Biozentrum, University of Basel, CH-4056 Basel, Switzerland
The orientation of signal-anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH2-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7-25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.
Most proteins are targeted and inserted into the
endoplasmic reticulum membrane by a mechanism involving signal recognition particle, single
recognition particle receptor, the Sec61 translocation complex, and translocating chain-associating membrane protein (TRAM;1 for review see Walter and Johnson, 1994 The most prominent feature that determines which end of
the signal is translocated is the distribution of charged residues flanking the signal anchor sequence. Statistically, positive charges are enriched on the cytosolic side and depleted
from the exoplasmic side of signal-anchor sequences (the
"positive inside" rule; von Heijne, 1986; von Heijne and
Gavel, 1988). For eukaryotic proteins, the charge difference between the two flanking segments (more positive on
the cytosolic side) rather than the positive residues per se
has been shown to correlate with protein orientation
(Hartmann et al., 1989 Another topologically important feature is the folding
state of the NH2-terminal hydrophilic domain. This segment is already synthesized when the signal-anchor sequence emerges from the ribosome. For the ASGP receptor H1 it was shown that this domain needs to be unfolded
for translocation and that translocation of the NH2 terminus is hindered or even prevented by a rapidly and stably folding domain but is facilitated by destabilizing mutations
(Denzer et al., 1995 An influence of the hydrophobic segment on the function of signal and signal-anchor sequences was suggested
by in vitro experiments by Sato et al. and Sakaguchi et al.
Short deletions within the hydrophobic segment of the
Nexo/Ccyt signal-anchor sequence of cytochrome P-450 resulted in translocation of the COOH-terminal reporter sequence for a fraction of the product proteins (Sato et al.,
1990 In the present study we tested in vivo the role of the hydrophobicity and the length of the apolar domain of a type
II signal-anchor sequence, either close to the NH2 terminus or in an internal position and in the context of altered
flanking charges. We found that long, hydrophobic signal
domains promote NH2-terminal translocation, whereas short
ones promote translocation of the COOH-terminal sequence. This effect adds to the topogenic influence of mutations in the charged flanking residues. By combining
appropriate flanking charges and apolar domains it is possible to design polypeptides with essentially unique Ncyt/
Cexo or Nexo/Ccyt topology.
DNA Constructs
The plasmids encoding the wild-type ASGP receptor subunit H1 (Spiess
et al., 1985 H1 H1 H1Leu#.
The KpnI site immediately preceding the initiation codon in
the cDNAs of H1 H1-4Leu#.
The plasmids encoding H1 H1-4gLeu#.
The plasmid pSA1-4g (Beltzer et al., 1991 In Vivo Expression and Analysis
of Receptor Constructs
Cell culture reagents were purchased from GIBCO BRL (Gaithersburg,
MD). COS-7 cells were grown in MEM with 10% fetal calf serum at 37°C
with 7.5% CO2. The media were supplemented with 2 mM L-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin. Transfection of COS-7
cells was performed according to Cullen (1987) For analysis with endo- For protease protection assays, the transfected cells were scraped into
200 µl PBS per well and pipetted up and down 10 times. Equal aliquots
were incubated with or without 0.1 vol of 0.5 mg/ml TPCK-treated trypsin
for 30 min on ice in the presence or absence of 1% Triton X-100. The reaction was terminated by addition of 0.1 vol of 1 mg/ml chicken egg white
trypsin inhibitor and 10 mM PMSF. The samples were then subjected to
immunoprecipitation using anti-H1C antiserum in the presence of 0.1 mg/ml
trypsin inhibitor and 1 mM PMSF. The half lives of the glycosylated and unglycosylated forms of H1 H1 The ASGP receptor subunit H1 is a type II membrane protein with an internal signal-anchor sequence. The hydrophobic core of the signal consists of 19 mostly apolar residues starting after arginine-40 and followed by 8 mainly
polar but uncharged amino acids and two glutamic acid residues (positions 68 and 69). Upon transfection of H1 cDNA
into COS-7 cells, 30 min labeling with [35S]methionine, and
immunoprecipitation, a single polypeptide of 40 kD was
recovered (Fig. 1 A, lane 1). Truncation of the NH2-terminal domain to only 4 residues in H1
In a first construct to analyze the topogenic importance
of the hydrophobic domain, the 19-amino acid apolar domain of H1 Lack of glycosylation could be due to a reduced efficiency of the mutated signal sequence in targeting the protein to the ER membrane, resulting in a soluble, cytosolic
polypeptide. Alternatively, all products may still be integrated in the membrane, but some with the opposite Nexo/
Ccyt orientation leaving the COOH-terminal portion with
the glycosylation sites in the cytosol. Membrane association of the unglycosylated form was analyzed by saponin and alkaline extraction of expressing COS-7 cells. After labeling with [35S]methionine, the cells were incubated with
0.1% saponin for 30 min at 4°C to release soluble proteins
into the supernatant. Extracted and membrane-associated
proteins were then immunoprecipitated from the saponin
extract (S) and from the residual cells (C), respectively
(Fig. 2 A). A secretory form of the exoplasmic portion of
H1 with a cleavable signal sequence (HC) was efficiently
extracted (Fig. 2 A, lanes 10-12), whereas wild-type H1
and H1
The disposition of the H1 If H1 Expression of all the constructs with a truncated NH2terminal domain produced a small amount of protein of
~33 kD, with an electrophoretic mobility intermediate between that of the twice glycosylated and the unglycosylated forms (indicated by asterisks in Figs. 1, 2, and 4).
