(Received for publication, August 16, 1994; and in revised form, December 22, 1994)
From the
The Neurospora plasma membrane
H-ATPase belongs to a family of cation-motive porters
called P-type ATPases. Putative transmembrane segments of these enzymes
contain one or more charged residues. Conditions were determined by
which a transmembrane segment with charged residues is integrated into
its cognate membrane. We constructed fusion proteins flanked by the
hydrophilic domains of the amino and carboxyl termini of the
H
-ATPase that contained either one or two
transmembrane segments. Neurospora in vitro translation system
supplemented with homologous microsomes was programmed with RNA
transcripts of these constructs. When transmembrane segment number one
(M1) or number two (M2) of the H
-ATPase was engineered
into the construct, the resultant protein did not integrate into
microsomes. When M1 and M2 were placed in tandem, the resultant protein
integrated into microsomes as judged by the criteria of resistance to
extraction at pH 11.5 and protection from protease digestion. The
integration event depended on ATP and GTP and on microsomal protein(s).
We posited that membrane topology of the amino-terminal third of the
H
-ATPase, and perhaps of other P-type ATPases is
achieved by inserting transmembrane segments into membrane in pairs.
The Neurospora plasma membrane electrogenic,
proton-translocating ATPase (H-ATPase) (
)transduces the chemical energy of ATP hydrolysis into a
transmembrane proton-motive force (Addison and Scarborough, 1981). The
latter functions to drive the uptake and extrusion of various ions and
molecules through chemiosmotic coupling devices called porters
(Mitchell, 1967). The Neurospora H
-ATPase
belongs to a family of P-type cation-motive ATPases that includes the
Na
/K
, the
H
/K
, and the Ca
motive ATPases from higher eukaryotic cells (Addison, 1986). We
know a great deal about the reaction mechanisms and the physiological
roles of these cation-motive ATPases. Their primary structure is known
mainly because of the methods of gene cloning and DNA sequencing. Yet
little is known about the structure-function relationships of these
remarkable energy transducers.
Knowledge of the membrane topology of P-type ATPases is essential to gaining insights into their structure-function relationships. Such knowledge, unfortunately, is not easy to obtain. Membrane topological models of P-type ATPases are based almost exclusively on hydropathy analysis of their primary structure. There is confusion about the actual number of transmembrane segments. Most agree these enzymes contain either 8 or 10 segments. Because of these uncertainties, a method is needed to verify if a predicted segment is within the membrane. One method is to study the topogenesis of P-type ATPases. The molecular mechanisms by which polytopic integral membrane proteins achieve their membrane topology are poorly understood, however. Topogenesis of membrane proteins is presumed to occur by sequential insertion of topogenic signals into the endoplasmic reticulum (Blobel, 1980). Transmembrane segments serve as the topogenic signals; odd-numbered segments initiate translocation of the carboxyl-terminal residues across the membrane, while the even-numbered segments stop the translocation process.
Using the Neurospora H-ATPase as a model P-type ATPase, we are
investigating the integration of this P-type ATPase into its cognate
membrane (Addison, 1990, 1991, 1993; Lin and Addison, 1994). We
constructed fusion proteins flanked by hydrophilic amino acids of the
amino and carboxyl termini of the H
-ATPase containing
putative transmembrane segments. RNA transcripts of these constructs
were translated in a Neurospora in vitro system supplemented
with homologous microsomes (Addison, 1987). The results demonstrate
that transmembrane segments of Neurospora H
-ATPase, when assayed out of context, have
various degrees of effectiveness as a topogenic signal and provide
functional evidence for a novel integration signal that is composed of
two transmembrane segments. In considering the results obtained, a
model is presented of how the amino-terminal third of the
H
-ATPases achieves its membrane topology.
Figure 1:
Constructing fusion proteins.
