(Received for publication, October 25, 1995; and in revised form, December 19, 1995)
From the
The length requirement for a functional uncleaved signal/anchor
(S/A) domain of the paramyxovirus hemagglutinin-neuraminidase (HN) type
II glycoprotein was analyzed. HN mutants with progressive
NH-terminal S/A deletions or insertions were expressed in
HeLa cells, and the membrane targeting, folding, tetramer assembly, and
intracellular transport of the proteins were examined. Changing the
length of the S/A by two residues resulted in HN mutants that displayed
aberrant endoplasmic reticulum (ER) membrane targeting or
translocation. This phenotype did not simply reflect upper or lower
limitations on the size of a functional S/A, because normal signaling
was restored by further alterations involving three or four residues.
Likewise, ER-to-Golgi transport of mutants containing deletions of one
or two S/A residues was delayed (
30% of WT) or blocked, but
transport was restored for a mutant with a total of three deleted
residues. HN mutants with S/A insertions of three or four Leu residues
differed from wild-type HN by having heterogeneous Golgi-specific
carbohydrate modifications. Differences in ER-to-Golgi transport of the
mutants did not strictly correlate with defects in either native
folding of the ectodomain or the assembly of two dimers into a
tetramer. Together, these data suggest that efficient entry into and
exit from the ER are sensitive to changes in the HN S/A that may
reflect alterations to a structural requirement along one side of an
-helix.
The synthesis and assembly of eukaryotic integral membrane
proteins differs in several ways from the biosynthesis of other types
of cellular proteins. A specific requirement for the biosynthesis of a
membrane protein is that the nascent polypeptide chain must first be
targeted to the endoplasmic reticulum (ER) ()through
interactions with a dedicated cellular machinery (reviewed in Walter
and Lingappa(1986)). Once the nascent chain has been inserted into the
lipid bilayer, additional unique aspects of the biosynthesis of
membrane proteins involve the folding and assembly of distinct
extracellular, cytoplasmic, and lipid-imbedded regions of the protein.
Thus, individual segments of a single polypeptide chain must undergo
maturation steps in very different environments, including the ER
lumen, the cytosol, and the hydrophobic lipid bilayer.
Proper folding of these individual domains and subunit assembly are important factors that influence the intracellular transport of membrane proteins. An ER-localized mechanism appears to act on newly synthesized integral membrane proteins to control ER-to-Golgi transport (reviewed in Doms et al.(1993), Hammond and Helenius (1995), and Pelham(1989)). For a large number of membrane proteins, progress has been made in our understanding of some of the factors and biosynthetic steps involving the ER-lumenal ectodomain that dictate the formation of a transport-competent protein, and ER proteins that assist in these steps have been identified (Hammond and Helenius, 1995; Pelham, 1989). Likewise, a number of studies on the cytoplasmic tail of membrane proteins have identified a variety of distinct signals in this domain that act during biosynthesis and intracellular transport (e.g. Casanova et al.(1991), Nilsson et al.(1989), and Parks and Lamb(1993)). In contrast to the ecto- and cytoplasmic domains, the questions of how lipid-imbedded segments influence membrane protein biosynthesis and the nature of structural requirements in this domain remain largely unanswered.
For the majority of known
integral membrane proteins, an NH-terminal cleavable
sequence serves to signal the nascent chain to the ER membrane and a
separate COOH-terminal transmembrane (TM) domain acts as a membrane
anchor. By contrast, the type II integral membrane proteins lack an
NH
-terminal cleavable signal sequence, but contain an
internal hydrophobic signal/anchor (S/A) domain that serves a dual
function: the signaling of the nascent polypeptide to the ER membrane
and the subsequent anchoring of the polypeptide in the lipid bilayer.
Examples of type II proteins include the asialoglycoprotein receptor
(Spiess and Lodish, 1986), the influenza virus neuraminidase (Bos et al., 1984), signal peptidase (Shelness et al.,
1993), the Golgi-resident glycosyltransferases (Paulson and Colley,
1989), and the paramyxovirus hemagglutinin-neuraminidase (HN)
glycoprotein (Hiebert et al., 1985). The HN protein of the
paramyxovirus simian virus 5 has served as a model type II integral
membrane protein for the analysis of the membrane insertion, assembly,
and intracellular transport (Ng et al., 1989, 1990; Parks and
Lamb, 1990a, 1990b, 1993). The predicted structure of the simian virus
5 HN protein includes a 17-residue cytoplasmic tail, a 19-amino acid
uncleaved S/A, and a large 523 residue COOH-terminal ectodomain
(Hiebert et al., 1985). Newly-synthesized HN folds into a
structure that is recognized by conformation-specific antibodies and
oligomerizes into a tetramer (t
25-30
min) before transport from the ER to the medial Golgi (t
90 min; Ng et al.(1989)).