Upon endo H digestion, this material shifted to the position of the 30-kD unglycosylated form (Fig. 1 B); and in a
protease protection assay, it was equally resistant as the twice glycosylated polypeptides (Fig. 2 B). These results
indicate that the 33-kD species corresponds to type II polypeptides that were glycosylated only once. Incomplete glycosylation was generally not observed for constructs with
the complete NH2-terminal domain of H1. Most likely, glycosylation at the site near the membrane (position 79 of
the wild-type sequence) is slightly influenced by the presence or absence of the NH2-terminal domain.
Long Hydrophobic Signal Domains Near the NH2
Terminus Promote Nexo/Ccyt Orientation
Replacing the transmembrane domain of H1 by a sequence of 19 leucine residues increased its hydrophobicity
without altering the physical length of the domain. To further explore the topogenic role of the hydrophobic domain, we prepared mutant constructs of H1 and H1
For the constructs with a short NH2-terminal domain of
only four residues (H1 Combination of a Long Hydrophobic Domain with a
Reduced NH2-Terminal Charge Produces Unique Nexo/
Ccyt Insertion
The best documented determinant for the topology of membrane spanning proteins are the charged residues flanking
the transmembrane domain. To test whether the length/
hydrophobicity and the charge difference of the signal anchor affect the insertion process in an additive manner, the
NH2-terminal arginine directly preceding the hydrophobic
core of the signal anchor was mutated to glutamine. In the
resulting constructs H1 For H1 These results show that both the flanking charges and
the length/hydrophobicity of the signal anchor contribute
to the topology decision. Reducing the NH2-terminal positive charge and increasing the length/hydrophobicity of
the signal anchor were sufficient to achieve unique type III
(Nexo/Ccyt) insertion of an NH2-terminally truncated form
of H1.
Membrane association of the products of the H1Leu#,
H1 The observed ratio of type II and type III products after
30-min labeling with [35S]methionine also depends on the
degradation rate of the two species. As determined by
pulse-chase experiments for selected constructs (not
shown), the type II species displayed half-times of degradation of up to 5 h. In contrast, type III products, which
cannot form disulfide bonds in their cytosolic COOH-terminal portion, were more rapidly degraded with half-times
of only ~0.5 h. For the extreme example of H1 A Long Internal Signal and Inverted Flanking Charges
Are Sufficient to Completely Convert H1 from a Type II
to a Type III Protein
In the constructs H1Leu#, described above, the leucine sequences had no effect on the topology of the proteins in
the ER membrane. This could be due to the charge difference of
As is shown in Fig. 6 A and quantified in Fig. 7 (open
squares), transmembrane segments >13 leucine residues
showed increased translocation of the NH2 terminus and
were thus topogenically active even in an internal position.
Virtually unique type III insertion was observed for H14Leu22 and H1-4Leu25. Whereas the longer leucine sequences favored type III insertion, shorter ones did not shift
the balance towards type II insertion. H1-4Leu13, H14Leu10, and H1-4Leu7 did not insert as type II proteins to
any larger extent than H1-4. Shortening the oligo-leucine
domain more effectively increased the fraction of Ncyt/Cexo
polypeptides in constructs with the signal domain located
close to the NH2 terminus than further inside the protein.