Single-letter code is used for the amino acids. Numbersabove each correspond to its position in the primary
sequence of the H-ATPase. Residues marked by an asterisk were introduced with restriction endonuclease
recognition sequences of oligonucleotide primers used in PCR. Protein
containing transmembrane segment was designated pM. Number
following M represents the position of the segment in the
H
-ATPase. The yeast invertase fragment is represented
by the thickerbar with verticallines, putative glycosylation sites, in
p
M.
pM1InM2 was made by inserting between the
DNA fragments (2) and (3) of the construct for pM
a 69-bp fragment encoding amino acid residues 116-138 (M1) of the
H
-ATPase and between DNA fragments (3) and (4) a 60-bp fragment encoding amino acid residues 141-160
(M2) of the H
-ATPase. pM3InM4 was made by inserting
between the DNA fragments (2) and (3) of the construct
for p
M a 69-bp fragment encoding amino acid residues 292-314
(M3) of the H
-ATPase and between the DNA fragments (3) and (4) a 69-bp fragment encoding amino acid
residues 322-354 (M4) of the H
-ATPase. The
overhang ends of DNA fragments were either filled-in using Klenow
fragment or deleted using mung bean nuclease to maintain the correct
reading frame of some inserts. All constructions introduced DNA
fragment encoding amino acid residues Gly-Ser-Ile at the 3` end of M2
and M4, DNA fragment encoding Ser-Ala-Glu at the 5` end of M2, and DNA
fragment encoding Ser-Ala-Ala at the 5` end of M4.
For the
energy-dependent studies, we used RNA transcripts transcribed from DNA
constructs for pM1M2 and pM3M4 without a stop codon based on the reason
described under ``Results.'' These were generated by
replacing DNA fragment (5) in the constructs with a 126-bp
fragment that codes for the last 42 amino acid residues of the
H-ATPase. The recombinant plasmids were restricted
with PvuII. The resultant transcripts contained no stop codon
and encoded an additional 45 amino acid residues derived from the
plasmid.
For all constructs, DNA fragments that encoded for the
amino acids indicated in Fig. 1by asterisks were
introduced with the oligonucleotide primers for the PCR. The single
strand DNA that served as template for the PCR was generated from the
recombinant phagemid pBluescript II KS as described by
the supplier (Stratagene, La Jolla, CA). The PCR products were
generated using the Perkin Elmer's GeneAmp kit and DNA Thermal
Cycler 480 according to protocols from supplier (Perkin Elmer). Primers
were removed from the PCR mixtures using the PrimeErase Quick push
column from Stratagene. The resultant products were digested with
restriction endonucleases. The resultant mixtures were resolved on an
agarose gel; the product of the expected size was excised and eluted
from the gel using Glassmilk or Glassfog (Bio 101, La Jolla, CA). The
PCR product was cloned into the plasmid as outlined above. All plasmid
constructs were verified by dideoxy sequencing (Sanger et al.,
1980).
Topological models of the Neurospora plasma membrane
H-ATPase are based mainly on hydropathic plots of the
primary sequence that predict either eight (Hager et al.,
1986), 10 (Addison, 1986), or 12 (Rao et al., 1991)
transmembrane segments. This method predicts transmembrane segments by
scanning the primary sequence for stretches of hydrophobic residues
long enough to span the lipid bilayer as
-helices. The eight- and
10-segment models agree that the amino-terminal third of the
H
-ATPase contains four transmembrane segments. The
relative position of these transmembrane segments is supported by
trypsin digestion of the reconstituted Neurospora H
-ATPase (Rao et al., 1991). Integration
of the H
-ATPase and truncated forms of the
H
-ATPase into microsomes suggests the presence of
internal signal sequences (Addison, 1990, 1991). Also, a fusion protein
with transmembrane segment number 3 (M3) of the
H
-ATPase integrated into microsomes as a type II
integral membrane protein (Lin and Addison, 1994). The objective of
this report is to determine how M1, M2, and M4 of the
H
-ATPase are integrated into the membrane.
Using
the PCR (Scharf, 1990), fusion proteins were constructed containing
sequences of putative transmembrane segments. The hydrophilic amino
acid residues of the amino and carboxyl termini of the
H-ATPase flanked the fusion proteins (Fig. 1).
