From the
Transmembrane segments of proteins generally consist of a long
stretch of hydrophobic amino acids, which can function to initiate
membrane insertion (start-stop sequences), initiate translocation
(signal-anchor sequences), or stop further translocation of the
following polypeptide chain (stop-transfer sequences). In this study,
we have taken Escherichia coli alkaline phosphatase, a
transported and water-soluble protein, and examined the requirements
for converting it into a transmembrane protein with particular
orientation. Since the wild type enzyme is transported, there is no
predisposition against membrane translocation, yet it is not a membrane
protein, so it does not possess any intrinsic membrane topogenic
preferences. A series of potential transmembrane segments was
introduced into an internal position of the enzyme to test the ability
of each to initiate translocation, stop translocation, and adopt a
particular orientation. For this purpose, cassette mutagenesis was used
to incorporate new structural segments composed of polymers of alanines
and leucines. The threshold value of hydrophobicity required to
function as a stop-transfer sequence was determined. For a
transmembrane segment of typical length (21 residues), this value is
equivalent to the hydrophobicity of 16 alanines and 5 leucines.
Interestingly, much shorter segments will also suffice to stop
translocation, but these must be composed of more highly hydrophobic
residues (e.g. 11 leucines). When the wild type amino-terminal
signal peptide is deleted or made dysfunctional, sufficiently
hydrophobic internal segments can initiate translocation of the
following polypeptide and function as a signal anchor. Furthermore, in
so doing, the orientation of the protein is changed from
N
For integral membrane proteins, the problem of obtaining
structural data has proven to be very complex, and only a handful of
structures have been solved to high resolution (Roth et al.,
1989; Henderson et al., 1990; Schertler et al.,
1993). At the same time, the identification of new membrane proteins
and their corresponding primary sequences is moving at a rapid pace.
Consequently, construction of topological models often relies on
sequence analysis to identify hydrophobic stretches of about 20
residues as putative transmembrane domains and clusters of positively
charged residues to predict orientation. While this approach has proven
remarkably consistent with experimentally defined topological data (von
Heijne and Gavel, 1988; von Heijne, 1992), nonclassical transmembrane
domains may be overlooked because little is known regarding the limits
of variation in membrane protein structure.
Theoretically,
hydrophobic
Positive
charges in the region flanking a transmembrane segment have been shown
to have significant effects on the orientation of a transmembrane
segment. The ``positive inside'' hypothesis (von Heijne,
1986, 1992) suggests that positive charges (or the charge differential,
Hartmann et al., 1989) surrounding the transmembrane segments
are the predominant topogenic signals that determine the orientations
of the transmembrane segments (Popot and Engelman, 1990; Andersson and
von Heijne, 1994). The positive charges are assumed to present an
energy barrier to translocation and, therefore, hold the transmembrane
segment in such an orientation that the positive charges remain inside
the cytoplasm (Andersson and von Heijne, 1994). The orientation of many
natural transmembrane segments is consistent with this hypothesis (von
Heijne and Gavel, 1988), yet counterexamples have also been observed
(Audigier et al., 1987; Hartmann et al., 1989; Parks
and Lamb, 1991; Andrews et al., 1992; Locker et al.,
1992). In addition to the effect of charges in flanking regions, there
is also evidence that the amino-terminal (Andrews et al.,
1992), carboxyl-terminal (Bibi et al., 1991), or the
transmembrane regions themselves (Parks et al., 1989; Locker
et al., 1992) may influence or direct the orientation of a
transmembrane segment.
In this study, we have taken E. coli alkaline phosphatase, a normally transported and water-soluble
protein, and examined the requirements for converting it into a
transmembrane protein with particular orientation. This approach has
several advantages: the globular form of the enzyme is active in the
periplasm but not the cytoplasm of E. coli (Michaelis et
al., 1986), which provides a useful indicator of the extent to
which mutants become anchored to the membrane. The structure of the
enzyme has been determined to high resolution (Kim and Wyckoff, 1989,
1991), and this provides the basis for choosing locations to insert
potential transmembrane segments. In E. coli, the wild type
form is periplasmic, so there should be no predisposition against
membrane translocation, and since it is not a membrane protein, it does
not possess any intrinsic membrane topogenic preferences. By inserting
polymeric sequences of different length and hydrophobicity into an
internal position of the mature protein, we have defined a threshold
value of hydrophobicity required to stop protein translocation and
function as a membrane anchor. Surprisingly, very short highly
hydrophobic segments can provide stable membrane anchors. Furthermore,
an internal hydrophobic segment of appropriate hydrophobicity can adopt
different orientations, depending on the export competence of the
amino-terminal signal peptide.