To obtain direct evidence for the translocation of the
NH2 terminus in those forms that were not glycosylated in
their COOH-terminal domain, we constructed the series
H1-4gLeu# which contains an additional glycosylation site
in the NH2-terminal portion. Like for H1-4g, the corresponding construct with the original apolar sequence of
H1 (Beltzer et al., 1991 The best established determinant of the orientation of
signal-anchor sequences is the distribution of charged residues flanking the hydrophobic transmembrane domain
(Dalbey, 1990 In the context of the wild-type sequence of H1, where
the charge distribution and the sizable NH2-terminal domain favor translocation of the COOH-terminal sequence,
replacing the transmembrane segment by sequences of 7-25
consecutive leucines (H1Leu#) did not affect the insertion
behavior. Truncation of the NH2-terminal domain (H1 The topogenic effect of long polyleucine transmembrane
domains is not limited to proteins with very short NH2-terminal hydrophilic portions. An inverted charge distribution flanking the apolar segment of H1 resulted only in a
partial change of orientation in the membrane (H1-4). In
combination with a segment of The topology decision of signal-anchor sequences is thus
a result of the combined effects of at least three different
features: the charge distribution in the vicinity of the signal
sequence, the presence or absence of folded NH2-terminal
segments, and the length and hydrophobicity of the apolar
segment of the signals. Each of these features may be involved in additional functions besides membrane insertion. For example, charged residues close to the membrane surface might be involved in functional interactions of the protein with particular lipids, and the length of the
membrane-spanning domain has been proposed to affect
intracellular protein sorting (Munro, 1995 Changing the number of leucine residues in the signal
anchor sequence of a protein varies both the length of this
domain and its total hydrophobicity. It is therefore not
possible to distinguish between these two factors with respect to their influence on topogenesis. The wild-type transmembrane segment of H1 has the same length as the Leu19
sequence and the same hydrophobicity as the Leu16 sequence (as estimated by summing up the hydropathy indices of each residue; Kyte and Doolittle, 1982 Cleaved signals of secretory and type I membrane proteins resemble type II signal anchors, since they promote
translocation of the COOH-terminal sequence through the
membrane and typically have a more positive NH2 terminus.
Cleaved signals differ from signal anchors by consistently
shorter apolar segments (7-17 vs. 17-28 residues, respectively; Nilsson et al., 1994).
Three types of single-spanning membrane proteins are
generated by this machinery (von Heijne and Manoil,
1990; Spiess, 1995
). Insertion of type I membrane proteins
is initiated by an NH2-terminal, cleavable signal sequence that directs the transfer of the COOH-terminal sequence
across the membrane. Translocation is terminated by a hydrophobic stop-transfer sequence that anchors the protein
in the membrane with an exoplasmic NH2 terminus and a
cytoplasmic COOH terminus (Nexo/Ccyt orientation). Typically, cleaved signals are composed of a short, positively charged hydrophilic segment followed by a hydrophobic
domain of 7-15 uncharged residues. In proteins of types II
and III, the signal is not necessarily located at the very
NH2 terminus, it is not cleaved, and it anchors the protein
in the membrane. These hydrophobic domains are longer,
usually 19-27 residues (Nilsson et al., 1994
). Type II signal
anchors, like cleavable signals, initiate translocation of
their COOH-terminal sequence generating an Ncyt/Cexo
orientation of the protein. In contrast, type III signal-anchor
sequences (or "reverse signal anchors") promote translocation of their NH2-terminal sequence and produce an
Nexo/Ccyt topology.
). The topogenic role of the flanking charges has been experimentally confirmed by sitedirected mutagenesis. The topology of bacterial membrane
proteins (e.g., von Heijne, 1989) as well as of eukaryotic
ones was shown to be affected by charge mutations. The
type III protein cytochrome P-450 was converted to a type
II protein by insertion of positively charged residues into
its short NH2-terminal domain (Monier et al., 1988
; Szczesna-Skorupa et al., 1988
; Szczesna-Skorupa and Kemper,
1989
; Sato et al., 1990
). The asialoglycoprotein (ASGP)
receptor subunit H1 and the paramyxovirus hemagglutinin-neuraminidase, two type II proteins, were induced to
insert partially in the opposite type III orientation by mutation of flanking charges (Beltzer et al., 1991
; Parks and Lamb, 1991
, 1993
). Positive charges had a stronger effect
on topogenesis than negative ones and were more effective the closer they were to the hydrophobic segment.