Carboxyl-terminal to the putative transmembrane segment was engineered
a segment of invertase that contains three consensus N-linked
glycosylation sites (Taussig and Carlson, 1983) to serve as a reporter
of translocation. In constructing the fusion proteins, we used primary
sequence of transmembrane segments as predicted by the 10-segment model
(Addison, 1986). Complementary DNA of fusion proteins was transcribed in vitro by T3 RNA polymerase; the resultant transcripts were
translated in a Neurospora in vitro system with or without
homologous microsomes. The proteins were identified by
immunoprecipitation using rabbit polyclonal antibodies directed against
carboxyl-terminal residues of the H
-ATPase.
Integration of proteins into microsomes was determined by monitoring
formation of glycosylated products, cosedimentation of products with
microsomes after alkaline treatment, and digestion of products by
protease.
Figure 2:
Translation and membrane integration of in vitro synthesized
[S]methionine-labeled p
M, pM1, pM2, pM3,
and pM4. Transcripts were translated in Neurospora in vitro system with microsomes (nRM) at 3 A
units/ml as indicated. Translation mixtures were extracted with
0.1 M Na
CO
, pH 11.5, and fractionated
into soluble (S) fraction and membrane pellet (P) as
described under ``Experimental Procedures.'' Proteins were
resolved on 10% polyacrylamide gel with SDS and visualized by
fluorography. Glycosylated protein is indicated by an asterisk.
In the
presence of microsomes, there was no change in the relative molecular
mass of pM1, pM2, or pM4 (Fig. 2, lanes1 and 3) as well as no significant increase in the quantity of
proteins found in the pellet fraction after alkaline treatment (Fig. 2, lanes2 and 4). In the
absence of microsomes, a small percentage of product is found in the
pellet fraction (lane2). This is caused most likely
by aggregate forms of the products that sediment under these conditions
(Addison, 1990). Fractionation of products using sucrose step gradients
containing 0.15 M KOAc gave similar results (data not shown),
precluding the possibility that pM1, pM2, and pM4 were bound to but not
integrated into microsomes. Only pM3 showed a change in its relative
molecular mass in the presence of microsomes; one product had a M of 36,100, and the other had a M
of 42,700 (Fig. 2, lane4). All of the M
42,700 product and about 60% of the M
36,100 product were found in the pellet fraction
after extracting the microsomes with 0.1 M Na
CO
, pH 11.5, and then fractionating the
products through a sucrose step gradient containing 0.1 M Na
CO
, pH 11.5 (Fig. 2, lanes3 and 4). Alkaline extraction converts closed
microsomes into opened sheets concomitantly extracting proteins from
the exposed faces of the sheets (Fujiki et al., 1982).
Peripheral membrane and lumenal proteins are found in the supernatant,
but integral membrane proteins sediment with the sheets or membrane
remnants. When the translational mixture with pM3 was treated with
N-glycosidase F, an amidase that cleaves between the innermost GlcNAc
of oligosaccharide chains and the asparagine residue, only the M
36,100 product was observed on SDS-PAGE (data
not shown). This demonstrated that the increase in the relative
molecular mass of pM3 was caused by adding N-linked
oligosaccharide side chains to the invertase segment as it was
translocated into the lumen of the microsomes.
