(Received for publication, September 13, 1994)
From the
The plasma membrane Ca-ATPase isoform 4b
(PMCA4CI) with truncations in the cytoplasmically exposed COOH-terminal
tail was expressed in COS and HeLa cells and in Sf9 cells using the
baculovirus system. The truncated protein terminating with the acidic
sequence Glu
-Arg
was retained
within the endoplasmic reticulum (ER), whereas mutants lacking this
sequence or having it at a distance from the COOH terminus were
delivered to the plasma membrane. Although the truncated protein
retained in the endoplasmic reticulum was still able to form a
Ca
-dependent phosphoenzyme, it underwent partial
degradation. Substitution of glutamic and aspartic residue(s) in the
acidic region promoted rescue of the protein to the plasma membrane.
The results suggest that the sequence Glu
-Arg
encodes a masked signal for ER retention and for the degradation
of the protein. However, its presence at the COOH terminus was not
sufficient to induce ER-retention and degradation; when the sequence
was attached to the full-length PMCA protein, normal plasma delivery
was observed. Evidently, ER retention and degradation required the
presence of the sequence in its specific location within the PMCA
structure. The degradation of the protein retained in the endoplasmic
reticulum occurred through the proteolytic attack at cytoplasmically
exposed residues (amino acid sequence 720-750) by a cytoplasmic
PEST sequence-related protease different from calpain.
The plasma membrane Ca-ATPase (PMCA) (
)is an essential component of eucaryotic cells. It pumps
Ca
to the extracellular space against a
10
-fold ionic concentration gradient at the expense of ATP
hydrolysis. The pump belongs to the family of P-type ATPases (for
reviews, see Pedersen and Carafoli (1987a, 1987b) and Inesi and
Kirtley(1992)) and is composed of a single polypeptide chain of
molecular mass about 134 kDa. It contains 10 putative transmembrane
segments and two main hydrophilic loops, protruding to the cytoplasm
between transmembrane segments 2 and 3 and segments 4 and 5 (a
molecular model of the membrane architecture of the pump is shown in Fig. 1). The intramembrane segments necessarily contain the
Ca
ion-transporting channel, while the
cytoplasmically exposed peptide loops are the locus of ATP hydrolysis,
of phosphoaspartate formation, and of Ca
ion binding.
One striking feature of the PMCA structure that distinguishes it from
other P-type ATPases, e.g. the sarcoplasmic reticulum
(SR)/endoplasmic reticulum (ER) Ca
pump, is the
extended COOH-terminal tail, which protrudes into the cytoplasm with
about 160 residues.The COOH-terminal portion is the target site for
most regulators of the pump: Ca
ions, calmodulin,
protein kinases A and C, and partially acidic phospholipids (James et al., 1988, 1989a; Wang et al., 1991; Brodin et
al., 1992) (for a review, see Carafoli(1991)).
Figure 1:
A model for the transmembrane
organization of the human plasma membrane Ca-ATPase
isoform 4b with indication of the truncation sites. The amino acid
sequence of the COOH-terminal portion has been expanded in the circle
and indicated in full. The sequences of the acidic regions
Glu
-Glu
and
Glu
-Asp
are enclosed in boxes. The calmodulin-binding domain is shown in bolditalicletters. The sites of truncations are
indicated by arrows. An arrow in the first
hydrophilic loop shows the site of the NH
-terminal
truncation (mutant
PMCA
). D465 is
the site of phosphoaspartate formation, i.e. the active site
of the ATPase. The epitopes for the JA9 and 5F10 antibodies (residues
17-75 and 724-783, respectively) are marked with thicklines. The numberedboxes indicate
putative transmembrane segments of the
pump.
cDNA cloning work has revealed that the PMCA is a product of a multigene family with additional isoform diversity linked to alternative mRNA splicing (for recent reviews, see Carafoli and Guerini (1993) and Carafoli(1994)). The full-length cDNA coding for a quantitatively predominant mammalian isoform PMCA4CI has been constructed and expressed in COS cells (Adamo et al., 1992a; Heim et al., 1992; Enyedi et al., 1993), baculovirus/Sf9 cell (Heim et al., 1992), and vaccinia virus/HeLa cell (Zvaritch et al., 1992).