However, in these and other studies (e.g., Andrews et al.,
1992
) the asymmetric distribution of flanking charges in
recombinant proteins was not sufficient to generate a
unique topology. Additional requirements must be met to
efficiently direct the polypeptide to insert with a single topology as generally observed for natural membrane proteins.
).
). Similarly, an artificial signal sequence with a hydrophobic segment of fewer than 12 leucines and a negative
NH2-terminal net charge was found to translocate the
COOH-terminal reporter sequence, whereas with longer
hydrophobic segments of 13 or 15 leucines, a fraction of
the polypeptides was anchored in the microsomal membrane from the cytosolic side as type III proteins (Sakaguchi et al., 1992
).
Materials and Methods
; Spiess and Lodish, 1986
) and the mutant constructs H1-4
(Beltzer et al., 1991
), HC (Schmid and Spiess, 1988
), H1
(= H1[
2-37]),
and H1
Leu19 (Wahlberg et al., 1995
) have been described previously.
Leu#.
The sequence encoding Leu19 in H1
Leu19 ends in a
BamHI site. The 5
sequence was sequentially shortened or extended by
three codons at a time by PCR using antisense oligonucleotide primers that corresponded to part of the Leu19 sequence and the BamHI site either lacking several leucine codons or containing additional ones. A second primer corresponding to a sequence in the plasmid vector was used.
PCR products were digested with HindIII and BamHI and ligated to the
3
BamHI-EcoRI portion of the H1 cDNA in the expression vector pECE
(Ellis et al., 1986
).
QLeu#.
The arginine codon CGC preceding the transmembrane
portion of the H1
Leu# constructs was mutated to CAG for glutamine by
PCR using the mutagenic primer CGGGTACCATGGGACCGCAGCTGTTGC, which also encodes a 5
KpnI site for subcloning. H1
Q was
similarly generated using the primer CGGGTACCATGGGACCTCAGCTCCTCC and H1
cDNA as the template.
Leu# was blunted and ligated to the 5
HindIII-NruI fragment of pSA11/5 (Spiess and Handschin, 1987
) to extend the 5
end by
that of wild-type H1.
Leu# were used as templates to
amplify the DNAs encoding the transmembrane and COOH-terminal
segments with a 5
GAT codon for aspartic acid and an overlapping BglII site, using the mutagenic primer GACCAGATCTGTTGCTTTTGCTGCTG. The DNA encoding the cytosolic portion of H1-4 with a matching
BglII site was amplified using the primer CAACAGATCTGGTCCGGAGCAGAGAT. The fragments were combined at the BglII site and
subcloned into pECE. Finally, the 3
BamHI-EcoRI fragments were replaced by the corresponding fragment of pSA1-4 (Beltzer et al., 1991
).
) encoding H1-4
having a potential glycosylation site in the cytoplasmic domain was used
as a template to amplify the DNAs encoding the NH2-terminal hydrophilic segment with a 3
BglII site, using the mutagenic primer CAACAGATCTGGTCCGGAGCAGAGAT. The resulting HindIII-BglII fragment was ligated to the BglII-EcoRI 3
portion of pSA1-4Leu# followed
by subcloning of the combined cDNA (HindIII-EcoRI) into pECE. The
sequence of the initial H1
Leu# constructs was confirmed by sequencing
and of the derivatives by diagnostic restriction analysis.
in 6-well clusters, and the
cells were processed the second day after transfection. For in vivo labeling, transfected cells were incubated for 30 min in methionine-free medium, labeled for 30 min at 37°C with 100 µCi/ml [35S]methionine in starvation medium, transferred to 4°C, and washed twice with PBS. To extract
cytoplasmic proteins, the cells were incubated with 500 µl of 0.1% saponin
in PBS for 30 min at 4°C. The saponin extract was removed, and the cells
were lysed. Both fractions were immunoprecipitated using a rabbit antiserum directed against a synthetic peptide corresponding to residues 277-
287 near the COOH terminus of the ASGP receptor H1 (anti-H1C). The immune complexes were isolated with protein A-Sepharose (Pharmacia, Upsala, Sweden) and analyzed by SDS-polyacrylamide gel electrophoresis. Alkaline extraction was essentially performed as described by Gilmore
and Blobel (1985)
.