For the CN-LA1, CN-LA2, and CN-LA3 mutants, a linker
containing the unique sites XhoI and HpaI was
inserted into the NcoI site of the WT CASS3 plasmid. The
resulting plasmid, CN-B, was then digested with XhoI and
HpaI to remove the intervening linker sequence, and the large
vector was purified. Oligonucleotide inserts coding for various
combinations of alanine and leucine residues were then ligated to this
vector. The mutants CN-LA1, CN-LA2, and CN-LA3 have an additional
serine and threonine residue at the beginning of the inserted
hydrophobic segment relative to the CN vector-derived mutants.
XhoI and SalI produce cohesive protruding ends with
the same sequence, and, therefore, oligonucleotides designed to ligate
to SalI-treated vectors can also be ligated to this
XhoI-treated vector.
The DS-CN and DS-LL mutants were
generated by deleting 16 amino acids in the amino-terminal signal
peptide, including the entire hydrophobic core and cleavage regions. CN
and CN-LL were digested with SalI and BssHII. The
recessed 3` termini that were generated were then filled in with
Klenow, and the blunt ends were ligated. The resulting mutants have the
initiating methionine, lysine, glutamine, serine, and threonine
followed by the sequence of the mature region of alkaline phosphatase.
The 7A-LL, 6A-LL, and 2A-LL mutants were constructed by replacing the
amino-terminal signal peptide hydrophobic core region of CN-LL with
various ratios of alanine to leucine residues (7A3L, 6A4L, and 2A8L).
These signal peptide mutant sequences were previously generated (Doud
et al., 1993) and only needed to be exchanged with the
complementary wild type signal peptide region of the CN-LL vector.
Two molecular weight markers, TLF and TSF, were also constructed for
this study. The TLF marker was generated by digesting the WT CASS3
plasmid with NcoI, filling the recessed 3` termini with Klenow
and then ligating the blunt ends. This resulted in a frameshift and
introduced five new amino acids after amino acid 278 followed by a stop
codon. To generate TSF, equal amounts of the CN vector and mutant 10L
(Chou and Kendall, 1990) were digested together with HpaI and
BamHI, extracted, and then ligated together. A product was
isolated in which the amino acids from -6 to 278 of alkaline
phosphatase were deleted. The truncated alkaline phosphatase (TSF)
contains most of the signal peptide and the remaining carboxyl-terminal
tail of the enzyme (original residues from 278 to 449).
To examine the hydrophobicity and length requirements for a
segment to halt polypeptide translocation and function as a stop
transfer sequence, segments composed of different lengths of
polyalanine, polyleucine, or mixtures of alanine and leucine residues
were inserted into the mature region of alkaline phosphatase. Alanine
and leucine were chosen for this study because both have a high
By converting a normally soluble, periplasmic protein into an
artificial membrane protein, the hydrophobicity and length requirements
for a segment to stop translocation and anchor the protein to the
membrane were examined. For a segment of typical transmembrane length
(21 residues), a polymer of contiguous alanine residues was not
sufficiently hydrophobic for membrane retention; rather, the protein
was transported and retained substantial enzyme activity. In contrast,
the incorporation of a polymer of leucines of the same length resulted
in stable membrane association and stopped the translocation of the
remaining carboxyl-terminal portion of the polypeptide chain. The
transition point for stop transfer activity was defined as a segment
with a total of 16 alanines and 5 leucines (mutant CN-LA2). In future
studies, it will be interesting to determine if the threshold value is
influenced by the position of the most highly hydrophobic residues
within the transmembrane segment and to examine the extent to which the
flanking net charge may modulate this threshold value.
Interestingly, a short segment of only 11 total leucine residues
(mutant CN-L) also serves as a stop transfer segment and, as judged by
NaOH extraction, is more membrane stable than the longer CN-LA3 mutant.
Hydropathy analysis (using scales described in Kyte and Doolittle
(1982), Eisenberg et al.(1982), and Engelman et
al.(1986)) indicates that the total hydrophobicity of the segments
in mutants CN-L and CN-LA2 (11 leucines versus 16 alanines and
5 leucines, respectively) is similar, suggesting that overall
hydrophobicity as opposed to length is the critical feature.