Alterations to the HN COOH-terminal ectodomain can result in a block in
ER-to-Golgi transport (Ng et al., 1990), consistent with an ER
quality control mechanism governing HN transport (Hammond and Helenius,
1995).
Recent work has shown that the uncleaved S/A domain serves multiple functions in the biosynthesis of a type II protein, including ER signaling, membrane anchoring, subunit assembly, and intracellular targeting. A typical S/A domain is longer than the hydrophobic core of a cleavable signal sequence (von Heijne, 1988), suggesting that subdomains may exist within the S/A that direct these individual functions. Previous attempts to identify by deletion analysis the subdomains that are responsible for signaling and anchoring have led to the proposal that these two functions are encoded by redundant and overlapping segments within the S/A (Brown et al., 1988; Lipp and Dobberstein, 1988; Spiess and Handschin, 1987; reviewed in Nayak and Jabbar, 1989). In contrast, mutational analysis of the HN protein has identified residues at the COOH-terminal end of the S/A that are important for the assembly of a tetramer from two dimers (McGinnes et al., 1993; Parks and Pohlmann, 1995), suggesting that the tetramer assembly function may reside in a subdomain of the HN S/A.
It has been shown that S/A alterations can also influence the
intracellular transport of a type II protein (reviewed in Machamer,
1993; Nayak and Jabbar, 1989), but to date no unifying theory has been
developed for the role that the S/A domain serves in the formation of a
protein that is competent for transport from the ER. Although it is
proposed that 20 amino acids are sufficient to span a lipid
bilayer as an
-helix, comparisons of known type II proteins have
shown a wide range of S/A lengths (Hartmann et al., 1989;
Bretscher and Munro, 1993). In some cases, the length of a S/A and not
the specific amino acid sequence per se may be an important
aspect of intracellular targeting of type II proteins (Masibay et
al., 1993; Munro, 1991). In this report, a systematic approach has
been taken to analyze the influence of S/A length on the biosynthesis
and intracellular transport of HN, a model type II plasma membrane
protein. The results suggest that efficient entry into and exit from
the ER are sensitive to changes in the conformation of the HN S/A that
may reflect alterations to a structural requirement along one side of
an
-helix.
Cells were washed in phosphate-buffered saline, lysed in 1% SDS,
boiled for 5 min, and clarified by centrifugation. Where indicated,
media were removed from cells and clarified by centrifugation (5 min,
15,000 g). Immunoprecipitations were carried out as
described previously (Ng et al., 1989; Lamb et al.,
1978), using rabbit polyclonal antiserum raised to SDS-denatured HN (HN
antisera, Ng et al.(1990)). Alternatively, cell lysates were
immunoprecipitated with mouse monoclonal antibodies (mAbs) 1b and 4b
that recognize epitopes in the fully mature form of HN (Randall et
al., 1987). Following immunoprecipitation, samples were treated
with endo-
-N-acetylglucosaminidase H (endoH) or
peptide:N-glycosidase F (N-glycanase) as described by
the manufacturer (New England Biolabs, Beverly, MA). Samples were
analyzed by SDS-PAGE on 10% polyacrylamide gels followed by
autoradiography (Lamb and Choppin, 1976). Radioactivity in dried gels
was quantitated using an Ambis radioanalytic imaging system (San Diego,
CA). Acquisition of endoH-resistant carbohydrate residues was
calculated as the ratio of radioactivity in the endoH-resistant form
divided by the total HN protein in both the endoH-resistant and
-sensitive forms. The data are the average of at least two experiments
(±10%), and are expressed as a percentage of that obtained with
WT HN assayed in parallel.
Figure 1:
Schematic diagram of HN S/A mutants.