Eucaryotic cells contain five P-type pumps: the
Na/K
-ATPase, the
H
/K
-ATPase (in the gastric mucosa),
the H
-ATPase (yeast cells), and the two
Ca
-ATPases: those of the plasma membrane and of the
sarco/endoplasmic reticulum (for reviews, see Pedersen and Carafoli
(1987a, 1987b)). Of these ATPases, all but one are delivered to the
plasma membrane; thus, it was interesting to perform a study of the
structural determinants responsible for this strict sorting pattern.
Previous work from this laboratory has shown that a
NH
-terminally truncated PMCA product missing the two first
intramembrane segments and the hydrophilic loop connecting segments 2
and 3, although functionally inactive, was still delivered to the
plasma membrane (Heim et al., 1992). It was somewhat
surprising that a molecule lacking about one-fourth of its mass was not
recognized by the intracellular quality control machinery as abnormal
and was not retained in the ER for degradation.
Selective retention and subsequent degradation within the ER have been demonstrated for many misfolded and mutated proteins (for reviews, see Klausner and Sitia(1990), Bonifacino and Lippincott-Schwartz(1991), Renaud et al.(1991), Tsao et al.(1992), and Wikström et al.(1992)). The signals for the ER retention/degradation are believed to be masked in properly folded molecules and to become exposed in mutated proteins (for reviews, see Klausner and Sitia(1990) and Bonifacino and Lippincott-Schwartz (1991)). These motifs are intensively searched; however, only a few have been detected so far. One example, represented by a hydrophobic intramembrane sequence containing charged residues, has emerged from studies on the intracellular fate of T cell antigen receptor subunits (Bonifacino et al., 1990, 1991). Another, the juxtamembranous hydrophilic sequence Glu-Gly-His-Arg-Gly, was detected in asialoglycoprotein receptor subunits (Lederkremer and Lodish, 1991).
In this study, the structural features in the PMCA
molecule recognized by the ER editing machinery were explored by
following the intracellular fate of truncated ATPase variants expressed
in COS, HeLa, and Sf9 cells. The results show that the COOH-terminal
sequence Glu-Arg
represents a
masked structural signal for the ER retention of mutated PMCA molecules
and that the retention is accompanied by proteolytic degradation of the
pump.
Trypsin from
bovine pancreas and soybean trypsin inhibitor were from Boehringer
Mannheim GmbH, Mannheim, Germany. The vectors were from Stratagene
GmbH, Zürich, Switzerland (pSG5) and from USB
Biochemical, Lucerna-Chem, Switzerland (pTZ18-19). The restriction
enzymes were from Pharmacia, Uppsala, Sweden. Taq polymerase
and Taq buffer were from HT Biotechnology Ltd, Cambridge,
United Kingdom. The oligonucleotide primers were provided by MWG
Biotech, Ebersberg, Germany. Nitrocellulose (pore size 0.45 µm) was
from Schleicher & Schuell, Dassel, Germany.
[-
P]dATP (1500 Ci/mmol) was from Amersham
International, United Kingdom. All other chemicals were of the highest
quality commercially available.
The 3` primer
for PCR amplification was 5`-TCA ATC GGA TCC AGT CCC TCG GCA
TCC TT-3`, which codes for the sequence from
Lys-Asp
followed by the BamHI restriction site (italic). The 5` primer, 5`-GTG CAG ATG
TTG TGG-3`, was the same as that employed in the preparation of the
truncated mutants.
Plasmids for the generation of recombinant baculoviruses cDNA
corresponding to PMCA and PMCA
were obtained from the pSG5 plasmids prepared as described above
and inserted in the pVL1393 vector digested with BamHI and KpnI.
The PCR-derived fragments were verified by dideoxy
sequencing of double-stranded DNA (Sanger et al., 1977) using
the procedure suggested by Zimmermann et al.(1990), modified
to accommodate the use of [-
P]dATP. The
plasmid constructs used in the expression experiments were purified on
Qiagen columns (Qiagen, Chatworth, CA) according to the
supplier's protocol.
COS-7 cells plated in 100-mm dishes (60% confluent) were
transfected with 20 µg of supercoiled DNA by the calcium
precipitation method (Chen and Okayama, 1987) at 6% CO.
For the expression using the hybrid vaccinia virus/T7 polymerase system (Fuerst et al., 1986), HeLa cells (80% confluent) were infected with the T7 recombinant vaccinia virus at a multiplicity of infection of 10. After adsorption for 30 min at 37 °C, the inoculum was replaced with DMEM/FCS and the cells were immediately transfected with plasmid DNA by the calcium precipitation method. The cells were harvested 16 h after infection.