Nevertheless, few native membrane proteins have such short segments,
and this may be because of the unfavorable perturbation of lipid
bilayer structure that would be necessary to maintain favorable
hydrophobic contacts (Tahara et al., 1992).
Based on
theoretical considerations of the energy involved in membrane
integration of transmembrane segments, Engelman et al. (1986)
suggested that a segment of 20 amino acids with a total transfer energy
of 20 kcal/mol (based on the scale described therein) may be the
minimum value required for a segment to stop and anchor a protein to
the membrane. This is equivalent to about 20 glycine residues and is
well below the value for the corresponding polyalanine segment, which
did not stop alkaline phosphatase transport. This could reflect a
difference between polytopic membrane proteins and single-segment
membrane-spanning proteins. Analysis of the amino acid composition of
transmembrane segments from these two classes of proteins (von Heijne
and Gavel, 1988) is consistent with this notion. Furthermore, the total
hydrophobicity of individual transmembrane segments within a polytopic
protein, such as the bacterial photoreaction center and
bacteriorhodopsin, does vary considerably, and some highly hydrophobic
segments may compensate for others that are only weakly so. In
addition, these proteins may be further stabilized in the membrane
through interactions between transmembrane segments (Khorana et
al., 1979; Rees et al., 1989).
In this study, the
orientation of the membrane-anchored alkaline phosphatase was found to
depend on the export competence of the amino-terminal signal peptide as
depicted in the model in Fig. 8. When a functional signal peptide
is present, it is cleaved as usual, the internal transmembrane segment
functions as a stop transfer sequence, and the protein is anchored in
an N
We thank Linda Randall for sharing her expertise and
teaching us flotation gradient centrifugation.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-C
to N
-C
-helical segments of at least 20 residues are required
to provide a hydrophobic surface of sufficient length to traverse the
normal 30-Å thickness of the hydrophobic interior of a bilayer
(Tanford, 1978). Indeed, the natural transmembrane segments of
glycophorin A (Tomita et al., 1978) and of the coliphage f1
gene II protein (Davis et al., 1985) are 23 residues in
length, and that of the vesicular stomatitis virus glycoprotein (G)
contains 20 residues (Adams and Rose, 1985). However, truncated natural
transmembrane segments and their replacement with artificial sequences
with as few as 12 nonpolar residues can be effective stop-transfer
sequences (Davis et al., 1985; Davis and Model, 1985; Spiess
and Handschin, 1987). Conversely, some long hydrophobic segments fail
to stop translocation, such as the fusion-related hydrophobic domains
from eukaryotic viruses, which can be passed through the host cell
membranes and the cytoplasmic membrane of Escherichia coli (Hsu et al., 1983; Davis and Hsu, 1986).
Bacterial Strains and Media
E. coli AW1043 (lac galU galK
(leu-ara)phoA-E15 proC::Tn5) was
used for the generation and replication of mutant forms of alkaline
phosphatase and for all transport and membrane association analyses.
For mutagenesis and general propagation of bacterial strains, LB medium
(Sambrook et al., 1989) supplemented with 50 µg/ml
kanamycin and 250 µg/ml ampicillin was used. For transport and
membrane association analyses, cells were cultured in MOPS
(
)
medium containing 50 µg/ml kanamycin and 250 µg/ml
ampicillin.
DNA Manipulations and Mutagenesis
The
CN-A, CN-AA, CN-L, CN-15L, and CN-LL mutants were generated using the
plasmid, CN. CN is a modified form of WT CASS3, which is a pBR322
derivative containing the alkaline phosphatase gene (phoA) and
its natural promotor (Kendall and Kaiser, 1988). A unique NcoI
site is located at codons 275-277 of the region encoding mature
alkaline phosphatase. An additional unique site, HpaI, was
introduced at the original NcoI site by ligating a small
double-stranded oligonucleotide fragment carrying the HpaI
site to NcoI-digested CASS3. The resulting plasmid, CN, has
four additional codons (encoding Leu, Thr, His, and Gly) at that
position and contains an additional NcoI site. Mutant
sequences were generated by ligation of oligonucleotides with
HpaI-digested CN vector as previously described (Chou and
Kendall, 1990). For the leucine series, single or multiple copies of
oligonucleotides coding for five leucines were inserted at the
HpaI site. For the alanine series, an oligonucleotide insert
coding for five alanines and carrying a unique SacII site was
first introduced in the HpaI site. This removed the
HpaI site, but longer alanine sequences were generated by
inserting additional copies of five alanine segments into the
SacII site. The design and sequences of the oligonucleotides
used here are described in Chou and Kendall(1990) and Doud et
al.(1993).