The HN protein is shown schematically as a rectangle with a stippled box and vertical forks denoting the
membrane-spanning S/A domain and the four sites for N-linked
glycosylation in the COOH-terminal ectodomain, respectively. The amino
acid sequence of the HN S/A is listed below the stippled box along with five residues flanking the amino- (NH, left) and carboxyl- (COOH, right) terminal sides of
the S/A. Numbers represent the position of residues from the
NH
-terminal methionine. Arrows below the S/A
indicate the direction of progressive single residue deletions from the
NH
-terminal (mutants Del N1-4) or COOH-terminal (Del
C1-4) ends of the S/A. The box above the S/A indicates
the site of insertion of 1 to 4 leucine (Leu) residues between R17 and
T18 that created mutants Ins N1-4. The asterisk near the
NH
terminus indicates the location of a new site for N-linked glycosylation that was added to create mutant Ins N2*
as described in the text.
WT HN was synthesized
as a single major polypeptide of 68 kDa (Fig. 2, WT
lane) and was converted to a faster migrating form after
deglycosylation by treatment with N-glycanase (HN
, D lane). HN mutants containing deletions of one or two
residues from the NH
-terminal end of the S/A (Del N1 and
Del N2) were detected as polypeptides with an electrophoretic mobility
matching that of WT HN (Fig. 2, deletion lanes 1 and 2). In the case of Del N2, however, approximately 20% of the
newly-synthesized protein was detected as an additional form that had a
mobility on gels matching that of deglycosylated HN
(deletion panel, lane 2). Digestion of Del N2 protein
with N-glycanase resulted in a single HN
species
on SDS gels (data not shown, but see below), indicating that the HN and
HN
forms of Del N2 were a single polypeptide chain that
migrated differently on SDS gels due to the presence and absence of
carbohydrate chains, respectively. As the only sites for N-linked glycosylation of HN are in the COOH-terminal
ectodomain, the presence of the unglycosylated HN
form of
mutant Del N2 indicates that this protein is partially defective in
some aspect of ER targeting or translocation across the ER membrane. To
determine if the aberrant glycosylation of mutant Del N2 reflected a
lower limit on the size of a functional HN S/A, two addition mutants
were constructed that contained progressive NH
-terminal S/A
deletions totaling three and four residues (Del N3 and N4, Fig. 1). Del N3 and Del N4 polypeptides were expressed as the
major HN form, and relative to Del N2, only trace amounts of
unglycosylated HN
was detected (Fig. 2, deletion
panel, lanes 3 and 4). These data indicate that
the aberrant glycosylation seen with mutant Del N2 was not simply a
reflection of a lower limit on the size of a functional HN S/A.
Figure 2:
Expression of HN NH-terminal
S/A mutants. HeLa T4 cells were infected with vTF7.3 and then
transfected with plasmid DNA encoding wild type HN (WT lane),
one of the NH
-terminal deletion mutants Del N1-4 (Deletion lanes 1-4, respectively) or one of the HN Leu
insertion mutants Ins N1-4 (Insertion lanes 1-4,
respectively). After radiolabeling cells with Tran
S-label
for 30 min, proteins were immunoprecipitated from cell lysates with HN
antiserum before analysis by SDS-PAGE. The positions of WT (HN) and
deglycosylated (HN
) forms of HN are indicated by arrows. M, molecular weight marker lane with
indicated sizes in kDa; D, marker lane of deglycosylated HN
protein.
To
determine if S/A insertions had a similar effect on HN biosynthesis, HN
mutants Ins N1-4 (Insertion, N-terminal, 1-4 residues, Fig. 1) were constructed in which
one to four Leu residues were inserted into the NH-terminal
end of the S/A. When expressed using the vaccinia virus/T7 system
described above, all of the insertion mutant produced a protein that
comigrated on SDS gels with WT HN (Fig. 2, Insertion panel,
lanes 1-4). Most importantly, the insertion of two
additional Leu residues into the S/A resulted in a mutant (Ins N2) in
which nearly half of the newly-synthesized protein was detected as the
unglycosylated HN
form (Insertion panel, lane 2).
In contrast, mutants with insertions of one, three, or four Leu
residues produced relatively little of the HN
form (<5%
of total HN protein). The finding that insertions or deletions of two
S/A residues results in an unglycosylated form of HN suggests a
phase-specific requirement in the HN S/A for efficient ER targeting or
translocation (see below). Treatment of membranes isolated from cells
expressing the Ins N2 mutant protein with pH 11.0 buffer did not
extract the HN
polypeptide into a soluble form (data not
shown), suggesting that this protein species was stably associated with
membranes (Steck and Yu, 1973). It is possible that the deletion or
insertion of two residues into the S/A leads to molecules that are
partially defective in ER targeting or translocation, such that the
COOH-terminal domain of these molecules, which contains the only sites
for glycosylation, had not been translocated into the ER lumen.