-N-acetylglucosaminidase H (endo H; Boehringer
Mannheim Corp., Mannheim, Germany), the immune complexes were
isolated with protein A-Sepharose and boiled for 5 min in 50 µl, 50 mM
Na-citrate, pH 6, 1% SDS. Aliquots were incubated with 1 mU endo H for
2 h at 37°C. Finally, samples were boiled in SDS-sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis. The gels were fixed,
soaked in 1 M Na-salicylate containing 1% glycerol, dried, and fluorographed on Kodak BioMax MR films. To quantify the relative abundance
of polypeptides with different topologies, the fluorographs were densitometrically scanned, and the intensities of differently glycosylated species
were expressed as percentages of the total of all forms.
Leu19, H1
Leu22, and H1
Leu25 were determined by pulse labeling for 30 min followed by a chase of up to 5 h.
Products were immunoprecipitated and analyzed by gel electrophoresis
and fluorography.
Results
with a Leu19 Sequence Replacing Its
Hydrophobic Domain Is Partially Inserted in an
Inverted Nexo/Ccyt Orientation
resulted in a product
of ~36 kD (Fig. 1 A, lane 2) consistent with its reduced
size. Digestion of an aliquot of the immunoprecipitates
with endo H showed that H1
, like wild-type H1, was glycosylated at two sites in the COOH-terminal portion of
the protein (Fig. 1 B, lanes 1-4; Beltzer et al., 1991
). Deletion of the NH2-terminal portion thus had no effect on the topology of the protein in the membrane. This was to be
expected according to the charge difference criterion
(Hartmann et al., 1989
): the charge difference between the
15 COOH-terminal and the 15 NH2-terminal flanking residues,
(C-N), was
3 for both H1 and H1
, statistically
correlating with an Ncyt/Cexo orientation.
Fig. 1.
Mutant H1Leu19
is expressed as two differently glycosylated forms. (A)
COS-7 cells were transfected
with cDNAs of H1, H1
, H1Leu19, H1
Leu19, and
HC (the exoplasmic portion
of H1 equipped with the
cleavable signal sequence of
influenza virus hemagglutinin) as indicated. The transfected cells were labeled for
30 min with [35S]methionine,
solubilized, and subjected to
immunoprecipitation using
an antiserum directed against the COOH-terminal sequence of
H1. The immunoprecipitates were analyzed by gel electrophoresis and fluorography. (B) Immunoprecipitates were treated without (
) or with endoglycosidase H (E) before analysis by gel
electrophoresis. The positions of marker proteins are shown with
their molecular weights indicated in kD. The band indicated by
an asterisk represents a partially glycosylated species (see text).
[View Larger Version of this Image (54K GIF file)]
was replaced by a generic sequence of 19 leucine residues. Upon expression of this mutant H1
Leu19,
two major forms were generated: one of 36 kD, with the
same electrophoretic mobility as H1
, and a smaller one
of ~30 kD (Fig. 1 A, lane 4). This latter form was resistant
to endo H treatment and indistinguishable from the endo H digestion product of the higher molecular weight form
(Fig. 1 B, lanes 5 and 6). This indicated that the 30-kD expression product was an unglycosylated form of H1
Leu19.
were completely retained with the cellular membranes (Fig. 1 A, lanes 1-6). Both the glycosylated and the
unglycosylated forms of H1
Leu were clearly membrane
associated in this assay (Fig. 1 A, lanes 7-9). For alkaline extraction, the transfected and labeled cells were homogenized, exposed to pH 11.5, and then separated into soluble
fraction (S) and membrane pellet (P) by centrifugation
through a sucrose cushion (Fig. 2 B). Like H1
(Fig. 2 B,
lanes 4-6), both forms of H1
Leu19 were recovered in the
pellet fraction (Fig. 2 B, lanes 7-9). These results indicate
that the unglycosylated form of H1
Leu19 is integrated in
the membrane and argues against a reduced ability of the
Leu19 domain to function as a signal sequence.
Fig. 2.
The unglycosylated form of mutant H1Leu19 is inserted in the membrane in an inverted orientation. (A) Saponin
extraction: cells were transfected with the indicated constructs,
labeled, and extracted with 0.1% saponin. The saponin extract (S)
and the remaining cells (C) were separately immunoprecipitated
and analyzed by gel electrophoresis and fluorography. Untreated
cells were solubilized and immunoprecipitated as a measure of
the total material (TOT). (B) Alkaline extraction: transfected
and labeled cells were homogenized and incubated at pH 11.5. The samples were either immunoprecipitated directly (TOT) or
after separation into pellet (P) and supernatant fractions (S). (C)
Protease protection: transfected cells were labeled, homogenized,
and incubated without (
) or with trypsin (T) or with trypsin in
the presence of detergent (TD). Immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. The band indicated by an asterisk represents the partially glycosylated species. (D) Schematic representation of the membrane orientation
of constructs H1
and H1
Leu19. The H1 sequence is shown in
black and the Leu19 domain as an empty rectangle. The glycosylation sites as indicated by open circles and N-linked glycans by
closed squares.