Induction of Alkaline Phosphatase
Expression
Overnight cultures grown in LB medium containing
250 µg/ml ampicillin and 50 µg/ml kanamycin were subcultured
1:20 in the same medium. Cells were then grown at 37 °C with
vigorous shaking to logarithmic phase and harvested at
A 1.0-1.2. An aliquot of cells was then
taken and washed twice with MOPS containing no phosphate and
resuspended in 2 ml of the same medium supplemented with amino acids
(20 µg/ml, minus methionine). Cells were incubated at 37 °C for
15 min with shaking to induce the expression of alkaline phosphatase.
Alkaline Phosphatase Activity
Assays
Alkaline phosphatase is enzymatically active only
when it is transported to the periplasm or membrane bound with the bulk
of the enzyme in a periplasmic orientation (Michaelis et al.,
1986; Manoil and Beckwith, 1985). After induction of alkaline
phosphatase, cells were lysed and treated with the substrate
p-nitrophenyl phosphate as described by Brickman and
Beckwith(1975) and Michaelis et al.(1986). The absorbance at
410 nm was used to determine the extent of hydrolysis of
p-nitrophenyl phosphate to p-nitrophenol by alkaline
phosphatase.
Cell Fractionation
Following the
induction of alkaline phosphatase, cells were radiolabeled with 40
µCi/ml L-[S]methionine for 30 s at
37 °C, then chased with cold methionine at a final concentration of
4 mg/ml for 30 s, and then quickly cooled on ice. One-half of the cells
were precipitated with trichloroacetic acid to give a ``whole
cell'' sample. The remaining cells were pelleted. The supernatant
was discarded, and the pellet was resuspended in 0.1 M Tris,
pH 8.2, 0.5 M sucrose, 0.5 mM EDTA and treated with
freshly prepared lysozyme. Immediately following, an equal amount of
ice-cold water was added and mixed well, and the mixture was incubated
for 5 min on ice. To stabilize the forming spheroplasts, Mg
was added to a final concentration of 10 mM during the
incubation (Thom and Randall, 1988). The cells were then centrifuged to
separate the periplasmic contents from the spheroplasts. The
periplasmic fraction was precipitated with trichloroacetic acid and
subjected to immunoprecipitation of alkaline phosphatase as described
by Kendall et al.(1986).
NaOH Treatment
Following labeling as
described above, an aliquot of cells was removed to an ice-cold tube,
and an equal volume of ice-cold 0.1 M NaOH solution was added
(Russel and Model, 1982). The mixture was vortexed hard and then
centrifuged at 4 °C for 10 min in a microcentrifuge to separate the
soluble fraction (supernatant) and membrane-associated proteins
(pellet). The protein in each fraction was then precipitated with
trichloroacetic acid, and alkaline phosphatase was immunoprecipitated
as described.
Flotation Gradient
Centrifugation
Flotation experiments were performed as
described by Thom and Randall(1988) on
[S]methionine-labeled cells. After
centrifugation, the contents of the tube were separated into six
150-µl fractions, each taken successively from the top of the
gradient. The protein was then precipitated with trichloroacetic acid
and analyzed by autoradiography.
Protease Accessibility Analysis
After
labeling and converting cells to spheroplasts as described above, cells
were divided into three fractions. One fraction of cells was
precipitated with trichloroacetic acid to provide an untreated control.
Proteinase K (final concentration, 25 µg/ml) was added to the other
two fractions. CHAPS (final concentration, 0.2%) was added to one of
the protease-treated fractions. Both fractions were incubated on ice
for 15 min. Protein was precipitated with trichloroacetic acid after
the incubation. Alkaline phosphatase and its partially digested
fragments were then immunoprecipitated as described by Kendall et
al. (1986).
Electrophoresis and Quantitation of Protein
Bands
Immunoprecipitated proteins were separated by
electrophoresis on 7.5-10% Laemmli SDS-polyacrylamide gel
electrophoresis gels (Laemmli, 1970). The pattern was visualized by
autoradiography as described by Kendall and Kaiser(1988), and protein
was quantified by scintillation counting.