Previous work has demonstrated that alterations to charged residues
flanking the HN S/A can lead to molecules that are inserted into the ER
membrane but have an inverted transmembrane topology (Parks and Lamb,
1993). Thus, a possible explanation for the presence of the faster
migrating HN form of the Del N2 and Ins N2 mutants is that
they represent molecules whose transmembrane orientation has been
reversed, such that they are now anchored in the membrane with the
NH
-terminal domain in the ER lumen. A prediction of this
outcome is that translocation of the NH
-terminal domain of
the inverted molecules across the ER membrane should expose the
NH
terminus to the glycosylation machinery that resides in
the ER lumen. To test this possibility, a consensus site for N-linked glycosylation (Asn-Ala-Thr; Parks and Lamb(1993)) was
added by site-specific mutagenesis to the NH
-terminal
domain of mutant Ins N2 to create Ins N2* (see asterisk in Fig. 1for location). A previously characterized HN mutant
containing substitutions of glutamate for arginine 13 and arginine 17
on the cytoplasmic side of the S/A (see Fig. 1) was expressed as
a positive control for inversion of HN membrane topology, as this
mutant adopts two opposing transmembrane topologies due to these amino
acid changes (HN 18*, Parks and Lamb(1993)). Following expression in
HeLa cells and radiolabeling with Tran
S-label, HN proteins
were immunoprecipitated, treated with endoglycosidase, and analyzed by
SDS-PAGE. The addition of carbohydrate to the NH
-terminal
glycosylation site of the inverted HN 18* molecules (Fig. 3, lane 2, HN
) results in a slower
electrophoretic mobility than deglycosylated HN (+lane
2). The mobility of the HN species is not altered by the presence
of the new glycosylation site, because the NH
terminus is
in the cytoplasm.
Figure 3:
The
NH terminus of HN mutant Ins N2 is not translocated into
the ER lumen. vTF7.3 infected HeLa T4 cells were transfected with
plasmid DNA encoding a modified version of mutant Ins N2 (Ins N2*, lane 1) or with a charge-altered HN mutant that adopts two
opposing transmembrane orientations (mutant HN18*, lane 2).
Both proteins contained a site for the addition of N-linked
carbohydrate at the NH
terminus. Following a 30-min
radiolabeling with Tran
S-label, cells were lysed and
proteins immunoprecipitated with anti-HN serum. Immune complexes were
incubated with (+lanes) or without
(-lanes) N-glycanase before analysis by
SDS-PAGE. The positions of WT (HN), deglycosylated (HN
),
and inverted (HN
) forms of HN are indicated by arrows.
Ins N2* was detected in equal amounts as two major
polypeptide species (Fig. 3, lane 1): one form with an
electrophoretic mobility matching fully glycosylated WT HN and a second
form with a mobility matching the deglycosylated protein
(HN). The comigration of the faster migrating Ins N2*
species with deglycosylated HN indicates that the new
NH
-terminal glycosylation site in the Ins N2* mutant was
not utilized. Given the caveat that alterations to the S/A may have
prevented glycosylation of a fully-translocated NH
-terminal
domain of Ins N2* molecules, these data indicate that the HN
form seen with mutants Del N2 and Ins N2 is not due to bona fide
inversion of the normal HN transmembrane orientation. While the nature
of the unglycosylated form of these mutants was not explored further,
the insertion or deletion of two residues at the
NH
-terminal end of the HN S/A results in polypeptides that
are aberrantly targeted to or translocated across the ER membrane.
As shown in Fig. 4(WT + lane), approximately half of the WT HN
polypeptides had acquired carbohydrate residues that were resistant to
endoH digestion by 90 min after synthesis. The electrophoretic mobility
of the endoH-resistant form of HN (Fig. 4, R arrow) is
not the same as that of the undigested HN (- lane),
because only two of the four COOH-terminal carbohydrate residues are
processed from a simple (endoH-sensitive, S) to complex
(endoH-resistant, R) form after transport to the Golgi apparatus (Ng et al., 1989, 1990). In contrast to the acquired endoH
resistance seen with WT HN, mutants Del N1 and N2 accumulated much
lower levels of endoH-resistant glycans by 90 min after synthesis (Fig. 4A, compare WT, 1 and 2 + lanes). Quantitation of gels from multiple experiments indicated
that the fraction of the total Del N1 protein that had accumulated
endoH-resistant carbohydrates (i.e. the ratio of the resistant
form to total radiolabeled protein) was only 30% of the value
obtained for WT HN. In the case of Del N2, only trace amounts of
protein with endoH-resistant glycans were detected. These data indicate
that decreasing the HN S/A by one or two residues can reduce or block
ER-to-Golgi transport.