Membrane fractions of the transfected cells were prepared by several cycles of freezing and thawing in 10 mM Tris-HCl, pH 7.4, as described by Heim et al.(1992). Protein concentration was determined by the Bio-Rad adaptation of Bradford's dye-binding assay (Bradford, 1976).
Sf9 cells were maintained in TNM-FH medium (Summers and Smith, 1987) supplemented with 10% FCS and 50 µg/ml gentamicin. The cells were transfected using BakPak baculovirus DNA (Clontech, Palo Alto, CA), and recombinant viruses were purified by two rounds of plaque purification (Summers and Smith, 1987).
For expression the Sf9 cells were
infected at a multiplicity of infection of 5 and collected 48 h
post-infection. After washing in 25 mM Tris-HCl, pH 7.5, 130
mM NaCl, the cells were homogenized on ice in 10 mM Tris-HCl, pH 7.5, and centrifuged at 6500 g for
10 min. The supernatant was sedimented twice at 100,000
g for 45 min at 4 °C. The membrane pellet was resuspended at
2-3 mg of protein/ml in 5 mM Tris-HCl, pH 7.8, 10%
sucrose and frozen at -70 °C.
Procalpain purified
from human erythrocytes was converted to calpain by incubation for 2
min at 25 °C in the presence of 0.2 mM Ca. Calpain activity was assayed as described by
James et al. (1989b). The membrane-bound polypeptides were
digested by calpain at 25 °C using 1 unit of calpain/10 µg of
total membrane protein in 50 mM Tris-HCl, pH 7.0, and 0.2
mM CaCl
. Aliquots were withdrawn, immediately
boiled for 2 min in the presence of electrophoresis sample buffer, and
analyzed by SDS-PAGE followed by immunoblotting.
A scheme of the
membrane architecture of the full-length ATPase with the truncation
sites used is shown in Fig. 1. The COOH-terminal protruding tail
of the pump is composed of at least three structurally different
regions: 1) the acidic stretch
Glu-Glu
, 2) the stretch of
interspersed basic and hydrophobic residues
Leu
-Ser
(the calmodulin-binding
site; James et al.(1988)), and 3) a second acidic stretch
Pro
-Asp
. The mutants were designed
to terminate just before the first acidic stretch at Lys
(PMCA
mutant), immediately after it at
Arg
(PMCA
), and after the second
acidic stretch at Lys
(PMCA
). The
cleavage sites are supposedly located at junctions between structural
domains (James et al., 1989b; Zvaritch et al., 1990),
and therefore the truncations were expected to have only minimal
perturbing influence on the folding of the expressed polypeptides. The
mutant called PMCA4CI-21 carried the
Glu
-Arg
sequence just after
Val
, the last amino acids in the wild type pump.
Figure 2:
A, expression of the full-length
PMCA4CI and of its COOH-terminally truncated mutants in the COS-7 cell
system. COS-7 cells were transfected with the cDNA plasmid constructs
coding for the indicated polypeptides, or with the control plasmid
without insert. The cells were harvested 40 h after transfection, and
aliquots (10 µg of total protein) of the membrane fractions (see
``Experimental Procedures'') were subjected to SDS-PAGE (7%
polyacrylamide) followed by Western blotting using a mixture of the
5F10 and JA9 antibodies. The expressed polypeptides are marked with asterisks. The antibody-positive protein band of 82 kDa is
indicated. The migration of the molecular mass standards is shown at right. B, membrane insertion of the expressed
polypeptides. Membrane samples (50 µg of total protein) of COS
cells were treated with 0.1 M sodium carbonate, pH 11.5, on
ice for 30 min. Membrane-integral proteins (P) were separated
from those extractable (S) by centrifugation. Both fractions
were subjected to 7% SDS-PAGE and analyzed by immunoblotting with the
JA9 and 5F10 antibodies. C, expression of the truncated
mutants in Sf9 insect cells. PMCA4CI, PMCA, and
PMCA
were expressed with the help of recombinant
baculoviruses. 48 h after the infections, the cells were collected and
membranes were prepared as described under ``Experimental
Procedures.'' 1.8 µg of membrane proteins of cells expressing
PMCA4CI (lanes1 and 4) and
PMCA
(lanes2 and 5) and
5.4 µg of membrane proteins of cells expressing PMCA
(lanes3 and 6) were separated by
SDS-PAGE, blotted to nitrocellulose, and incubated with the monoclonal
antibodies JA9 (lanes1-3) and 5F10 (lanes
4-6). D, formation of the phosphoenzyme
intermediate from ATP. 30 µg of membrane proteins from cells
expressing PMCA
(lanes 1-3),
PMCA
(lanes 4-6), or infected with a
control virus (a virus carrying a cDNA for the SERCA2b in the opposite
orientation) (lane7) and PMCA4CI (lane8) were incubated in the presence of 0.3 µM [
-
P]ATP (150-200 Ci/mmol) as
described under ``Experimental Procedures.'' The following
additions were made: lanes 1 and 4, 20 µM Ca
; lanes 2, 3, and 5-8, 20 µM Ca
and 20
µM La
. After stopping the reaction, the
samples in lanes 3 and 6 were resuspended in 200
mM hydroxylamine, pH 7.0, for 20 min at room temperature.