-helical propensity (Ferretti and Paolillo, 1969; Arfmann et
al., 1977; Chou and Fasman, 1978), and both have alkyl side
chains, which are nonpolar but to very different degrees. Amino acid
position 278 was chosen as the location for the insertions because it
is in a loosely folded loop region, which is removed in the tertiary
structure from the core of the enzyme and the catalytic site (Kim and
Wyckoff, 1989, 1991). We anticipated that this region may be able to
accommodate the introduction of relatively long segments with minimal
disruption to the integrity of the globular structure and the activity
of transported alkaline phosphatase. On the other hand, the generation
of a transmembrane form should inhibit final folding and enzymatic
activity. Fig. 1shows the amino acid sequences of the resulting
mutants. Note that the mutants all contain an additional leucine
immediately carboxyl to the insert and therefore contain a total of 11
or 21 contiguous hydrophobic residues. The ten-residue segments
flanking the inserts have net charges of +2.5 (amino-terminal
side) and +0.5 (carboxyl-terminal side), counting histidine as
+0.5.
Figure 1:
Amino acid sequences of the relevant
regions of mutant alkaline phosphatases. Wild type CASS3 (Kendall and
Kaiser, 1988) and CN, a derivative with one additional leucine residue,
are also included for comparison. A, sequences of the mutants
that have segments of polyalanine, polyleucine, or mixtures of leucines
and alanines inserted at the HpaI site of the CN vector or the
XhoI and HpaI sites of the CN-B vector. All mutants
retained wild type signal peptides. The inserted hydrophobic residues
are shown in boldfacetype. B, sequences of
the mutants that have both internally inserted hydrophobic segments and
a mutated signal peptide. Hydrophobic core regions of the signal
peptide and the inserted hydrophobic segments are shown in boldfacetype. For mutants with deleted signal peptides, the
deleted region is indicated by brackets. In all cases, an
arrow marks the signal peptide cleavage site, and the
numbersabove the sequences indicate the position of
the amino acid in the entire mature wild type alkaline phosphatase
sequence.
The transport properties of the mutants with polyalanine
and polyleucine segments were first assessed by a cell fractionation
experiment. As shown in Fig. 2, insertion of 10 or even 20
alanine residues did not stop protein translocation; the mutated
proteins were mostly translocated and, like wild type alkaline
phosphatase, released to the periplasm. In contrast, mutants containing
insertions of either 10 or 20 leucine residues were not released to the
periplasm, although their signal peptides were cleaved. Alkaline
phosphatase activity levels are consistent with these findings. Wild
type alkaline phosphatase is not active in the E. coli cytoplasm, but it becomes active subsequent to translocation,
disulfide bond formation, and folding (Boyd et al., 1987;
Derman and Beckwith, 1991). Mutants with the polyalanine inserts were
found to be active, while no enzymatic activity was detected for the
leucine series (Fig. 2).
Figure 2:
Cell fractionation of mutants with various
polyalanine or polyleucine segments. Cell fractionation studies were
performed as described under ``Materials and Methods.'' Whole
cell (W) and periplasmic fractions (P) are shown for
each mutant. All bands correspond to the mature form of the protein.
This was verified by the observation of precursor in the presence of
azide. The aberrant mobility of CN-L likely reflects atypical SDS
binding as has been seen before (Kendall et al., 1986; Vlasuk
et al., 1984). The intensity of each band was quantified by
liquid scintillation counting, and the percentage of alkaline
phosphatase found in the periplasmic fraction is listed under each
mutant. Also given are the enzymatic activities of each mutant. These
were measured as described under ``Materials and Methods''
and normalized to CN, which was assigned a value of
100%.
Membrane localization of mutants
containing the leucine segments was indicated by both NaOH extraction
and flotation gradient centrifugation. The method of Russel and
Model(1982) utilizes NaOH treatment of cells to separate membrane
proteins (pellet) from cytoplasmic and periplasmic proteins
(supernatant). This analysis (Fig. 3A) indicated that
mutants with additional 10 or 20 leucines partitioned with the membrane
fraction (84 and 94%, respectively), while those containing polyalanine
segments remained associated with the soluble fraction (each only 2% in
the membrane fraction). Gradient centrifugation of lysates from cells
expressing the polyleucine mutants further revealed that these proteins
float to the top of the gradient with the membranes, while a control
mutant with neither a signal peptide nor a membrane-spanning segment
(mutant DS-CN) largely remained at the bottom of the gradient with
cytoplasmic proteins (Fig. 3B).