Figure 4:
Acquisition of endoH resistance for HN S/A
mutants. HeLa T4 cells were infected with vTF7.3 and then transfected
with plasmid DNA encoding wild type HN (WT lanes), one of the
NH-terminal deletion mutants Del N1-4 (panel A,
lanes 1-4, respectively) or one of the HN Leu insertion
mutants Ins N1-4 (panel B, lanes 1-4, respectively). Cells were radiolabeled with
Tran
S-label, and incubated in nonradioactive chase medium
for 90 min. Proteins were immunoprecipitated from cell lysates with HN
antiserum. Immune complexes were then divided into two aliquots which
were incubated with buffer alone (-lanes) or with endoH
(+lanes) before analysis of samples by SDS-PAGE. R, endoH-resistant HN species; S, endoH-sensitive HN
species. The asterisk in panel B indicates the
position of a variant form of endoH-resistant
HN.
Surprisingly, however, a further deletion
that removed a total of three consecutive residues from the
NH-terminal end of the S/A resulted in a mutant protein
(Del N3) that was competent for transport from the ER (Fig. 4A, + lane 3). Quantitation of gels from
multiple experiments indicated that the fraction of the total Del N3
protein that had accumulated endoH-resistant carbohydrates was on
average
70% of the value found with WT HN analyzed in parallel.
The removal of a total of four S/A residues resulted in a mutant that
accumulated only trace amounts of endoH-resistant glycans (+ lane 4). Thus, transport of HN from the ER is reduced or blocked
by removal of one, two, or four consecutive residues from the
NH
-terminal end of the S/A, but transport can be restored
to levels similar to that obtained with WT HN when a total of three
residues are deleted from this region.
The effect of extending the
length of the HN S/A on ER-to-Golgi transport was examined as described
for the deletion mutants above. HN mutants containing insertions of one
or three Leu residues into the NH-terminal end of the S/A
accumulated endoH-resistant glycans to approximately half the level
seen with WT HN (Fig. 4B, + lanes 1 and 3). In the case of mutants with insertions of two or four
consecutive Leu residues, the fraction of properly translocated HN
molecules that had accumulated endoH-resistant glycans was greater than
that seen for mutants with insertions of one or three Leu residues
(+ lanes 2 and 4). As transport of these
insertion mutants was not blocked, these data suggest that the overall
effect of insertions into the S/A was less disruptive to ER-to-Golgi
transport than were the deletions described above.
Close inspection
of the products resulting from endoH digestion of mutants Ins N3 and
Ins N4 showed an additional protein species that migrated between the R
and S forms of HN (asterisk, Fig. 4B). By
comparison to the products of a partial digestion of WT HN produced by
using limiting amounts of endoH, the mobility of this endoH-resistant
variant form was consistent with HN polypeptides in which only one of
the four carbohydrate chains had been processed to an endoH-resistant
form (not shown). A time course of acquisition of endoH resistance was
carried out for Ins N3 and Ins N4 to determine the kinetics of
carbohydrate processing. As shown in Fig. 5, the variant form of
endoH-resistant HN was first detected by 30 min after synthesis for
both Ins N3 and Ins N4, and this species accumulated with approximately
the same kinetics as the fully processed R form. In comparison, WT HN
was processed throughout the time course into two distinct forms (R and S arrows, Fig. 5, WT panel), with no
detectable levels of the variant endoH-resistant form. Thus, in
contrast to the S/A deletions described above, insertions into the
NH-terminal end of the S/A do not block ER-to-Golgi
transport. However, insertions of three or four residues appear to
alter the processing of HN COOH-terminal carbohydrates by Golgi
enzymes.
Figure 5:
Time
course of acquisition of resistance to endoH digestion for HN mutants
Ins N3 and Ins N4. Dishes of vTF7.3-infected HeLa T4 cells were
transfected with plasmid DNA encoding Leu insertion mutants Ins N3, Ins
N4, or WT HN. Following radiolabeling for 30 min with
TranS-label, cells were incubated in chase medium for the
times indicated at the top of the figure (in h). Immune complexes
isolated from cell lysates were incubated with endoH before analysis by
SDS-PAGE. -Lane, pulse-labeled HN protein treated with
buffer alone. The asterisk indicates the position of a variant
form of endoH-resistant HN as discussed in the
text.
