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(Received for publication, June 19, 1995; and in revised form, July 28, 1995) From the
The hydrophobic core or h region of both prokaryotic and
eukaryotic signal sequences is the predominant structural domain that
controls the efficiency of protein translocation across membranes.
Characteristically, hydrophobic cores appear to assume
The N-terminal signal sequences of prokaryotic and eukaryotic
polypeptides, required for protein export to extracytoplasmic sites,
are characterized not by a conservation of primary amino acid sequence
but rather by the maintenance of three structural domains(1) .
At the N terminus, each signal sequence contains a hydrophilic or n
region composed of 1-5 residues, several of which are positively
charged. This is followed by the hydrophobic core or h region that is
typically 7-15 amino acids in length and highly hydrophobic. At
the C terminus, a more polar region (c region) of 3-7 residues
defines the signal peptidase cleavage site that is used once the signal
sequence has traversed the membrane. Numerous genetic and biochemical
studies in prokaryotes and eukaryotes have demonstrated that the
predominant feature required for signal sequence function is the
hydrophobicity of the core
structure(2, 3, 4) . Another conserved
structural feature of signal sequences is the We have been
conducting a genetic analysis of the gC (
Figure 4:
Helical wheel plot of the gC signal
sequence. The entire 22-amino acid signal sequence is shown, beginning
with the initiator methionine at the top position of the plot.
Each residue is indicated using the single-letter amino acid code and a
subscript to denote its position within the signal sequence.
Hydrophilic amino acids are shown in bold. Residues that were
substituted with proline in this study are highlighted by shaded
squares, and the translocation efficiency of the gC mutant
containing a proline in a particular position is given in parentheses. The diametric line divides the plot and
was empirically determined by noting those residues (to the left) that were sensitive to proline substitution and those
(to the right) that were not.
Figure 1:
Proline substitution
and single codon deletion mutants of the gC signal sequence hydrophobic
core. The nucleotide sequence of the sense strand and the predicted
amino acid sequence of the wild-type gC signal sequence are shown. The arrows below the sequence indicate the alterations made to
substitute a single proline into positions 9-14. The shaded
boxes demarcate the deletion of residue 10 and 11, whereas the
double mutant gC A10PL11A is shown at the bottom. In all
mutants, the strain number is listed below the substituted or deleted
amino acid. The downward pointing arrow above the wild-type
sequence denotes the predicted signal peptidase cleavage
site.
The export competency of each
mutant form of gC was evaluated by steady-state radiolabeling infected
cell proteins as described previously(8) . Wild-type gC was
found in the infected cell in two well characterized forms(8) :
as a 74-kDa ER-localized glycoprotein and as a 92-kDa Golgi-modified
species that resides in the plasma membrane and the mature virus
envelope (Fig. 2). An additional form of gC was evident in each
of the mutant-infected cells; this protein has been previously
demonstrated to be the nonglycosylated, 58-kDa precursor species
localized in the cytoplasm(10) . The extent of each
precursor's accumulation reflected the degree of the
translocation defect, ranging from almost no impairment for a proline
substitution at position 10 (strain PRV544) to a near complete block in
translocation across the ER membrane as a result of a proline
substitution for residue 14 (PRV548). Interestingly, a vaccine strain
of PRV (Bartha) contains greatly reduced levels of gC and has been
found to harbor a proline substitution at position 14(16) . In
general, mutants in which proline replaced a leucine residue exhibited
a more pronounced defect than those exchanging proline and alanine.
This was not unexpected because leucine is more hydrophobic than
alanine, and it has been established that the principal determinant of
gC signal sequence function is its overall hydrophobicity(4) .
Still, the concordance between loss of hydrophobicity and loss of
translocation proficiency was imperfect. For example, the gC signal
sequences of strains PRV543, 545, 546, and 548 were identical in
hydrophobic content, yet PRV548-encoded gC was far more defective than
the others for translocation across the ER membrane.
Figure 2:
Steady-state radiolabeling analysis of
wild-type and mutant forms of gC. PK15 cells were infected at a MOI of
10 with wild-type or mutant virus and incubated at 37 °C for 4 h in
Dulbecco's modified Eagle's medium containing 2% fetal
bovine serum. At that time, 50 µCi/ml of
[
All of the mutant strains and wild type were used
in pulse-chase experiments (8, 11) to accurately
determine the translocation defect of each gC signal sequence mutant.