[View Larger Version of this Image (53K GIF file)]
Leu19 products with respect
to the membrane was analyzed by protease digestion of
homogenized labeled cells. The glycosylated 36-kD form
of H1
Leu19 was resistant to trypsin, unless the membranes were disrupted with detergent (Fig. 2 C, lanes 4-6).
This confirms its type II topology with only four NH2-terminal residues exposed to the cytoplasm. In contrast, the
unglycosylated 30-kD protein was sensitive to trypsin also in the absence of detergent, indicating that the COOH-terminal portion is cytoplasmically exposed and that the protein was inserted into the membrane in an Nexo/Ccyt orientation. Thus, exchanging the apolar signal domain of H1
by an artificial, even more hydrophobic sequence of identical length resulted in a significant fraction of the polypeptides inserting as type III proteins (Fig. 2 D).
Leu19 was NH2-terminally extended by the NH2terminal hydrophilic portion of H1, the resulting construct
H1Leu19 was expressed uniquely as a glycosylated protein
of 40 kD like wild-type H1 (Figs. 1 A, lane 3, and 2 B, lanes
1-3), indicative of type II topology. This was confirmed by
trypsin digestion, which generated a resistant COOH-terminal fragment of 36 kD (Fig. 2 C, lanes 1-3). In an internal position, the Leu19 sequence thus did not affect the orientation of the protein.
Fig. 4.
Effect of different
hydrophobic domains on
membrane insertion of H1,
H1, and H1
Q. The constructs H1 (A), H1
(B), and
H1
Q (C) with the wild-type
transmembrane domain of
H1 (lane 1) or with hydrophobic segments consisting
of 7-25 leucine residues (lanes 2-8) were expressed in
COS-7 cells, labeled, immunoprecipitated, and analyzed
by gel electrophoresis and
fluorography. Membrane integration assessed by saponin
extraction (D) and protease
sensitivity (E) is shown for
the constructs with the shortest hydrophobic segments of
7 leucines (see legend to Fig.
2). The position of the
marker proteins of 29 and 35 kD are indicated.
[View Larger Version of this Image (38K GIF file)]
with
artificial transmembrane domains consisting of 7-25 leucine residues. Thus the overall hydrophobicity and the
length of the apolar domain were varied in parallel (Fig. 3).
Fig. 3.
Amino acid sequence of the signal-anchor domain of
H1 mutant constructs. The hydrophobic transmembrane segments
and their flanking sequences are listed. H1-4g and H1-4gLeu# are
identical to H1-4 and H1-4Leu# except for the insertion of the
tripeptide sequence MTM following asparagine-13 in the NH2terminal portion, which creates a potential glycosylation site.
[View Larger Version of this Image (25K GIF file)]
Leu#), the amount of the unglycosylated form increased with increasing number of leucines
in the signal anchor and reached ~80% of the total translation products for H1
Leu25 (Figs. 4 B, lanes 2-8, and
quantified in 5, closed squares). This form was essentially
absent in the constructs with transmembrane domains of
16 leucines or fewer. Increasing length and/or hydrophobicity of the signal anchor sequence thus promotes translocation of the NH2-terminal sequence across the ER membrane despite a negative charge difference
(C-N), when
the signal is positioned near the NH2 terminus of the protein. In contrast, the same constructs extended by the NH2terminal domain of H1 (H1Leu#) were all expressed exclusively as glycosylated type II membrane proteins of 40 kD (Figs. 4 A, lanes 2-8, and 5, open triangles). Even a hydrophobic core of 25 consecutive leucine residues in an internal position did not affect the protein's topology.
Q and H1
QLeu#, the NH2-terminal charge was reduced from +2 to +1, and the charge
difference changed from
3 to
2; this is still characteristic for many natural type II membrane proteins.
Q, which contains the wild-type transmembrane domain, this charge mutation resulted in ~25% of
type III insertion (Fig. 4 C, lane 1). For the H1
QLeu#
constructs (Figs. 4 C, lanes 2-7, and 5, closed circles), the
mutation resulted in an increased fraction of unglycosylated Nexo/Ccyt form in comparison to the H1
Leu# series.
