaryotes, where translocation
normally occurs co-translationally, shorter range folding interactions might be expected to influence the efficiency of the
process. Our observation that the effect of deletions in the
prolactin passenger gradually diminishes such that translocation efficiency returns to wild type levels by approximately
amino acid 22 is consistent with this prediction. Moreover,
deletions in the prolactin molecule beyond approximately
residue 22 in prolactin were shown not to significantly affect
signal function in a related series of mutants.'
Previous studies have demonstrated that a signal sequence
is both necessary and sufficient to mediate protein domain
translocation across the ER membrane (9). Such results led
to the conclusion that passenger domains do not contribute
significant information to theprocess. Indeed, it has generally
been presumed that theefficiency with which a given protein
domain is translocated is determined by the particular signal
sequence employed. The results presented here argue that the
specific sequence of the protein domain to be translocated
can have a marked effect on the efficiency of translocation.
In addition to the deleted amino acids four of the five
mutants also have a single amino acid substitution at the
deletion point. For the most part these substitutions appear
to be relatively conservative as they neither add nor delete
charges nor do they introduce proline residues. However, in
the molecule with the deletion end point s+' the substitution
does replace the proline normally found at residue +2 with a
glycine.
Although the precise explanation for the decreases in translocation efficiency observed remains to be elucidated, the
effects of the deletions on the interaction of the nascent
polypeptides with SRP and signal peptidase implicate preprotein folding in the process. All ofthe deletion mutants retained
the ability to interact with SRP to arrest translation,Fig. 9,
panel A. However, the extent to
which the resultant inhibition
of synthesis could be released by solubilized membranes suggests that the affinity of the different deletion mutants for
SRP varies dramatically. The percent release of the SRPdependent inhibition of synthesis for the deletion mutants
varied from 11 to 92%,Fig. 9, panel C.These same conditions
result in 65% release of the inhibition of synthesis for the
wild type molecule, sP. Values for percent release of inhibition
greater than 65% may be interpreted as suggesting the mutants, s+**pand s?p, have a lower affinity for SRP, whereas
S+"P appears to interact with SRP much more tightly. Although the effect is smaller, s+I4pmay also bind SRP such
that release by solubilized membranes is less efficient than
for sP. In the arrested state SRPhas been shown to interact
with the signal sequence of the nascent polypeptide directly
(22). The possibility that SRP also interacts with sequences
beyond the signal sequence has not yet been examined. However, the lack of either identifiable sequence homologies or
clusters of amino acids with similar properties, such as the
hydrophobic stretch characteristic of signal sequences, in this
region of secreted proteins ( 5 ) suggests that structure rather
than sequence recognition is required for efficient translocation. Moreover, in four of the five deletion mutants examined
here the signal sequences are identical, suggesting that the
differences in affinity for SRP are due to alterations in the
rate or patternof nascent preprotein folding.
Altered protein folding may also explain the differences we
observed for processing of the deletion mutants by signal
'D. W. Andrews and E. Perara, unpublished data.
15797
peptidase. Susceptibility to enzymatic degradation has been
used as a sensitive indicator of protein conformation (25). It
is possible to use signal peptidase in such degradation assays
because the enzyme can be released from microsomes in an
active form. Processing by solubilized signal peptidase is a
particularly relevant indicator of protein structure for the
deletion mutants employed here because the enzyme cleaves
the nascent polypeptide chains at a single site, the carboxyl
terminus of the signal sequence. As judged by this assay, the
deletion mutants fold such that the peptidase site is more
accessible to the enzyme for mutants s+I4pand s+"p than in
the wild type molecule and virtually inaccessible for s+'p and
s-p.
Examination of the precise sequence of the deletion mutants, shown in Fig. 1, reveals that the region between s+"p
and s+'p deleted in these molecules contains 3 proline residues, at positions +2, +5, and +8. The tantalizing possibility
suggested by the inaccessibility of these mutants to cleavage
by solubilized signal peptidase would involve one or more of
these prolines in directing molecular folding such that the
signal sequence cleavage site remainsaccessible to theenzyme
in this assay and is notmasked by the rest of the molecule.
Although the mutants s+'p and S - ~ Pare not cleaved when
solubilized signal peptidase is added to translation reactions
eitherco-translationally or post-translationally(datanot
shown),these molecules are efficiently processed during
translocation, compare Figs. 5 and 9. Moreover, we have not
determined the site of cleavage for the deletion mutants.
Therefore, we do not know if the same cleavage site is used
in all of the molecules. A different cleavage site must be
employed when s?p is cleaved when the original site was
removed by the deletion. For this reason alterations in signal
processing in solubilized signal peptidase assays cannot be
correlated directly to effects on translocation efficiency. One
interpretation for this difference in susceptibility to cleavage
is that the conformation of the nascent polypeptide is different when the molecule is undergoing translocation than during synthesis in the presence or absence of detergent-solubilized membranes. Other less likely explanations would require
a subtlechange in specificity of the enzyme when membranes
are solubilized.