The results are shown in Fig. 3, and a quantitation of
translocation efficiency, determined from the 15-min chase point, is
provided in Table 1. Wild-type gC was rapidly and efficiently
exported; the cytoplasmic 58-kDa form was not detected even at the
earliest chase times. Each of the mutants also exported a fraction of
the radiolabeled gC with a wild-type rate of conversion from the
74-92-kDa form. However, virtually all of the mutants produced a
precursor form of gC that remained throughout the 2-h chase period (i.e. no post-translational translocation or, in general,
substantial degradation of the cytoplasmic species was observed). In
some panels, a species just smaller than 74 kDa could be seen and may
have represented an under-glycosylated ER form of gC. A protein just
smaller than the 58-kDa precursor accumulated throughout the 2-h chase
period in PRV547-infected cells; the kinetics suggested that it was a
breakdown product of the cytoplasmic gC species. Additional minor bands
were also evident, but their relationship, if any, to gC was not clear.
Overall, the pulse-chase results were in good agreement with the
outcome of the steady-state analysis of each of the original proline
substitutions, with quantitated translocation efficiencies ranging from
5-98%. However, the inclusion of strains PRV549-551 yielded
additional results indicating that proline's negative effects on
signal sequence function were the result of more than just loss of
hydrophobicity. Deletion of codons 10 or 11 had little effect on the
translocation efficiency of gC, even though a proline substitution at
position 11 resulted in nearly a 70% reduction in the process.
Furthermore, the double mutant gC A10PL11A was somewhat more defective
than the gC A10P mutant alone (presumably due to the reduced
hydrophobicity at position 11) but remained far more translocation
competent than the similarly hydrophobic gC products encoded by strains
PRV545, 546, and 548.
Figure 3:
Pulse-chase analysis of wild-type and
mutant forms of gC. PK15 cells were infected at a MOI of 10 with
wild-type or mutant virus. At 4 h postinfection, cells were
radiolabeled for 2 min with 100 µCi/ml
[
Proline residues are rarely found in the hydrophobic cores of
prokaryotic or eukaryotic signal sequences(18) . This is likely
due to proline's nonhydrophobic character and its propensity to
disrupt A good example of where
proline, exclusively through its nonhydrophobic character, has a very
slight effect on translocation efficiency is position 10 of the gC
signal sequence. Regardless of whether the alanine at this position was
replaced with proline (hydrophilicity value of 0) or simply deleted,
the encoded gC polypeptide was translocated with near wild-type
efficiency. A similar pattern of highly efficient translocation was
found for proline substitutions at residues 9 or 13, suggesting that
proline's effect at these positions too was due to a reduction in
hydrophobicity alone. However, two observations in particular ruled out
the possibility that proline always exerts its effects solely by a
reduction in overall signal sequence hydrophobicity. First, the
deletion of codon 11 resulted in only a 10% loss of translocation
efficiency for gC. In contrast, the introduction of proline at this
position produced a gC species that was seven times more export
defective. Second, several of our mutants had identical hydrophobicity
values for their gC core structures, yet the translocation efficiencies
of these mutant gCs ranged from 5 to 89%. In addition, proline
substitutions at position 12, 13, or 14 resulted in similarly sized,
interrupted What
then may explain the positional effects on translocation that we
observe for the proline insertions in the gC signal sequence
hydrophobic core? Fig. 4shows a helical wheel plot of the
wild-type gC signal sequence. The These proposals raise the question of what
orients the gC signal sequence with respect to SRP, thus promoting a
sidedness to the hydrophobic core. This is a particularly pertinent
question given the inherent flexibility of SRP that allows it to
recognize a myriad of signal sequences differing in their primary amino
acid sequences and in the lengths of their three structural domains.
One possibility would be the hydrophilic N terminus whose positively
charged residues could provide an electrostatic anchor. We conducted a
preliminary experiment to test this by replacing arginine with glycine
at position 6 of the gC L12P signal sequence. We have previously shown
that a gC R6G mutant is exported with 94% efficiency(11) ; the
translocation efficiency of the gC R6GL12P double mutant was reduced by
an amount equal to the combined defects of each mutant alone. ( Previous analyses of sporadic disruptions of the
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U29121[GenBank]-U29129[GenBank].
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27876-27879
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helical
conformations, and studies in prokaryotes have indicated that this
conformation is necessary for efficient signal sequence function. To
address the conformational constraints of a eukaryotic signal sequence,
we have introduced a single proline in almost each position of the
signal sequence hydrophobic core of glycoprotein C (gC) of the swine
herpesvirus, pseudorabies virus. When the resulting mutant virus
strains were used to infect cells, we found that substitution of
proline at certain positions affected gC translocation greater than its
introduction at other sites within the hydrophobic core. The observed
positional effects did not completely correlate with reductions in
overall hydrophobicity or linear position within the hydrophobic core.
Rather, it appeared that one face of the gC signal sequence -helix
is far more sensitive to proline disruption than the other, potentially
indicating a functional asymmetry.