Finally the trichloroacetic acid-precipitated proteins were separated
by acidic gels, stained with Coomassie Blue, dried, and exposed for 3
days at -70 °C.
The
expression level of the truncated polypeptides, as judged from the
intensity of the staining with the mAbs, was similar to that of the
full-length ATPase (Fig. 2A). Their apparent molecular
masses: 130 kDa (PMCA), 124 kDa
(PMCA
), and 116 kDa (PMCA
) were
in good agreement with those predicted from the cDNA sequences. A
mAb-positive weaker band of about 140 kDa seen in all lanes
corresponded to the endogenous plasma membrane Ca-ATPase of COS cells.
Interestingly, in the samples containing the PMCA
, a
polypeptide of about 82 kDa reproducibly reacted with the mAbs. This
protein, which was never detected in membrane preparations of other
expressed polypeptides or in control samples of cells transfected with
the vector alone, was tentatively assumed to be a degradation product
of the PMCA
. The ratio between the 82-kDa fragment
and the PMCA
, as judged from the staining
intensities, remained constant at different expression times (24, 36,
40, and 48 h post-transfection), indicating that the formation of the
82-kDa fragment was not a result of the overproduction of the
PMCA
.
After 40 h of expression, the cells
transfected with the plasmid constructs were fixed, permeabilized with
Triton X-100, and processed for indirect immunofluorescence microscopy.
As shown in Fig. 3the cells expressing the full-length ATPase (A), PMCA (B), and
PMCA
(D) displayed diffuse fluorescence
over the whole cytoplasm with clearly visible cell edges. This pattern
is characteristic of cell surface protein expression (Munro, 1991;
Teasdale et al., 1992). By contrast, cells expressing
PMCA
(C) showed exclusively the
perinuclear, reticulate fluorescence staining characteristic of
proteins retained in the ER (Stafford and Bonifacino, 1991).
Figure 3:
Subcellular location of PMCA4CI and of its
truncated mutants expressed in COS-7 cells. Cells grown on coverslips
were transfected with plasmid constructs encoding the full-length
PMCA4CI (A) or the truncated mutants: PMCA (B), PMCA
(C), and
PMCA
(D). Forty hours after transfection,
cells were fixed with 4% paraformaldehyde, permeabilized with 0.1%
Triton X-100, and incubated with monoclonal antibodies followed by
FITC-conjugated goat anti-mouse IgG. Immunocomplexes were visualized by
fluorescence microscopy. Bar, 10
µm.
A
frequent reason for the mislocalization of an expressed protein is its
overproduction (Munro, 1991; Nilsson et al., 1991; Humphrey et al., 1993). The possibility was thus considered that the
abnormal location of PMCA was related to the level
of its expression. Cells expressing PMCA
and PMCA4CI
were stained for immunofluorescence at early times after transfection
(24 h). Since at this time point less protein was expressed, the
fluorescence signal was weaker, but the staining pattern for the
truncated fragment and for the full-length ATPase was similar to that
presented in Fig. 3.