Whereas the construct with the shortest apolar domain of
only 7 leucines still mostly translocated the COOH-terminal sequence across the ER membrane (Ncyt/Cexo), those
with the longest of 22 and 25 leucines almost exclusively
exposed their COOH-terminal domain to the cytoplasm
(Nexo/Ccyt).
Leu#, and H1
QLeu# constructs was confirmed by
saponin extraction, as is shown for those with the shortest
apolar segment of only 7 leucines in Fig. 4 D. The disposition of the COOH terminus was tested by protease sensitivity in Fig. 4 E for these constructs. The partial sensitivity
of H1Leu7 to exogenous trypsin indicates a transmembrane topology. Since seven residues are too short to span
a membrane as a helix, it is likely that the following eight residues in the sequence, which are largely polar but uncharged (Fig. 3), are also part of the membrane-embedded
segment.
Leu19 for
which the degradation rates of the two forms differ by a
factor of 10, the true insertion ratio of type II to type III
products, corrected on the basis of measured degradation
rates, were 59 to 41% in comparison to the apparent ratio
of 66 to 34% after 30 min pulse labeling. The different stabilities of the two forms thus cause a bias in favor of the more stable type II forms. However, the observed ratios in
our experimental setup are close estimates of the initial insertion ratios of the tested constructs.
4.5 (as calculated according to Hartmann et al.,
1989
), which is more negative than in the H1
Leu# series.
In addition, the presence of a sizable NH2-terminal hydrophilic domain is likely to disfavor Nexo/Ccyt insertion. To
test whether a long, hydrophobic signal domain in an internal position can influence insertion at all, we used H1-4, a previously described mutant of H1 in which the two
NH2-terminal and the two COOH-terminal charges flanking the transmembrane domain had been mutated to residues of opposite charge (Beltzer et al., 1991
; Fig. 3). Even
though the charge difference of this mutant was strongly
positive (+5), only half of the polypeptides were inserted
as type III proteins (Fig. 6 A, lane 1). In this mutant construct, the 19 hydrophobic amino acids of the signal anchor were replaced by 7-25 consecutive leucine residues, thus
generating the mutant series H1-4Leu#.
Fig. 6.
Effect of different hydrophobic domains on membrane
insertion of H1-4 and H1-4g. The constructs H1-4 (A) and H1-4g
(B) with the wild-type transmembrane domain of H1 (lanes 1 and
2) or with hydrophobic segments consisting of 7-25 leucine residues (lanes 3-14) were expressed in COS-7 cells, labeled, and immunoprecipitated. Samples were treated without () or with endoglycosidase H (E) before analysis by gel electrophoresis and
fluorography.
[View Larger Version of this Image (52K GIF file)]
Fig. 7.
Quantitation of the topology of the constructs H14Leu# and H1-4gLeu#. The insertion experiments including those
shown in Fig. 6 were quantified by densitometric scanning of the
fluorographs. The fraction of once glycosylated protein, i.e., with
Nexo/Ccyt orientation, is presented as percent of the total of all
forms as described in the legend to Fig. 5. The values for H14Leu# represent the mean of three or more experiments with
standard deviations; those for H1-4gLeu# represent single determinations performed simultaneously.
[View Larger Version of this Image (16K GIF file)]
Fig. 5.
Topology of signal-anchor mutants. Insertion experiments, including those shown in Fig. 4, were quantified by densitometric scanning of fluorographs. The fraction of unglycosylated
protein, i.e., with Nexo/Ccyt orientation, is presented as percent of
the total of all forms. The values for constructs with polyleucine domains are plotted as a function of the number of leucines in this segment (Leu#). Corresponding constructs with the transmembrane segment of wild-type H1 are shown to the right (wt).
The values represent the mean of three or more experiments with
standard deviations.
[View Larger Version of this Image (19K GIF file)]
), two types of products were expressed in transfected COS cells (Fig. 6 B): polypeptides
twice glycosylated in the COOH-terminal portion and polypeptides with electrophoretic mobility intermediate between the twice glycosylated and the deglycosylated proteins,
indicative of a single glycosylation in the NH2-terminal domain. For the shorter polyleucine transmembrane domains, the NH2-terminal glycosylation site resulted in an
increased type III insertion, as has previously been observed for H1-4g versus H1-4. A likely explanation is that
the tripeptide insertion to generate the glycosylation site
affected the folding of the NH2-terminal domain, thus facilitating its translocation through the membrane.