-helical nature of
the hydrophobic core(1) . This conservation has been
interpreted to indicate an important role for secondary structure in
the translocation process, a conclusion supported by studies of
prokaryotic signal sequences. In particular, genetic analysis of
suppressors of Escherichia coli LamB signal sequence mutants
and subsequent biophysical assessments of mutant LamB signal peptides
strongly suggest that an -helical conformation is necessary for
efficient LamB export(5, 6) .
)signal sequence
encoded by PRV, a swine herpesvirus. A 479-amino acid protein, gC
contains 8 N-linked glycosylation sites and is localized as a
mature species on the infected cell surface and in the virus
envelope(7, 8) . Importantly, gC is nonessential for
virus growth(9) , and it has been previously shown that a
complete deletion of the 22-amino acid signal sequence abolishes gC
export and renders it a cytoplasmic, nonglycosylated
protein(10) . We have also introduced more discrete alterations
into the hydrophilic and hydrophobic regions of the gC signal sequence
and have determined that it is typical of other characterized
N-terminal signal sequences(4, 11) . Here, we report
on the systematic introduction of proline, an accepted -helix
breaker (12) , throughout most of the hydrophobic core as a way
to evaluate the importance of secondary structure to gC signal sequence
function. Our results indicate that some positions within the
hydrophobic core are more sensitive to proline substitution than
others.
Cells and Virus Strains
Our wild-type strain,
PRV-Be, and the porcine kidney cell line PK15 have been previously
described (8) .Construction and Identification of gC Signal Sequence
Mutants
Site-specific mutagenesis using the vector pAXS (a
derivative of pALTER-1 of Promega Corp.) was performed as described
previously(11) . Plasmid-borne gC mutants were confirmed by DNA
sequencing and introduced into the PRV genome by calcium
phosphate-mediated cotransfection of plasmid and viral
DNA(11, 13) . Recombinant viruses were identified
using gC-specific antibodies in a peroxidase-linked immunoassay, the
black plaque assay(8, 14) . Recombinants were
plaque-purified and confirmed by Southern analysis of viral
DNA(9) .Radiolabeling and Quantitation of Infected Cell
Proteins
Steady-state or pulse-chase radiolabeling of infected
cell proteins was performed as described previously(8) . gC
species were immunoprecipitated with a polyvalent antiserum and
resolved on SDS-10% polyacrylamide gels followed by fluorography and
autoradiography(8) . Appropriately exposed autoradiograms of
each pulse-chase experiment were scanned using a PDI densitometer, and
the relative levels of the cytoplasmic and ER forms of gC were
determined for the 15-min chase point as described(4) .Computer Analyses
The predicted secondary
structure of wild-type and mutant gC signal sequences was determined
using the Peptidestructure program of the University of Wisconsin
Genetics Computer Group(15) . The Helicalwheel program of the
same package was used to generate Fig. 4.
Systematic Introduction of Proline Residues into the
Hydrophobic Core of the gC Signal Sequence
The hydrophobic core
of the gC signal sequence is composed of residues 7-15 and is
predicted to exist as an -helix(12) . Initially, six
mutant alleles were produced in plasmid-borne copies of gC such that a
single codon lying within positions 9-14 was replaced with a
proline codon (Fig. 1). Each of the mutant alleles was then
independently recombined into the wild-type PRV genome; this gave rise
to six different gC signal sequence mutants designated strains
PRV543-548 (Fig. 1). Collectively, the mutants introduced
a proline residue in each position of one complete turn of the
-helix of the hydrophobic core.
S]cysteine was added, and incubation was
continued for an additional 2 h. Each monolayer was then washed with
phosphate-buffered saline and lysed, and the gC species was
immunoprecipitated. The image is an autoradiogram obtained after
electrophoresis of the immunoprecipitates. The virus strain used to
infect cells is indicated at the top of each lane, and
molecular mass markers (in kDa) are indicated on the left.
Proline Substitutions Impact on gC Translocation
Efficiency Beyond Their Dimunition of Overall
Hydrophobicity
Proline substitutions reduce the hydrophobicity
of signal sequences owing to a net hydrophilicity value of 0 as
assigned by Hopp and Woods(17) . We therefore reasoned that a
simple deletion of one of the 9-14 codons should mimic an
introduction of proline at the same position if loss of hydrophobicity
was the only detrimental consequence of the substitution. Accordingly,
two additional mutant strains were generated; PRV549 contains a
deletion of the tenth gC codon, whereas PRV550 is deleted for the
eleventh codon. A final mutant, PRV551, was also produced. This mutant
encoded gC A10PL11A, an allele similar to that harbored by PRV544 but
in which the additional replacement of leucine at position 11 with
alanine resulted in an overall hydrophobicity identical to the gC
signal sequences encoded by strains PRV543, 545, 546, and 548 (Fig. 1).