In the absence of microsomes, the transcripts for pM1M2 yielded one product (Fig. 3, lane1). In the presence of microsomes, approximately 50% of the product cosedimented with microsomes after alkaline extraction (Fig. 3, lanes3 and 4). The presence of pM1M2 in the pellet fraction implies that this protein has topogenic information for integration. Less than 10% of pM1M2 is glycosylated by microsomes. When we engineered about 20 additional native residues carboxyl-terminal to M2 in pM1M2, glycosylation of the resultant protein by microsomes was not observed (data not shown). Clearly, combining M1 and M2 created a topogenic signal. Joining M1 and M2 may have created a stretch of hydrophobic amino acid residues. In the primary sequence of M1 and M2 (Fig. 1), the longest continuous stretch of hydrophobic amino acids is 10, and this is located in M2. As demonstrated, pM2 did not sediment with microsomes after alkaline extraction (Fig. 2). Alternatively, M1 and M2 may have created a structure that served as the topogenic signal. If so, this implies a crucial role for the proximity of the transmembrane segments. To disrupt this putative structure of the transmembrane segments, we inserted the invertase segment (see Fig. 1) between M1 and M2. Integration effectiveness of the resultant protein pM1InM2 was reduced to 20% of that of pM1M2 (Fig. 3, lanes3 and 4), and it did not become glycosylated. This was interpreted to imply that the topogenic information for integration is a function of the proximity of the transmembrane segments.
Figure 3:
Translation and membrane integration of in vitro synthesized
[S]methionine-labeled pM1M2, pM3M4, pM1InM2,
pM3InM4, and pM1M4. Transcripts were translated with microsomes (nRM) as indicated. Mixtures were extracted with 0.1 M Na
CO
, pH 11.5, and fractionated into
soluble (S) fraction and membrane pellet (P) as
described under ``Experimental Procedures.'' Proteins were
resolved on 10% polyacrylamide gel with SDS and visualized by
fluorography. Glycosylated protein is indicated by an asterisk.
pM4 did not sediment
with microsomes after alkaline extraction. M4 is a presumed
stop-transfer signal. To evaluate this possibility, we constructed
pM3M4 (Fig. 1). M4 partially blocked transfer of the invertase
segment into microsomes (compare Fig. 3, lane4, with Fig. 2, lane4). To make
charge differences in the residues flanking the segments in pM3M4
similar to that of the native protein, hydrophilic residues flanking
the segments were engineered into the construct (see Fig. 1).
Despite this precaution, about 30% of pM3M4 had an increase in the
relative molecular mass of 2,400, suggesting the addition of one N-linked oligosaccharide side chain to the invertase segment.
It is not apparent why only one site in the invertase segment was
glycosylated. The appearance of glycosylated pM3M4 demonstrated that
the four lysines carboxyl-terminal to M4 (Fig. 1) could not
completely block transfer of the invertase segment into the lumen of
microsomes. When 29 native residues (residues 364-392 of the
H-ATPase) were engineered carboxyl-terminal to M4 in
pM3M4, the resultant protein was still glycosylated. When native
residues 262-283 were engineered amino-terminal to M3 and
residues 364-392 carboxyl-terminal to M4, the resultant protein
was not glycosylated (data not shown). This was interpreted to suggest
that factors other than difference in the charge of residues flanking
the transmembrane segments and the presence of positively charged
residues dictate orientation of segments in the membrane. These results
were unexpected and apparently add an additional level of complexity as
to the nature of the factors controlling orientation of transmembrane
segments in the membrane.
Although M4 did not function
independently, it functioned as a stop-transfer signal when engineered
carboxyl-terminal to M3. Did the activity of M4 in pM3M4 depend on its
proximity to M3? To evaluate this, we inserted the invertase segment
between the segments in pM3M4 to give pM3InM4. In the presence of
microsomes, pM3InM4 gave a slower migrating product on SDS-PAGE with a M of 44,200 that sedimented with microsomes after
alkaline extraction (Fig. 3, lanes3 and 4). N-glycosidase F converted the slower migrating
product to a product with M
of 38,100 (data not
shown), implying that the invertase segment was glycosylated. To
determine if M4 blocked transfer of the carboxyl terminus into the
lumen of the microsomes, pM3InM4 was digested by proteinase K in the
absence or presence of microsomes (Fig. 4). The protected
fragment observed only when microsomes were present in the mixture was
not immunoprecipitated by polyclonal antibodies directed against the
amino or the carboxyl-terminal residues (Fig. 4, lanes4 and 5). These results demonstrate that pM3InM4
was integrated into microsomes as a polytopic IMP, implying that the
decoding of M4 in pM3InM4 is independent of its proximity to M3.