Alkali treatment of cell membranes strips off loosely bound proteins
and aggregates (Fujiki et al., 1982) and is routinely used to
distinguish between peripheral and membrane proteins (Bonifacino et
al., 1991; Wong et al., 1992). Fig. 2B shows that all the expressed proteins were tightly associated with
the pelleted membrane fractions, indicating their insertion into the
lipid bilayer. Interestingly, the proteolytic fragment of 82 kDa
present in the samples of the PMCA was also found
associated with the membrane fraction (Fig. 2B). The
folding state of the expressed mutants was further tested by controlled
trypsin digestion of membrane-bound polypeptides. The 81-, 76-, and
35-kDa limited fragments detected with mAbs in immunoblots (results not
shown) corresponded to those normally obtained upon digestion of the
membrane-bound human erythrocyte Ca
-ATPase (Enyedi et al., 1987; Sarkadi et al., 1986), suggesting that
the folding state of the expressed polypeptides, regardless of the
length of the COOH-terminal truncation, was similar to that of the
native ATPase.
Additional and, possibly, conclusive proof for the
correct folding of the ER-retained mutant came from activity
measurements on the truncated product PMCA expressed
in Sf9 cells (Fig. 2C) with the baculovirus system.
PMCA
, PMCA
, and the full-length
pump were able to form a Ca
-dependent phosphorylated
intermediate, which was canonically increased by La
(Fig. 2D, lanes 2, 5, and 8). The baculovirus system was chosen for the experiments
since the relatively large amounts of endogenous SERCA and PMCA
Ca
pump in COS cells greatly hampered the
demonstration of the phosphorylated intermediate formed by the newly
expressed pumps. Enyedi et al.(1993) had shown previously that
a similarly truncated PMCA expressed in the COS cells is functionally
active. It is worth mentioning that the expression pattern of
PMCA
in Sf9 cells was indistinguishable from that in
COS cells; the 124- and 82-kDa products were observed, the latter being
only stained with antibody JA9 (Fig. 2C, compare lane 3 with lane 6) and about 4 times more abundant
than the former. No phosphoenzyme intermediate was ever observed on the
82-kDa product (Fig. 2D).
Figure 4:
A,
schematic description of the amino acid substitutions in the
COOH-terminal portion of PMCA. The upperline shows the original peptide sequence
Glu
-Arg
. The lowerlines show the positioning and the nature of the
substitutions. The code names of the mutants are written to the left of the corresponding sequence. The numbers in brackets show the location of the NH
and COOH
termini of the peptide sequence in the structure of PMCA4CI. The numbers above the mutated amino acids indicate the residue
location in PMCA4CI. The asterisks indicate the residues
conserved in all PMCA isoforms. B, expression of
PMCA
and of its mutated variants. Membrane fractions
(10 µg of total protein) of COS-7 cells transfected with the
indicated plasmid constructs were subjected to SDS-PAGE (7%
polyacrylamide) and analyzed by immunoblotting with the JA9 and 5F10
antibodies. PMCA
is indicated as
118,
the mutated variants are described by the amino acid substitutions. The
control lane shows the immunostaining of membrane fractions of cells
transfected with the plasmid without inset. The migration of molecular
mass standards is indicated at right.
The variant
PMCA encodes a protein, in which 4 acidic
residues (Glu
, Asp
, Glu
,
and Glu
) of the peptide stretch were replaced by 1 Leu
and 3 Ala residues, respectively. The mutations would significantly
change the net negative charge of the region, rendering it much more
hydrophobic. In other variants the mutations were less dramatic; they
influenced the net charge of the peptide but were more conservative in
character. In variant PMCA
, 3 Glu
residues(1078, 1083, 1085) were substituted by Gln; in variants
PMCA
and PMCA
, the
substitution of Glu
by a Gln was accompanied by
mutations of Glu
to a Gln, or of Asp
to
an Asn, respectively. Variant PMCA
contained a
single Glu
Gln mutation.
The cells expressing
the mutants invariably showed a pattern of cell surface staining (Fig. 5), indicating that all of the mutants escaped ER
retention and were delivered to the plasma membrane. To facilitate the
comparison, the immunofluorescence staining pattern of cells expressing
the wild type PMCA in the same experimental group is
also presented in Fig. 5.
Figure 5:
Immunofluorescence microscopy of COS-7
cells transfected with PMCA (F) and with
its mutated variants: A,
118(A,L,L,L); B,
118(Q,Q,Q); C,
118(Q,Q); D,
118(Q, N); E,
118(Q). The mutants are described by the amino acid
substitutions. The transfected cells were fixed with paraformaldehyde
after 40 h of expression, permeabilized with Triton X-100, and
incubated with the mAbs followed by FITC-conjugated goat anti-mouse
IgG. Bar, 20 µm.