Figure 3:
Membrane association of mutant proteins
with internal polyleucine segments. A, NaOH extraction of
mutant proteins. Cells expressing mutant proteins were treated with
ice-cold NaOH immediately following the labeling. Pellets (M),
which contain proteins stably associated with the membrane, were
separated by centrifugation from the supernatant (S), which
contains cytoplasmic, periplasmic, or loosely membrane-associated
proteins. The intensity of the alkaline phosphatase bands in each
fraction was quantitated by liquid scintillation counting, and the
percentage of the membrane fraction was calculated with respect to the
total counts of both fractions. B, flotation analysis of the
mutant proteins. Spheroplasts of bacteria expressing the mutant
proteins were generated and separated from the periplasmic fractions.
Lysed spheroplasts were then subjected to centrifugation as described
under ``Materials and Methods.'' After centrifugation,
fractions 1-6 were removed sequentially from the top to the
bottom of the centrifuge tube. Fractions 1 and 2 correspond to a
1.15-1.22 g/ml density of metrizamide and are considered membrane
fractions. Fractions 5 and 6 correspond to a 1.3-1.4 g/ml density
of metrizamide and are considered cytosolic fractions. Whole cell
(W) and periplasmic contents (P) were also loaded for
comparison.
A protease
accessibility experiment (Fig. 4) revealed that the mutants with
polyleucine inserts were membrane bound in an N-C
orientation. Untranslocated alkaline phosphatase is sensitive to
proteinase K because it is in a partially unfolded state; in contrast,
the transported protein is resistant to protease because it is tightly
folded into a more compact form. The wild type complement used in this
study, mutant CN, has no hydrophobic segment insertion and, as
expected, was insensitive to protease digestion even in the presence of
detergent, which permeabilizes the cytoplasmic membrane. Mutants
containing polyleucine inserts were however, completely digested in the
presence of protease plus detergent, consistent with our earlier
finding that these were not periplasmically localized. In contrast,
when the protease was added in the absence of detergent, a partially
protected fragment was evident, and the relative electrophoretic
mobility of the fragment increased as the length of the inserted
polyleucine segment increased. The results suggest that the protein is
membrane-bound via the polyleucine segment in an
N
-C
orientation. A comparison of the
protected fragment and molecular weight markers revealed that the
protected fragment corresponds to about one-third of the protein plus
the length of the insertion (i.e. the carboxyl-terminal
segment of the protein), and this is consistent with the
N
-C
topological orientation depicted in
Fig. 4
(bottom). Since mutants with polyalanine
insertions were found to fractionate with the periplasm and retain
enzymatic activity, they should be resistant to protease digestion.
Indeed, the wild type domains of these mutants were resistant, but the
inserted polyalanine portion was largely proteolyzed; little fully
intact alkaline phosphatase was observed, but two fragments
corresponding to molecular weight markers representing two-thirds and
one-third of the protein (the lengths of the domains flanking the
insert) were protease resistant even in the presence of detergent.
Figure 4:
Orientation of membrane-associated
proteins. Protease accessibility experiments were performed as
described under ``Materials and Methods.'' Cytoplasmic or
cytoplasmically oriented proteins are protected from digestion by added
proteinase K in the absence of CHAPS but are exposed to the enzyme in
the presence of the detergent. Periplasmic or periplasmically oriented
proteins are exposed to proteinase K regardless of the presence of
CHAPS. Wild-type alkaline phosphatase is transported to the periplasm
and is tightly folded and resistant to proteinase K digestion. For the
purpose of discussion, CN can be regarded as wild type and is resistant
to proteinase K. CN-A and CN-AA are transported. However, the inserted
alanine segments are in an exposed region and are digested by
proteinase K (Chou and Kendall, 1990). The flanking domains of CN-A and
CN-AA retain their compactness and are resistant to degradation. These
are resolved essentially as two fragments that correspond with the
molecular weight markers. Mutants CN-L, CN-15L, and CN-LL are anchored
to the membrane in a N-C
orientation as
shown in the topology model at the bottom of the figure. The
periplasmically oriented amino termini (Peri) are not tightly
folded and are sensitive to proteinase K. The cytoplasmically oriented
C termini (Cyto) are protected from proteinase K in the
absence of CHAPS and digested when CHAPS is added. The molecular weight
of the protected small C termini increases as the length of the
inserted leucine segment increases. Molecular weight markers (TLF and
TSF) were loaded in the firstlane, and their
relative size with respect to wild type alkaline phosphatase is
schematically shown by the filledbars next to their
respective bands. Emptyarrowhead, TLF and the
proteinase K-generated large amino-terminal fragments. Filledarrowhead, TSF and the proteinase K-generated small
carboxyl fragments. All of the mutants exhibit signal peptide cleavage
and are present in the mature form
(m).