Figure 4:
Analysis of pM3InM4 by proteinase K
digestion. pM3InM4 was synthesized in the absence or presence of
microsomes (nRM) and then digested by proteinase K at 150
µg/ml as described under ``Experimental Procedures.''
Triton X-100 (TX-100) was present at 1% during the
proteinase K (lane3). Digested mixtures were either
precipitated by trichloroacetic acid (lanes1-3) or immunoprecipitated by polyclonal antibodies (Ab) directed against amino (N, lane4) or carboxyl (C, lane5)
terminal residues. Molecular mass markers are hen egg white ovalbumin,
bovine carbonic anhydrase, and soybean trypsin inhibitor with M of 42,700, 31,000, and 21,500, respectively.
Precipitates were resolved on 15% polyacrylamide gel with SDS; product
was visualized by fluorography.
The integration efficiency of pM1M2 was reduced by inserting amino acids between the segments. This suggests that decoding the segments in pM1M2 is a function of the proximity of M1 and M2. Does the putative association of M1 and M2 represent specific interactions between these segments? Can another segment in the proximity of M1 create the structural motif that is decoded as an integration signal? To answer this question, we replaced M2 with M4 to give the resultant protein pM1M4 (Fig. 1).
Without microsomes in the translation mixture, transcripts for pM1M4 yielded one product (Fig. 3, lane1). With microsomes in the translation mixture, there was no change in the relative molecular mass of pM1M4, but about 40% of the product sedimented with microsomes after alkaline treatment (Fig. 3, lanes3 and 4). This implies that M4, like M2, could form with M1 the structural motif that functioned as a topogenic signal, albeit not as effectively.
Figure 5:
Digesting pM1M2 and pM3M4 with proteinase
K. RNA transcripts for pM1M2 and pM3M4 were translated in the in
vitro system with microsomes (nRM) as indicated. pM1M2 or
pM3M4 was digested with 300 or 150 µg/ml of proteinase K,
respectively, as outlined under ``Experimental Procedures.''
Triton X-100 (TX-100) was present at 1% during the
proteinase digestion (lanes3 and 6). The
molecular mass markers are bovine trypsin inhibitor with a M of 6,200 and insulin with a M
of 3,000. Proteins were resolved on a 15% polyacrylamide gel
containing 8 M urea and 0.1% SDS; radioactive bands were
visualized by fluorography.
When digested by
proteinase K in the absence or presence of microsomes, pM3M4 gave
similar results (Fig. 5, lanes4-6). The
protected fragment had a relative molecular mass of 7,000 (Fig. 5, lane5). The number of residues in
M3M4 is 63. The expected relative M is about
8,000. The observed relative M
of the protected
fragment is close to the expected M
. This
protected fragment could not be immunoprecipitated by polyclonal
antibodies against amino- or carboxyl-terminal residues (data not
shown). As observed in Fig. 3, about 25% of pM3M4 is
glycosylated. Therefore, we expected to find a protected fragment of
about M
20,000. No protected fragment, however,
with this M
was observed. The inability to detect
a protected fragment of this size in the autoradiograms could be caused
by the fact that the quantity of proteinase K protected glycosylated
product is beyond the limit of detection by autoradiography.
Figure 6:
Integrating proteins into trypsin treated
microsomes. RNA transcripts for pM1M2, pM3M4, and pM3 were translated
in the in vitro system. Mock-treated nRM (nRM) and trypsin treated nRM (nRM
) were added co-translationally to
the in vitro system, as indicated. Samples were incubated and
fractionated as outlined under ``Experimental Procedures.''
The products were immunoprecipitated using antibodies against
carboxyl-terminal residues of the H
-ATPase. Samples
were prepared for and resolved on 10% polyacrylamide gel with SDS and
were visualized by fluorography.