S]cysteine in Dulbecco's modified
Eagle's medium lacking fetal bovine serum. Following the addition
of medium supplemented with 20-fold the normal amounts of cystine and
methionine, monolayers were lysed at specific times (indicated in min
across the top), and the gC species were immunoprecipitated.
The immunoprecipitates were resolved by electrophoresis and visualized
by autoradiography. The virus strain used to infect cells is indicated
to the side of each panel, and molecular mass markers (in kDa)
are indicated between panels.
-helices(12) . Thus, it is not surprising that the
introduction of proline residues into wild-type hydrophobic cores or
near truncated cores results in translocation defects (Refs. 5, 16, and
19 and this study). However, it has not been clear for all such mutants
whether the majority of signal sequence dysfunction should be
attributed to proline's reduction of hydrophobicity or to its
disturbance of secondary structure. Our results indicate that proline
affects signal sequence function via both properties, but certain
positions within the gC signal sequence are more susceptible to
secondary structure disruptions than others.-helices, but gC A13P was translocated 4.5- and
18-fold more efficiently than gC L12P or gC L14P, respectively. Thus,
the positional effects that we observed did not appear to be directly
related to the lengths of the -helices that resided on either side
of the inserted proline. This was a consideration because Emr and
Silhavy (5) isolated suppressors of an E. coli LamB
signal sequence deletion mutant in which proline or glycine residues
flanking the deletion were replaced with hydrophobic amino acids. They
concluded that suppression was accomplished through the restoration of
a suitably long -helix to the truncated hydrophobic core. In
follow up studies, Bruch and Gierasch (6) placed peptides
corresponding to the LamB signal sequence of the suppressor strains in
membrane mimetic environments and found that suppression efficiency
correlated with the stability and not necessarily the length of the
restored -helix. In the absence of biophysical data, we cannot
address the stability of the helices formed by our gC mutants.-helix has been empirically
divided into two halves that reflect the severity of the translocation
defect imposed by proline substitutions throughout the hydrophobic
core. Coincidentally, the same axis divides the helix into its most
hydrophobic (Fig. 4, left side) and least hydrophobic (Fig. 4, right side) faces. As depicted in Fig. 4, residues on the left side of the helix are much
more sensitive to proline substitutions than positions on the right
side. In fact, a proline substitution at position 12 is more
detrimental (resulting in a 19% translocation efficiency for gC) than
the introduction of a positively charged arginine at this site, which
has previously been shown to result in a 40% translocation
efficiency(4) . von Heijne (20) has noted that prolines
create kinks in the backbone of transmembrane helices. If this is
occurring with the gC signal sequence mutants, then the position of
certain kinks may sterically interfere with the interaction between the
hydrophobic core and SRP. But von Heijne additionally concludes that
the kinked helices appear to orient themselves such that the convex
sides reside near proteinaceous components, whereas the concave sides
face toward lipids. This seems necessary to provide hydrogen bonding
groups to the unpaired amide and carbonyl groups of the introduced
kink. Extending this proposal to the gC signal sequence, we would
suggest that residues lying on the right side of the helix in Fig. 4interact with another protein, possibly the 54-kDa subunit
(SRP54) of SRP, whereas those on the left side may not. As a
consequence, positions 9, 10, and 13 of the hydrophobic core would be
reasonably tolerant of a proline substitution that placed them on the
convex side of a kinked helix. The opposite would be true for residues
7, 8, 11, 12, 14, and 15, whose exposed amide and carbonyl groups of
the convex side would face away from any nearby protein sequences.
However, we should caution that if the gC signal sequence is uniformly
surrounded by protein, which may very well be the case given the number
of proteins implicated in ER membrane translocation(21) , the
von Heijne proposal for kinked helix-protein interactions is not
valid(20) .
)Thus, reducing the potential electrostatic interaction of
the N terminus of the signal sequence did not appear to alter the
orientation of the gC hydrophobic core. In fact, if anything, the
additive effect of the two mutations suggested that the hydrophilic and
hydrophobic domains of the gC signal sequence function independently. -helices of
hydrophobic cores have strongly suggested a role for secondary
structure in signal sequence function. Our work represents the first
systematic, genetic analysis of proline insertions in a signal sequence
hydrophobic core, prokaryotic or eukaryotic. Our results indicate that
the -helix of the gC signal sequence, although not strongly
amphipathic, may be functionally asymmetric.
))
We thank Shannon Flynn for critical reading of the
manuscript prior to its submission. All oligonucleotides were
synthesized by the Molecular Resource Center of the University of
Tennessee at Memphis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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