As shown in Fig. 6, WT HN was
detected on sucrose gradients as a major disulfide-linked species
sedimenting slightly slower than a 9 S marker protein (panel A,
fractions 5 and 6). The HN protein in the 9 S region of
the gradient migrates on SDS gels as a disulfide-linked dimer
(HN), consistent with the proposal that two dimers are held
in a tetramer by noncovalent interactions. A small amount of
disulfide-linked HN was detected near the bottom of the gradient
(fraction 1) and also as an aggregate in the pellet fraction that was
too large to effectively enter the gel (P fraction). HN mutants Del
N1-N4 were all detected on sucrose gradients as slower sedimenting
disulfide-linked dimers (panels B-E, respectively, fractions 8 and 9), indicating that each of these
mutants was defective in the assembly of two dimers into a stable
tetramer. It is noteworthy that the NH
-terminal deletion
mutants also produced significant levels of monomeric HN protein (e.g.Fig. 6, panels C and E), a
result that suggests an additional role for this region of the S/A in
the assembly of two HN monomers into a dimer. The finding that mutants
Del N2 and Del N3 both sedimented as dimers (panels C and D) and yet displayed very different ER-to-Golgi transport
phenotypes indicates that the efficiency with which a S/A mutant is
transported from the ER does not strictly correlate the assembly of two
dimers into a stable tetramer.
Figure 6:
Sucrose gradient sedimentation analysis of
NH-terminal S/A deletion mutants. vTF7.3 infected HeLa T4
cells were transfected with plasmid DNA encoding wild type HN (panel A), or HN mutant Del N1 (panel B), Del N2 (panel C), Del N3 (panel D), or Del N4 (panel
E). Cells were radiolabeled with Tran
S-label, and
incubated in nonradioactive chase medium for 90 min. Cell lysates were
analyzed by sucrose gradient sedimentation as described under
``Materials and Methods.'' Gradient fractions were analyzed
by immunoprecipitation and SDS-PAGE under nonreducing conditions. The P lane represents proteins immunoprecipitated from the pellet
fraction that was recovered from the bottom of the tube. Sedimentation
was from right (top fraction) to left (bottom fraction).
HN
, HN monomer; HN
, disulfide-linked HN dimer. Arrows in panel A denote the positions of 9 S and 4 S
sedimentation markers that were analyzed in a parallel
gradient.
The oligomeric form of the HN
insertion mutants Ins N1-4 was determined by sucrose gradient
sedimentation. For each insertion mutant, the major oligomeric form
detected on sucrose gradients was a disulfide-linked dimer (Fig. 7, fractions 8 and 9) that sedimented
more slowly than the WT HN tetramer. In addition, lesser amounts of
insertion mutants Ins N2-N4 were detected as higher order
disulfide-linked oligomers that sedimented to the bottom portion of the
gradient (panels B, C, and D, fractions
1-5). A portion of the mutant polypeptides displayed
aberrant electrophoretic mobilities under the non-reducing conditions
of the SDS gels (e.g.Fig. 7, panel D,
HN species), a result suggesting that disulfide bonding of
the large COOH-terminal ectodomain of these mutants was altered.
Together these results indicate that the formation of a stable HN
tetramer is sensitive to deletion or insertion of even a single residue
from the NH
-terminal end of the S/A, and that tetramer
assembly defects do not correlate with efficiency of transport from the
ER.
Figure 7:
Sucrose gradient sedimentation analysis of
HN Leu insertion mutants. vTF7.3 infected HeLa T4 cells were
transfected with plasmid DNA encoding HN mutants Ins N1-4 (panels A-D, respectively). Cells were radiolabeled with
TranS-label, and incubated in nonradioactive chase medium
for 90 min. Cell lysates were prepared and analyzed as described in the
legend to Fig. 6. HN
, HN monomer; HN
, disulfide-linked HN
dimers.
Figure 8:
EndoH analysis and secretion of
COOH-terminal S/A deletion mutants. vTF7.3-infected HeLa T4 cells were
transfected with plasmid DNA encoding WT HN or one of the COOH-terminal
deletion mutants Del C1-4 (lanes 1-4,
respectively) and radiolabeled for 30 min with
TranS-label. A, endoH analysis of cell-associated
HN proteins. Following radiolabeling and a 90-min incubation in chase
medium, cells were lysed and proteins were immunoprecipitated from the
extracts with anti-HN serum. Immune complexes were incubated in the
presence (+) or absence(-) of endoH before analysis by
SDS-PAGE. B, secretion of COOH-terminal deletion mutants.