Second, we
studied integration into microsomes post-translationally to determine
if this process is a function of chemical energy. Truncated forms of
the H-ATPase are integrated post-translationally into
microsomes when they remain associated with ribosomes. The exception to
this rule is pM3. This protein is integrated effectively into
microsomes with or without its association with ribosomes (data not
shown). Accordingly, RNA transcripts without stop codons that encode
pM1M2 and pM3M4 were used in these studies. Small molecules were
removed by passage of translation mixtures through a size fractionation
column. To the desalted mixtures, microsomes were added in the absence (Fig. 7, lanes3 and 4) or presence (lanes7 and 8) of an energy-regenerating
system. The data demonstrate that integrating fusion proteins into
microsomes depended on ATP and GTP. The amount of pM1M2 that integrates
post-translationally is lower than that observed for pM3M4 and for pM3.
The exact reason for this is not apparent. This may suggest that the
topogenic signal in pM1M2 had become less accessible to the integration
machinery. This could be caused by aberrant folding of nascent
polypeptide chain of pM1M2 after or during the desalting step.
Figure 7:
Integrating proteins into microsomes as a
function of chemical energy. pM3 was synthesized in the in vitro system. For pM1M2 and pM3M4, stop codonless RNA transcripts were
used. Protein synthesis was stopped with 0.2 mM emetine.
Mixtures were desalted on column containing Bio-Gel P-4. Desalted
fractions were divided into 20 µl and adjusted to 30 µl. Assay
mixture contained 20 mM Hepes, pH 7.5, 150 mM KOAc, 2
mM Mg(OAc), 6% glycerol, 1 mM dithiothritol, 0.2 mM emetine, and 1 µg/ml each of
chymostatin, antipain, leupeptin, and pepstatin. Energy-regenerating
system (ATP/GTP) contained 400 µM ATP, 100
µM GTP, 16 mM creatine phosphate, and 50
µg/ml creatine phosphate kinase. For samples in lanes5 and 6, 4 units of glycerol kinase (GK) were added to the desalted fraction (40 µl) and 1
unit of GK was added to nRM (10 µl) with 2.5% glycerol.
The resultant mixtures were incubated at 20 °C for 20 min. GK-nRM were added to GK-treated desalted fraction (lanes5 and 6). All samples were incubated at 20
°C for 20 min with or without nRM (3 A
units/ml) and then fractionated into soluble (S)
fraction and membrane pellet (P) as outlined under
``Experimental Procedures.'' Proteins were resolved on 10%
polyacrylamide gel in the presence of SDS and visualized by
fluorography. Amount of product in each lane is expressed as a
percentage of the total material recovered in the pellet and soluble
fractions of that reaction.
Even in the absence of the energy-regenerating system, a small percentage of products sedimented with microsomes after alkaline treatment (Fig. 7, compare lanes3 and 4 with lanes1 and 2). This suggests that either the fusion proteins can integrate spontaneously into microsomes or the post-translational assay that contained microsomes and desalted translation mixture were not depleted of nucleotides. To distinguish between these possibilities, the desalted translation mixtures and microsomes were incubated with glycerol kinase to remove endogenous nucleotides. This reduced the amount of product found in the pellet after alkaline extraction (compare lanes5 and 6 with lanes3 and 4). This was interpreted to imply that even without the energy regenerating system, integration into microsomes is a function of nucleotides. The nucleotides may have been added to the assay by microsomes or they could represent nucleotides that were refractory to the desalting step. The combination of ATP and GTP was essential for optimal integration. This suggests that integrating fusion proteins into microsomes requires an ATPase(s) and a GTPase(s).
We investigated conditions required for integrating
transmembrane segments from the amino-terminal third of the Neurospora H-ATPase into homologous
microsomes. The results allowed us to confirm the number of segments in
the amino-terminal third of the H
-ATPase and to gain
insights into how transmembrane segments containing charged amino acids
and proline residues are integrated into membranes.