Discussion
; von Heijne, 1994; Spiess, 1995
). In addition,
the NH2-terminal hydrophilic portion and its folding properties were shown to play a topogenic role, because the
polypeptide needs to be unfolded for translocation through
the membrane (Denzer et al., 1995
). Deletion constructs by Sakaguchi et al. (1992)
and Sato et al. (1990)
also suggested an influence of the hydrophobic segment on the
topology. Whereas these criteria were shown by site-directed
mutagenesis to direct the insertion process, they were not
generally sufficient to generate a unique topology in recombinant proteins. In the present study we have systematically analyzed the contribution of the hydrophobic segment of the signal on topogenesis. By combining these
different determinants it was possible to generate uniquely
one or the other orientation in the membrane.
)
alone also had no effect. However, when in addition the transmembrane segment was exchanged by a stretch of 19 or more leucines, significant fractions of the polypeptides
inserted in the opposite Nexo/Ccyt orientation (H1
Leu19/
22/25). This fraction increased with the length of the
polyleucine segment. The effect was further enhanced by
reducing the positive charge at the NH2 terminus. Even
though the NH2 terminus still carried a net positive charge,
significant Nexo/Ccyt products were generated by all constructs, including that containing the wild-type transmembrane segment of H1 (H1
Q) as well as those containing
stretches of 7-25 leucines (H1
QLeu#). Constructs with
polyleucine sequences of 19 or more residues inserted essentially with unique type III topology.
16 leucines, type III insertion was significantly enhanced, and with 22 or 25 leucines, unique type III insertion was achieved.
; Parks, 1996
).
Multiple determinants for protein orientation allow variability in each of them without compromising insertion in
a unique topology. This may explain how the exceptional proteins that do not conform to the charge distribution
rule are correctly inserted. Unlike the charge distribution,
the length and the total hydrophobicity of the transmembrane segment do not clearly correlate with the orientation of natural signal anchor proteins. This suggests that
the flanking charges are the most general criterion in determining signal anchor orientation.
). According
to our results, NH2-terminal translocation is promoted by
these transmembrane segments in the order Leu19 > Leu16 > wild type. It is thus neither the length of the apolar segment of the signal nor its hydrophobicity alone that is responsible for the observed effects on insertion behavior. It
is obvious that two different sequences of the same length
also differ in other properties, e.g., the shape of the molecule and the propensity to assume
-helical conformation.
). Since they generally lack sizable hydrophilic NH2-terminal domains, translocation of
the NH2 terminus is not sterically hindered. Our results
are consistent with a role of short apolar signal domains in
keeping the NH2 terminus cytosolic and can provide an explanation for those eukaryotic signal sequences that do
not conform to the "positive inside" and the charge difference rules. Short polyleucine stretches in an NH2-terminal
position (constructs H1
Leu# and H1
QLeu#) promoted
translocation of the COOH-terminal sequence. In contrast, short polyleucine sequences in an internal position
(constructs H1Leu# and H1-4Leu#) had only a limited
effect on the topology of the proteins. H1-4Leu7, H14Leu10, and H1-4Leu13 inserted to the same extent of
~50% with Nexo/Ccyt orientation. The translocation machinery thus seems to recognize the hallmarks of a cleavable signal: a short hydrophobic segment close to the NH2
terminus. If these characteristics are present, there is a
strong preference for COOH-terminal translocation. Studies by Nilsson et al. (1994)
have provided evidence that
Ncyt/Cexo signals with short apolar segments are positioned
differently in the translocation machinery. The minimal
distance between the apolar segment of a signal and a potential glycosylation site required to reach the active site of oligosaccharyl transferase was found to be different for
short apolar segments of up to 14 leucine residues than for
longer apolar segments. TRAM may be the component of
the translocation machinery involved in distinguishing
these signals, since in reconstitution experiments TRAM
independence appeared to correlate both with the size of
the NH2-terminal hydrophilic portion of a signal and with
the length of the hydrophobic segment (Voigt et al., 1996
).
Received for publication 20 December 1996 and in revised form 15 February 1997.
1. Abbreviations used in this paper: ASGP, asialoglycoprotein; endo H, endo-We thank Nicole Beuret for technical assistance, Drs. C. Köhler and S. Schröder, and the colleagues in the lab for critically reading the manuscript.
This work was supported by grant 31-43483.95 from the Swiss National Science Foundation.