It is evident from a comparison of the properties of the mutants
with polyalanine and polyleucine insertions that while there is
considerable flexibility with regard to length, a segment must be
sufficiently hydrophobic to function as a stop transfer segment. To
determine the minimum level of hydrophobicity required for a stop
transfer segment, a series of mutants was constructed, which varied in
the ratio of alanine to leucine residues yet which retained an overall
insertion length of 21 residues (see Fig. 1). The results of a
cell fractionation experiment with these mutants are shown in
Fig. 5
. Mutant CN-LA1 (3 leucines:18 alanines), which has the
least hydrophobic insert, was translocated and released to the
periplasm. Mutant CN-LA2 (5 leucines:16 alanines) represents a
transition point with a marked reduction in transported alkaline
phosphatase, and essentially no transported protein is detected for
mutant CN-LA3 (7 leucines:14 alanines). The trend from transported to
membrane-anchored forms was revealed further by NaOH extraction
(Fig. 6). Stable membrane association distinctly increased with
the higher leucine content. A protease accessibility experiment was
consistent with these results and with the membrane-anchored form in
the N-C
orientation (data not shown),
comparable with the mutants with the inserts of leucines only.
Figure 5:
Cell fractionation of mutants with
internal segments composed of alanine and leucine mixtures. Methods and
identifications are as described in the legend to Fig.
2.
Figure 6:
NaOH
extraction of mutants with internal segments composed of alanine and
leucine mixtures. Methods and identifications are as described in the
legend to Fig. 3A.
The
orientation of the membrane-bound form can be manipulated by changing
the nature of upstream sequences. In particular, the effect of the
amino-terminal signal sequence on the topology of an alkaline
phosphatase mutant containing a polyleucine transmembrane segment was
examined by signal peptide attenuation or deletion. Previous studies in
our laboratory demonstrated that the function of the alkaline
phosphatase signal peptide can be titrated by replacing the wild type
hydrophobic core with different combinations of alanines and leucines
(Doud et al., 1993). When the hydrophobicity of the signal
peptide falls below the equivalent of 6:4 (alanines to leucines),
transport is slow if it occurs at all, while at levels above the
equivalent of 5:5 (alanines to leucines), the signal peptide is
functional and transport is efficient. Mutants DS-LL, 7A-LL, 6A-LL, and
2A-LL were generated by deleting the signal peptide core region or
replacing it with different ratios of alanine to leucine (see
Fig. 1
). In each case, the mature region of the protein
corresponded to mutant CN-LL. The topological orientation of this
series was analyzed by the protease accessibility method
(Fig. 7). Interestingly, the molecular weight of the protected
fragment shifted from one corresponding to the carboxyl-terminal domain
(mutants 2A-LL and 6A-LL) to one corresponding to the amino-terminal
domain (mutants 7A-LL and DS-LL). This suggests that the orientation
changed from N-C
to C
-N
when the signal peptide was changed from a functional to a
dysfunctional one. In the presence of a functional signal peptide, the
internal hydrophobic segment functions only to stop transfer of the
translocating polypeptide chain (i.e. stop-transfer sequence).
However, in the absence of a functional signal peptide, the precursor
form is maintained, and the internal hydrophobic segment is itself
capable of initiating membrane insertion; it does so in the same
orientation that functional signal peptides do, and like a signal
peptide, the portion of the protein carboxyl-terminal to it is
translocated (i.e. it functions as a signal anchor).
Figure 7:
Orientation of membrane-bound mutants with
functional and dysfunctional signal peptides. The 6A-LL and 2A-LL
mutants are anchored to the membrane in the N-C
orientation as shown in the topology model at the bottom of the figure. The periplasmically oriented amino termini
(Peri) are sensitive to proteinase K and are digested. The
cytoplasmically oriented carboxyl termini (Cyto) are protected
from proteinase K in the absence of CHAPS and digested when CHAPS is
added. The DS-LL and 7A-LL mutants are anchored to the membrane in the
N
-C
orientation. The periplasmically
oriented carboxyl termini are sensitive to proteinase K and are
digested. The cytoplasmically oriented amino termini are protected from
proteinase K when no CHAPS is added and digested in the presence of
CHAPS. The molecular weight of the protected fragments is larger than
the molecular weight markers due to the internally inserted polyleucine
segment. The molecular weight markers (TLF and TSF) were loaded in the
firstlane. Emptyarrowheads, TLF
and the proteinase K-generated large amino-terminal fragments.