M3 initiated transfer of carboxyl-terminal residues of pM3 into the lumen of microsomes. Constructs containing either M1, M2, or M4 were not integrated into microsomes as judged by the fractionation of alkaline treated mixtures. When M4 was engineered downstream of M3 to give pM3M4, M4 blocked the glycosylation of the invertase segment. pM3M4 was integrated into microsomes as a polytopic integral membrane protein. When M2 was placed carboxyl-terminal to M1, the resultant protein pM1M2 was also integrated into microsomes as a polytopic integral membrane protein.
Using chimeric polytopic integral membrane proteins constructed by tandemly repeating the transmembrane segment of a bitopic integral membrane protein, researchers demonstrated that each transmembrane segment functioned independently and was inserted sequentially into microsomes (Wessels and Spiess, 1988; Rothman et al., 1988; Lipp et al., 1989). These chimeric polytopic membrane proteins, however, do not resemble P-type ATPases. P-type ATPases contain few, if any, residues on the exoplasmic side of the membrane. The studies presented herein suggest that transmembrane segments may function in pairs.
In vitro studies on the
biogenesis of the multidrug resistance protein (MDR1) suggest that
cooperative interaction between the first two transmembrane segments is
a prerequisite for integrating MDR1 into microsomes. When these
segments were assayed independently, each could direct protein
translocation, but not integration, into microsomes (Skach and
Lingappa, 1993). In contrast, the first two transmembrane segments of
the H-ATPase did not function independently. In
studies on the biogenesis of lac permease using a gene fusion
method, transmembrane segment number nine (M9) gave a low export
efficiency of the fused alkaline phosphatase. This was interpreted to
imply that Arg-302 in M9 caused the low export efficiency. The authors
suggested that M9 is stabilized in the membrane by forming an ion pair
with Glu-325, which is found in M10 (Calamia and Manoil, 1992). Forming
a salt bridge between M1 and M2 of the H
-ATPase cannot
explain the behavior of these segments in pM1M2, for M1 and M2 each
contain a negatively charged residue, but no positively charged residue
is present in either.
Most transmembrane segments of P-type ATPases
contain hydrophilic residues that are presumed to serve as specific
ligand for cations (Clarke et al., 1989). Proline residues are
found in some transmembrane segments of P-type ATPases also. When
hydrophilic and proline residues are introduced into transmembrane
segments, the resultant segments have a reduced hydrophobicity and
helical character (Chou and Fasman, 1978). The introduction also
of these residues by site-directed mutagenesis into the hydrophobic
core of signal sequences of presecretory proteins blocks or greatly
impairs the translocation of the resultant proteins across
translocation competent membrane (Emr et al., 1980; Emr and
Silhavy, 1983). And as demonstrated herein, the presence of hydrophilic
residues in transmembrane segments of H
-ATPase
impaired their ability to function individually as topogenic signals
when assayed out of context. Apparently, pairing of transmembrane
segments is essential for masking charged residues and for stabilizing
the integrated segments. This conjecture is supported by the following
observations. M3 contains no charged residue and functioned
independently as a topogenic signal. M1, M2, and M4 each contain a
negatively charged residue, and these functioned as a topogenic signal
only when combined.
Because M1 and M2 acted concertedly as a topogenic signal for integration, we will refer to this topogenic signal as an integration signal. Although we can not say with certainty what type of structure is formed by M1 and M2, a reasonable structure would be an antiparallel arrangement of the transmembrane segments held together by hydrophobic interaction to minimize interaction with water. This structure most likely would resemble a hairpin with the transmembrane segments serving as the arms. This arrangement would be favored by the short stretch of residues between the segments that acts as a physical constraint, analogous to disulfide bonds, and by the optimal arrangement of the helix dipoles of the segments.
Integration effectiveness of M1M2 was almost abolished by inserting
the invertase segment between the segments. This was interpreted to
imply that effective decoding of the integration signal depended on the
proximity of or of a crucial number of residues between the segments.