Following a 3-h chase period, samples representing all of the medium (medium lanes) or one-fourth of the cell-associated protein (cell lanes) were immunoprecipitated with anti-HN serum and
analyzed by SDS-PAGE.
To determine if the HN COOH-terminal
S/A deletion mutants were secreted from cells, infected/transfected
cells were pulse-labeled with TranS-label for 30 min and
incubated in chase medium for 3 h before analysis of cell-associated
proteins and media. HN protein was detected in extracellular media from
cells expressing mutants Del C2-C4, but not from cells expressing WT HN
or mutant Del C1 (Fig. 8B). The lack of intracellular
Del C2-C4 protein with endoH-resistant glycans (Fig. 8A) is consistent with the proposal that
ER-to-Golgi transport of HN is rate-limiting, but once HN reaches the
Golgi the transport to the cell surface (i.e. secretion) is
very rapid (Parks and Lamb, 1990b). Thus, it appears that Del C2-C4
proteins are synthesized in a soluble and secreted form due to S/A
deletions. Similar results have been reported previously for S/A
deletion mutants of the influenza A virus NA and the MHC-associated
invariant chain (Hogue and Nayak, 1994; Lipp and Dobberstein, 1986).
Previous work on type I integral membrane proteins has shown that changes in the length of a membrane anchor domain do not always disrupt stop-transfer function or intracellular transport. For example, the COOH-terminal membrane anchor of the vesicular stomatits virus G protein can be shortened from 20 to 14 residues without affecting anchoring or transport of the protein from the ER to the cell surface (Adams and Rose, 1985). For type II proteins, the length requirements for each of the S/A functions in biosynthesis and transport have not been extensively characterized. The in vivo data presented here indicate that changes in S/A length can have two general consequences on a biosynthetic step: a phase-specific effect for entry into and exit from the ER or a general effect on the structure of the ectodomain as seen for subunit assembly and carbohydrate processing.
Increasing or decreasing the length of the hydrophobic core of the
S/A by two residues resulted in polypeptides that displayed aberrant
targeting to or translocation across the ER membrane. This phenotype
did not simply reflect upper or lower size limitations on a functional
HN S/A, because normal signaling function could be restored by further
alterations involving greater than two residues. It is unlikely that
this phenotype results from disruption of a signal in the primary amino
acid sequence per se, since NH-terminal S/A
substitutions that do not change the length of the hydrophobic domain
show no detectable defect in these steps (Parks and Pohlmann, 1995).
Previous in vitro studies have led to the proposal that
hydrophobicity is the dominant feature of a functional S/A (Spiess and
Handschin, 1987; Zerial et al., 1987; reviewed in Nayak and
Jabbar, 1989). In the case of cleavable signal sequences, theoretical
considerations (reviewed in Engleman et al., 1986), as well as
results from mutational and biophysical studies, have suggested that an
additional critical feature of a functional signal sequence may be the
propensity of the hydrophobic core to form an
-helical
conformation (reviewed in Izard and Kendall, 1994). The data presented
here indicate that an optimum length of the hydrophobic core of a S/A
can greatly influence the efficiency with which a type II protein is
properly targeted to the ER in vivo, and suggest that there is
a functional requirement in the S/A along one face of an
-helix.
While alterations of two residues could prevent the formation of an
-helical conformation in the nascent chain, it is also possible
that these alterations change the relative position of critical
residues along one face of an
-helix.
Once inserted into the ER
membrane, changes in the length of a transmembrane domain could have
several possible consequences on S/A-lipid interactions:
membrane-flanking sequences could be pulled into or could extend out of
the lipid bilayer, the hydrophobic segment could adopt a compacted or
extended structure, or the lipid bilayer itself could adapt to
accommodate length changes in the hydrophobic segment of the
polypeptide (Adams and Rose, 1985). Theoretical models (e.g. Mouritsen and Bloom, 1984) as well as experiments with
bacteriorhodopsin in synthetic bilayers (Lewis and Engelman, 1983)
suggest that it is more likely that changes will take place in the
lipid bilayer to accommodate the length of the hydrophobic anchor
domain than to have changes occur in the structure of the
membrane-imbedded polypeptide segment. These putative changes in the
thickness of the bilayer induced by the polypeptide chain could prevent
transport from the ER by influencing the ability of the altered protein
to be incorporated into transport vesicles. While removal of one or two
residues from the NH-terminal end of the HN S/A resulted in
proteins that were delayed or blocked in transport from the ER to the
Golgi, transport of HN could be restored by a further deletion that
resulted in the removal of a total of three consecutive S/A residues.