Western blotting experiments on
membrane samples of cells expressing the mutants (Fig. 4B) demonstrated that the levels of expression
were similar to those of the wild type PMCA. As seen
in Fig. 4B, some of the mutated proteins had increased
electrophoretic mobilities (see, for instance,
PMCA
), which was assumed to be a
consequence of the amino acid substitutions. The proteolytic band of 82
kDa seen in the samples of the expressed wild type PMCA
was not observed in the samples of the variants. The results of
the mutation work were particularly impressive in the case of
PMCA
, where a single amino acid substitution
(Glu
Gln) was apparently sufficient for the
release of the polypeptide from the ER and for the prevention of its
proteolytic degradation.
The immunofluorescence experiments revealed two different patterns
of subcellular localization of the mutants (Fig. 6). Cells
expressing mutant PMCA
showed
reticular staining as in the case of PMCA
while
cells expressing mutant
PMCA
displayed plasma membrane staining.
Figure 6:
Intracellular location of PMCA
(A) and
PMCA
(B) proteins
expressed in COS-7 cells. The cells were fixed 40 h after transfection
and processed for indirect immunofluorescence microscopy as described
under ``Experimental Procedures.'' Bar, 20
µm.
Figure 7: Expression of PMCA4CI and of its truncated mutants in HeLa cells using the hybrid vaccinia virus system. HeLa cells were infected with the recombinant vaccinia virus and then transfected with cDNAs encoding the full-length PMCA4CI or the indicated mutants. At 16 h after infection, the cells were harvested and the membrane fractions were prepared. 2 µg of total membrane protein were subjected to SDS-PAGE (7% polyacrylamide) followed by Western blotting using the mAbs. The expressed proteins are marked with asterisks. The position of the 82-kDa fragment is indicated. The migration of the molecular mass standards is shown at right.
Figure 8:
A,
expression of the PMCA4CI-A21 mutant. Western blotting of 20-30
µg of membrane proteins from COS-7 cells transfected with
PMCA4CI-A21 (lane 1) and PMCA4CI (lane2)
and the pSG5-vector alone (lane3) were incubated
with a polyclonal antibody specific for the NH terminus of
PMCA4CI (T. Stauffer, D. Guerini, and E. Carafoli, manuscript in
preparation). Membranes were prepared by freeze and thawing as
described by Heim et al.,(1992). B, formation of the
phosphoenzyme intermediate from ATP. 20-30 µg of membrane
proteins from COS cells transfected with PMCA4CI-A21, PMCA4CI, and the
pSG5 vector alone were incubated in the presence of 0.3 µM ATP (150 Ci/mmol) for 30 s on ice in the presence of 200
µM CaCl
(lanes2, 4, and 6) and 200 µM CaCl
and 200 µM LaCl
(lanes1, 3, and 5). After separation on
acidic gels, staining with Coomassie Blue, and drying, the gels were
exposed for 4 days at -70 °C. The position of PMCA4CI-A21 is
indicated by the asterisk. C and D,
immunocytochemistry of COS cells transfected with PMCA
(C) and PMCA4CI-A21 (D). The procedure is
described under ``Experimental Procedures'' and in the legend
to Fig. 3. However, as in the case of panelA,
a polyclonal antibody against the NH
terminus of PMCA4CI
was used.
Figure 9: A, separate immunostaining of membrane fractions with JA9 and 5F10 antibodies. Membrane samples (10 µg of total protein) of COS cells transfected with the indicated plasmid constructs (lanes 1-4), or with the control plasmid without insert (lane 5) were analyzed on 7% SDS-PAGE, electroblotted, and stained with monoclonal antibody JA9 (lanes1, 3, and 5) and 5F10 (lanes 2 and 4). The positions of molecular mass standards are indicated at right. B, controlled calpain treatment of the expressed proteins. Membrane fractions of COS-7 cells transfected with the plasmid constructs indicated were digested with calpain as described under ``Experimental Procedures.'' At the time points indicated, aliquots (15 µg of total membrane protein) were withdrawn from the incubation mixture and analyzed by SDS-PAGE followed by immunoblotting with the mAbs. The zero time point aliquots were withdrawn from the proteolytic mixture immediately after the addition of calpain. The positions of molecular mass standards are shown at right.