Our assays of SRP binding and signal peptide cleavage
demonstrate that deletions in the mature portionof prolactin,
beyond the cleavage site of the signal sequence, alter functional recognition of the signal sequence. However, neither of
these alterations correlates with the translocation efficiency
measured for the deletion mutants. One interpretation of this
observed lack of correlation is more than one lesion was
introduced in the molecules as a result of the deletions. In
this view the differences in translocation efficiency observed
may be dueto thecombined effect of suboptimal interactions
with various components involved in translocation. An attractive aspect of this proposal is that it is consistent with
the altered pattern of translocation efficiency observed in
wheat germ when translocation depends on exogenous purified SRP rather than membrane-associated SRP, compare
Figs. 3 and 9, panel B. A different yet related interpretation
would suggest a single progressive lesion in the molecules
which inhibits an as yet uncharacterized step in the translocation process. In this view the alterations observed in SRP
binding and/or signal peptide cleavage are symptomatic but
unrelated to theprimary defect.
Nevertheless, taken together these assays provide compelling evidence that deletions beyond the signal cleavage site in
prolactin alter preprotein folding andthereby reduce the
translocation efficiency of the molecules. Furthermore, rela-
15798
Function
Peptide
Signal
tively small deletions in the normally secreted portion of the
molecule, onthe order of 10 amino acids, can profoundly alter
the recognition of the signal sequence by SRP and signal
peptidase. For example, deleting the amino acids between
s+”p and s+’p reduces signal peptidase accessibility by over
70%.
Our results provide some insight intothe mechanism
whereby selectivity is maintainedina
system utilizing a
degenerate recognition system. Protein function is intimately
linked to molecular structure. Such structural requirements
place constraints on the accessibility of signals encoded in the
amino acid sequence of the molecule. Although amino-terminal location and subsequent cleavage might be expected to
minimize such constraints itseems likely that over the course
of evolution a particularsignal sequence is tailored to achieve
optimal translocation of the appropriate passenger domain,
in this case the rest of the molecule.
Support for this model comes from two recent studies on
the role of the pro sequence in the translocation of human
preproapolipoproteins AI and AI1 (26, 27). The pro sequences
in these molecules are immediately carboxyl to the signal
sequences. Deletion of the pro sequence from preproapolipoprotein AI1 results in redirection of the site of signal peptidase
cleavage to a position two amino acids into the mature molecule (26). In contrastdeletion of the prosequence in preproapolipoprotein AI generates a molecule which is cleaved by
signal peptidase faithfully but translocates with reduced efficiency (27). These results lead Folz and Gordon (26, 27) to
suggest that the role of the prosequence may be to mediate
folding of the preproteins to conformations compatible with
efficient translocation. The results described here, while consistent with this view, suggest an alternate interpretation.
The signal sequence of preproapolipoprotein AI mayhave
evolved to most efficiently translocate the molecule beginning
with the pro sequence. Deleting this prosequence creates a
protein context which leads to less favorable signal sequence
presentation. In this model normal presequence function remains unspecified. Systematic mutagenesis of a defined subset
of signal sequences and passenger domains may reveal rules
by which signal sequences have become optimized and permit
these questions to be addressed.
It has long been suggested that all signal sequences present
some common secondary or tertiary structural features
to
receptors on the membrane. The evolutionary selection proposed here would account for why variation in primary sequence is the rule rather than the exception. The particular
amino acid sequence of a signal is, in part, a result of accommodation to optimize translocation of the passenger protein
domain. Appropriate segregation of cytosolic and ER lumenally disposed proteins may also rely to some extent on evolutionary pressure in a complementary fashion for cytosolic
proteins. Inthis view cytosolic proteins that containsequences which fortuitously resemble signal sequences and
could therefore potentially display signal function may mask
such sequences by protein folding. This wouldbe directly
analogous to thecryptic mitochondrial import signal recently
unmasked at the amino terminus of the cytosolic protein
dihydrofolate reductase (11).The evolution of a system for
discrimination of cytosolic proteins from those destined for
ER translocation need provide only enough specificity to
achieve that goal through the sum of both positive (signal
sequence) and negative (masking through folding) influences.
For example, if bona fide signal sequences, either aminoterminal or internal, must be exposed on the surface of the
protein to be functional while latent signals are found folded
into the interior of protein domains, then relatively degenerate interactions would be sufficient to accomplish discrimination. In such a scenario, an important influence on whether
or not “random” sequences can serve to direct protein translocation is location at theamino terminus of (as described by
Kaiser et al. (10)) or between otherwise independently folding
protein domains.
Finally, from these results we see that the most useful
definition of a signal sequence is one that is both functional
and operational: a sequence capable of directing a given
protein domain across microsomal membranes. Moreover,
commentary on signal function must include an evaluation of
the efficiency of function since it seems likely that signal
sequences of secretory proteins have been evolutionarily tailored to achieve optimum translocation of a particular passenger in uiuo. Further studieswill allowus to extend and specify
these influences.
Acknowledgments-Special thanks are dueAlison Cowie and Larry
Mirels for many helpful discussions and critical reading of the manuscript.
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