Assuming the lipid bilayer changes to accommodate S/A length, the
efficient transport of this mutant from the ER is not consistent with a
control mechanism that is based on partitioning of membrane proteins by
lipid bilayer thickness alone. The easiest interpretation of these
results is that HN S/A length is less important for efficient
ER-to-Golgi transport than is overall conformation, and that the
deletion of three residues restores a transport requirement along one
face of an
-helix.
Several lines of evidence are consistent
with a requirement for a critical structure along one face of a TM
-helix. First, sequence comparisons between the TM domains of
various strains of the influenza virus HA (Lazarovits et al. 1990) and Newcastle disease virus HN proteins (Sakaguchi et
al., 1989) have shown conservation of sequence at intervals that
cycle every fourth or seventh position within the TM domain,
respectively. Second, extensive mutagenesis of the TM domain of M13
coat protein and of glycophorin A has identified positions that are
sensitive to assembly-disrupting mutations. These positions occur with
a periodicity of
4 residues, suggesting that these critical TM
residues might compose a face of an
-helix that is involved in
subunit interactions (Deber et al., 1993; Lemmon et
al., 1992). In the case of the
subunit of CD3, ER retention
and degradation can be affected by changes in TM domain length,
consistent with a requirement for correct positioning of a charged
residue along a face of a TM
-helix (Lankford et al.,
1993). Finally, the structure of the photosynthetic reaction center TM
segments shows that residues forming helix-helix contact points are
spaced at intervals of
4 residues (Rees et al., 1989).
It is possible that the phase-sensitive transport defect seen with
the HN S/A deletion mutants results from a defect in proper subunit
helix-helix interactions in the lipid bilayer. Assuming the HN S/A
exists as an -helix in the membrane, the removal of three S/A
residues might restore the correct face of an
-helix relative to
neighboring subunits and allow transport whereas other alterations
might prevent proper subunit interactions. However, it is important to
note that HN S/A mutants with very different ER-to-Golgi transport
characteristics were all dimeric, indicating that a defect in tetramer
assembly does not always correlate with reduced transport from the ER
(Parks and Pohlmann, 1995). A limitation of the sedimentation assay is
the inability to distinguish between two types of assembly-defective
mutants: those that have matured only to the point of forming dimers
and those that sediment as dimers as a result of unstable tetramers
which have dissociated during centrifugation (Parks and Pohlmann,
1995). Thus, all dimers detected by sedimentation analysis may not be
equivalent, having inapparent differences in assembly that account for
the varying transport phenotypes.
For a subset of insertion mutants Ins N3 and Ins N4, only one of the four COOH-terminal carbohydrate residues was processed to an endoH-resistant form by Golgi-specific enzymes, contrasting with the two processed chains seen for WT HN (Ng et al., 1989). The link between processing of N-linked oligosaccharides and transport is not absolute, as examples of surface-expressed membrane proteins with endoH-sensitive N-linked carbohydrates have been described (Williams and Lamb, 1988). Furthermore, heterogeneity in carbohydrate processing can occur when membrane proteins are expressed in a soluble form from cDNA plasmids (e.g. influenza NA protein, Paterson and Lamb(1990)) or may be a natural feature of some membrane proteins (Hubbard, 1988). Oligosaccharide processing can be influenced by local secondary structure around an N-linked carbohydrate site or by subunit interactions by a mechanism that may involve stearic inaccessibility (Ashford et al., 1993; Hubbard, 1988). Thus, the differential processing of Ins N3 and Ins N4 oligosaccharides can be viewed as indicative of changes in the ectodomain conformation or subunit interactions that may influence access to one of the COOH-terminal carbohydrate chains.
In summary, these data demonstrate that changes
in S/A length can differentially affect various steps in the
biosynthesis and transport of a type II protein. Entry into and exit
from the ER are sensitive to S/A length changes in a phase-specific
manner, suggesting a requirement in the S/A along one side of an
-helical configuration to function efficiently in these steps. The
positioning of subunits may be a critical step during the formation of
a disulfide-linked dimer or during interactions with cellular proteins
involved in promoting assembly and intracellular transport.