The family of P-type ATPases now comprises a dozen enzymes, five of which have been detected in eucaryotic cells (for reviews, see Pedersen and Carafoli (1987a, 1987b)). As mentioned in the Introduction, of these five only one is retained in the SR/ER, the other four being strictly sorted to the plasma membrane. This difference in membrane targeting is somewhat surprising, since all these enzymes share catalytic and, especially, membrane architecture properties, i.e. they all have an even number of transmembrane helices and protrude with two large domains in the cytoplasmic space.
In this work the role of the protruding COOH-terminal portion of the PMCA in the protein sorting has been investigated, essentially because this portion represents the most striking difference with the otherwise structurally and functionally similar SR/ER pump. Interestingly, this region was shown to be highly susceptible to proteolytic degradation and has been proposed to be the site of activation of the pump by calpain in vivo (Carafoli, 1992).
The view that the ER is not only the compartment for protein synthesis and folding, but also the organelle that performs protein editing, has recently gained favor (for reviews, see Klausner and Sitia(1990), Bonifacino and Lippincott-Schwartz(1991), Renaud et al. (1991), Tsao et al.(1992), and Wikström et al.(1992)). The ER is proposed to possess a quality control system, recognizing aberrant structures among newly synthesized proteins and often rerouting them to degradation, presumably within the same (ER) compartment. The retention/degradation signals recognized by the ER editing machinery are apparently encoded in the protein structure but are masked in correctly folded molecules or protein complexes. The exposure of these signals due to the improper protein assembly leads in many cases to the protein retention within the ER and to its subsequent degradation. The identification of the ER retention/degradation determinants would obviously be essential for the understanding of this physiological process. A hydrophobic intramembrane sequence containing potentially charged amino acid residues was recently shown to determine the intracellular fate of T cell antigen receptor subunits (Bonifacino et al., 1990, 1991). The same type of signal was later suggested to be involved in the elimination of some other proteins, i.e. HMG reductase and apolipoprotein B (Bonifacino and Lippincott-Schwartz, 1991) and is presently believed to be of common use in membrane proteins. As mentioned above, another structural signal has emerged from the studies on the assembly and degradation of asialoglycoprotein receptor subunits (Lederkremer and Lodish, 1991); it is the exoplasmic pentapeptide sequence Glu-Gly-His-Arg-Gly, located next to the single membrane-spanning domain of the 2Ha subunit of the receptor. The relevance of this signal to other protein systems is still unknown.
The results presented in this paper suggest that the sequence
spanning residues Glu-Arg
of
PMCA4CI is a masked structural signal (or portion of it) for the
intracellular retention of mutated PMCA molecules. However, when this
sequence was added to the COOH terminus of the full-length pump,
neither ER retention nor degradation was observed. This finding implies
either that the ER retention signal sequence was still incomplete (i.e. it could require a larger portion at the NH
terminus) or that the signal was positional (i.e. it
would only be operational when located in its (tridimensional) site).
The sequence was highly sensitive to the mutation of its acidic amino
acids, a procedure preventing both ER retention and degradation.
Although a systematic analysis of all the amino acids involved has not
been performed, the mutations so far have performed indicated that the
signal was highly specific. The subcellular distribution of the
truncated mutants and their susceptibility to proteolysis were
independent of the cell type used for protein expression: monkey kidney
(COS), human epithelial (HeLa), and insect (Sf9) cells exhibited
essentially the same sorting and proteolysis pattern. The results on
Sf9 have cells indicated that the protease involved in the degradation
process was conserved from insect to mammalian cells.
The sequence
Glu-Arg
is located downstream to
the last putative transmembrane segment of the PMCA4CI and is
distinctly acidic. The structural features underlying the selective
recognition of the Glu
-Arg
sequence
are not yet clear. The properties of the sequence are very similar to
those of the sequence Pro
-Asp
: both
are highly acidic, have a rather high PEST score (Wang et al.,
1989), and are presumably implicated in Ca
binding
(Hofmann et al., 1993). However, the latter sequence does not
contains ER retention signals; the polypeptide PMCA
,
which has the sequence Pro
-Asp
at
the COOH terminus, was not trapped within the ER. It is also probably
important to mention that the Glu
-Arg
domain contains the sequence
Glu-Ile-Asp-His-Ala-Glu-Met/Arg-Glu-Leu-Arg, which is conserved in all
ATPase isoforms (Strehler, 1991).