In contrast, transmembrane segments in pM3InM4 functioned individually
and as predicted by the sequential insertion model. Nevertheless, in
pM3M4 as well as in the H-ATPase, M3 and M4 are
separated by seven residues. In this close association will M3 and M4
act concertedly or independently? Because M4 formed an integration
signal with M1, this suggests that the putative integration machinery
recognizes some general structural feature of the paired segments. In
pM1M4, only eight residues lie between the transmembrane segments (see Fig. 1). It is likely that M3 and M4 in pM3M4 and in the
H
-ATPase form a structural motif similar to that
formed by M1 and M2 in pM1M2. If this is true, then integrating the
amino-terminal third of the H
-ATPase into microsomes
uses just one type of topogenic signal and not multiple topogenic
signals.
Additional evidence supporting this conjecture is obtained
from the following. Recognition of the signal sequence by the signal
recognition particle (SRP) varies with the length of the nascent
polypeptide chain. With some nascent presecretory proteins, effective
binding of SRP with signal sequences requires that approximately
70-140 residues be synthesized. Depending on the length of the
signal sequence, this suggests that 20-60 residues
carboxyl-terminal to the signal sequence be in the cytosol (Siegel and
Walter, 1988). Integration of the Ca motive ATPase, a
P-type ATPase, from rabbit sacrcoplasmic reticulum into heterologous
microsomes is SRP-dependent (Anderson et al., 1983). It is
likely that SRP acts as a general adapter in the targeting of most
nascent secretory proteins to the endoplasmic reticulum. If so,
targeting of the Neurospora H
-ATPase to the
endoplamic reticulum could be SRP-mediated also. If this is true, and
taking into consideration SRP recognization of signal sequences, this
would imply that in the nascent H
-ATPase when M1 and
M3 are in the cytosol the 20-60 residues carboxyl-terminal to
these segments would contain M2 or M4, respectively. Once in the
cytosol, these hydrophobic segments will interact to minimize their
interaction with water (Engelman and Steitz, 1981). This implies that
closely juxtaposed transmembrane segments in nascent polypeptide chain
must interact in vivo. Assuming that this is true, juxtaposed
segments would form hairpin-like structures before the decoding process
begins. Alternatively, the looped segments could be disrupted by an
effector exposing the individual segments, which are then decoded. This
interpretation of the data is not consistent with results obtained with
pM1 or pM2. Also, this process would require the use of energy to
disrupt a thermodynamically stable structure. Furthermore, experimental
observations suggest that the hydrophobic core of signal sequences,
which consists of eight or 12 amino acids, exists in a looped structure
resembling a hairpin (Nouwen et al., 1994; Rizo et
al., 1993). If this is true, it is possible the SRP recognizes
topogenic sequences in a hairpin-like configuration. Therefore, the
hairpin-like structure of paired transmembrane segments may actually
represents a common structural motif of topogenic signals.
From the
above considerations, we propose that membrane topology of the
amino-terminal third of the H-ATPase is achieved by
inserting into the membrane-paired transmembrane segments in a
receptor-mediated, energy-dependent event. Paired transmembrane
segments would be used as an integration signal only by polytopic
integral membrane proteins that do not have large exoplasm domains.
This model implies that for a region containing four transmembrane
segments, there would be two integration events. This conjecture is
consistent with the results of Homareda et al.(1989). This
group studied integration of truncated forms of the
-subunit of
the Na
/K
ATPase into heterologous
microsomes; the results imply that for the first four transmembrane
segments there are two topogenic signals causing two insertion events.
Clearly, the structural analysis of transmembrane segments in an
aqueous milieu will provide valuable knowledge about the nature of
integration signals for polytopic integral membrane proteins.
In conclusion, the results presented herein provide functional evidence for a novel type of topogenic signal for integrating a polytopic integral membrane protein into membrane. This signal consists of two transmembrane segments. The data also provide evidence that transmembrane segments, when assayed out of context, have various degrees of effectiveness as topogenic signals. This method can serve as a paradigm for identifying transmembrane segments functioning as integration signal.