Filledarrowheads, TSF and the proteinase K-generated
small carboxyl fragments. Mutants DS-LL (made without a signal
peptide), 2A-LL, and 6A-LL are predominantly in the mature form
(m), while mutant 7A-LL retains the signal peptide and is
mostly in the precursor (p) form.
-C
orientation (mutants 6A-LL, 2A-LL,
and CN-LL). However, when the amino-terminal signal peptide is
attenuated or deleted, alkaline phosphatase remains in the precursor
form, and the internal segment functions as a signal anchor in a
C
-N
orientation (mutants 7A-LL and DS-LL).
It is not surprising that in the former case, the functional signal
peptide is more favored than the internal segment as a translocation
initiation signal since it can engage the transport pathway even before
the internal segment is synthesized. In contrast, when the signal
peptide is deleted or made dysfunctional, the mutant proteins must
initiate translocation at a later stage of translation or do so
post-translationally. Regardless, it is remarkable that the internal
segment, about 280 amino acids from the amino terminus, directs export
and topogenesis. Furthermore, the translocation of these mutants, as
well as that of the series with the wild type signal peptide, was
inhibited by azide (Oliver et al., 1990), suggesting that they
function via SecA, a primary component of the secretory pathway (data
not shown).
Figure 8:
Model of the orientation of membrane-bound
alkaline phosphatase with competent and incompetent (or deleted) signal
peptides. A, model for mutants 2A-LL and 6A-LL, which have a
functional amino-terminal signal peptide (filledbox). Translocation of mutant proteins is initiated by
the amino-terminal signal peptide, which is later cleaved. The
translocation is stopped by the internal transmembrane segment
(shadedbox), and the protein is anchored to the
membrane in an N-C
orientation. B,
model for mutants with a deleted (DS-LL) or a dysfunctional (7A-LL)
amino-terminal signal peptide (emptybox). Since the
amino-terminal signal is absent or not functional, translocation of
mutant proteins is initiated by the internal transmembrane segment
(shadedbox). The carboxyl-terminal polypeptide chain
is translocated, and the proteins are anchored to the membrane in a
C
-N
orientation. The amino terminus remains
in the cytoplasm (Cyto), and the mutant protein is mainly in
the precursor form. Peri,
periplasm.
Both statistical and experimental analyses have
suggested that the charge of the regions flanking transmembrane
segments plays a dominant role in determining the orientation of the
transmembrane segment with the more positively charged region remaining
cytoplasmic (von Heijne and Gavel, 1988). In this study, the internal
transmembrane segment is flanked by a net amino-terminal +2.5
charge and a net carboxyl-terminal +0.5 charge. Nevertheless, the
transmembrane segment was able to adopt both N-C
and N
-C
orientations, similar to the
observations of Andrews et al.(1992). It is very likely that,
in addition to charge, other factors, such as the presence of a
cleavable signal peptide, may influence the orientation of a
transmembrane segment. Blobel(1980) proposed that the orientation of a
transmembrane segment is determined by its relative position with
respect to the most amino-terminal signal peptide or signal anchor and
that a membrane protein is incorporated into the membrane by the
sequential actions of signal anchors and stop-transfer sequences. The
orientation of the most amino-terminal signal anchor, or signal
peptide, may well be determined by the surrounding positive charges
according to the positive inside rule (Hartmann et al., 1989;
Lipp et al., 1989), but the orientation of subsequent segments
is then influenced by this first transmembrane segment. Our results are
consistent with this model. In the presence of a functional signal
peptide, protein translocation is initiated by the amino-terminal
signal peptide, and the internal transmembrane segment functions as a
stop transfer sequence; it stops further translocation and anchors the
protein to the membrane in an N
-C
orientation. When the amino-terminal signal peptide is deleted or
dysfunctional, the internal transmembrane segment can initiate protein
translocation and function as a signal anchor. The orientation of the
transmembrane segment agrees with the positive inside rule, and the
protein is anchored to the membrane in a C
-N
orientation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.