A sequence with homology to the
Glu-Arg
stretch is absent in the
other ATPases of the P-type family, even in the
Ca
-ATPase of the SR/ER. The retention signal detected
in this work is thus probably specific for the plasma membrane
Ca
-ATPase, although it is probably not the only one
encoded in its structure.
The cleavage site of the ER-retained
PMCA protein was tentatively located at residues
720-750. In the current model of the transmembrane organization
of the ATPase this site is located in the COOH-terminal portion of the
hydrophilic loop protruding into the cytoplasm between transmembrane
segments 4 and 5. Little is so far known on the proteases mediating the
degradation of proteins within the ER and on their intracellular
localization (Stafford and Bonifacino, 1991;
Wikström and Lodish, 1992; Tsao et al.,
1992). The results presented in this study indicate the involvement of
cytosolic/membrane-bound protease. The sensitivity of the proteolysis
to changes in the PEST score of the
Glu
-Arg
sequence suggests an
involvement of a PEST-related protease (for reviews, see Rechsteiner
(1988) and Wang et al.(1989)), however, most probably not
calpain.
All amino acid changes in the sequence
Glu-Arg
prevented the proteolytic
degradation of PMCA
. The sensitivity of the protease
to amino acid substitutions in a region remote from the presumed site
of proteolytic attack (residues 720-750) could be rationalized in
two ways: 1) a protease molecule could interact with the region
Glu
-Arg
and cleave the protein at a
site distant in sequence but spatially close; 2) the sequence
Glu
-Arg
could be recognized by some
other protein, which then would present it to a protease; this would be
a mechanism resembling that of the ubiquitin degradative machinery (for
a recent review, see Ciehanover and Schwartz(1989)). The sequence
Glu
-Arg
is evidently recognized by
the ER machinery only when positioned at the very COOH terminus of the
polypeptide chain. In vivo such a COOH-terminal location of
the signal could, for instance, result from the premature termination
of protein translation.
The reason why of all possible versions of
prematurely terminated PMCA polypeptides only those with the
carboxyl-terminal sequence Glu-Arg
should be selectively retained in the reticulum and degraded
remains obscure and deserves further study. It is worth mentioning that
no solid information is currently available on the principles
underlying the selectivity of the ER retention/degradation machinery;
some aberrant proteins are not recognized by the quality control
machinery and are still sorted to their normal sites of destination
(Garoff et al., 1983; Murre et al., 1984; Puddington et al., 1986; Doyle et al., 1986; Renaud et
al., 1991; see also this study). Possibly, the failure in protein
folding and assembly is not the only feature that induces retention and
degradation within the ER. As an example, one could quote the
dramatically different intracellular fate of the alternatively spliced
asialoglycoprotein receptor subunits: the 2Hb subunit is expressed
almost quantitatively on the cell surface, while subunit 2Ha is
predominantly retained within the ER and degraded (Lederkremer and
Lodish, 1991). The selective retention of the PMCA
product is unlikely to have been caused by dramatic folding
perturbations. In fact PMCA
was phosphorylated from
ATP in a manner similar to that for the PMCA.
On a more speculative
line, it could be suggested that the selective ER retention and
degradation of the PMCA polypeptide with the COOH-terminal sequence
Glu-Arg
could have evolved to
prevent the release of the prematurely terminated protein with
undesirable functional properties. PMCA
corresponds
precisely to the calpain-produced 124-kDa fragment of the pump, which
has been shown to be fully active at physiological concentrations of
Ca
in the absence of calmodulin (James et
al., 1989b). In addition, a COOH-terminally truncated polypeptide
similar to PMCA
118 (this peptide is just 2 amino acids shorter)
has been expressed recently in COS cells and shown to be fully active
(Enyedi et al., 1993). Since this region of the pump is easily
proteolyzed by other proteases in vitro (see above), the
possibility of its cleavage in vivo seems realistic. If the
fully active truncated protein were delivered to the plasma membrane,
it would pump calcium out of the cell in an uncontrolled manner,
resulting in cell damage.
More work is required to assess the
possibility that this signal could work as retention signal in other
proteins. First, it will be important to determine if the 21 amino
acids (Glu-Arg
) described here are
sufficient for retention in the ER or if other ones (located
NH
-terminally to Glu
) are needed. Second, it
will be also important to determine if the location of this sequence is
critical to its capacity to cause retention the endoplasmic reticulum.