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.

EXPERIMENTAL PROCEDURES

Materials

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.

Plasmid Constructions

PMCA

PMCA and PMCA

PMCA Construct with Point Mutations

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.

PMCA and PMCA

PMCA4CI-21

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.

Cell Culture and Transfections

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.

Gel and Immunoblot Analyses

Indirect Immunofluorescence Microscopy

Phospoenzyme Intermediate

Alkali Treatment and Proteolysis

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.

RESULTS

Design and Description of the Mutants

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.

COS Cell Expression of COOH-terminally Truncated PMCA Fragments

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.

The PMCA Is Retained in Endoplasmic Reticulum

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.

Testing of the Folding State of the Mutants

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).

Point Mutations at the COOH Terminus of the PMCA Promote Its Rescue from the ER

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 Targeting of NH- and COOH-terminally Truncated PMCA4CI Is Affected by Mutations in the Sequence Glu-Arg

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.

Expression of Mutants in the Hybrid Vaccinia Virus/T7 RNA Polymerase System

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.

The Glu-Arg Sequence Is Not a Universal ER Retention Signal

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.

The 82-kDa Fragment Originates from the NH-terminal Portion of PMCA

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 Nature of the Endogenous Protease

DISCUSSION

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 PMCA118 (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.

FOOTNOTES

*

The work was supported in part by Swiss National Science Foundation Grant 31.30858.91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Recipient of a FEBS long term fellowship.

¶

To whom correspondence should be addressed: Biochemie III, ETH-Zentrum, Universitätstr. 16, CH-8092 Zürich, Switzerland. Tel.: 41-1-632-30-11; Fax: 41-1-632-12-13.

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The abbreviations used are: PMCA, plasma membrane Ca-ATPase; PEST region, region rich in proline (P), glutamic acid (E), serine (S), and threonine (T); SR, sarcoplasmic reticulum; PCR, polymerase chain reaction; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; FCS, fetal calf serum; DMEM, Dulbecco's medium; FITC, fluorescein isothiocyanate; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

ACKNOWLEDGEMENTS

REFERENCES

1. Adamo, H. P., Verma, A. K., Sanders, M. A., Heim, R., Salisbury, J. L., Wieben, E. D., and Penniston, J. T. (1992a) Biochem. J. 285, 791-797
2. Adamo, H. P., Caride, A. J., and Penniston, J. T. (1992b) J. Biol. Chem. 267, 14244-14249 [Abstract/Free Full Text]
3. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250 [CrossRef][Medline] [Order article via Infotrieve]
4. Bonifacino, J. S., Suzuki, C. K., and Klausner, R. D. (1990) Science 247, 79-82 [Abstract/Free Full Text]
5. Bonifacino, J. S., Cosson, P., Shah, N., and Klausner, R. D. (1991) EMBO J. 10, 2783-2793 [Medline] [Order article via Infotrieve]
6. Bonifacino, J. S., and Lippincott-Schwartz, J. (1991) Curr. Opin. Cell Biol. 3, 592-600 [CrossRef][Medline] [Order article via Infotrieve]
7. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
8. Brodin, P., Falchetto, R., Vorherr, T., and Carafoli, E. (1992) Eur. J. Biochem. 204, 939-946 [Medline] [Order article via Infotrieve]
9. Carafoli, E. (1991) Physiol. Rev. 71, 129-153 [Free Full Text]
10. Carafoli, E. (1992) J. Biol. Chem. 267, 2115-2118 [Free Full Text]
11. Carafoli, E. (1994) FASEB J. 8, 993-1002 [Abstract]
12. Carafoli, E., and Guerini, D. (1993) Trends Cardivasc. Med. 3, 177-184
13. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752 [Abstract/Free Full Text]
14. Ciehanover, A., and Schwartz, A. L. (1989) Trends Biochem. Sci. 14, 483-488 [CrossRef][Medline] [Order article via Infotrieve]
15. Doyle, C., Sambrook, J., and Gething, M.-J. (1986) J. Cell Biol. 103, 1193-1204 [Abstract/Free Full Text]
16. Enyedi, A., Flura, M., Sarkadi, B., Gardos, G., and Carafoli, E. (1987) J. Biol. Chem. 262, 6425-6430 [Abstract/Free Full Text]
17. Enyedi, A., Verma, A. K., Filoteo, A. G., and Penniston, J. T. (1993) J. Biol. Chem. 268, 10621-10626 [Abstract/Free Full Text]
18. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8122-8126 [Abstract/Free Full Text]
19. Fujiki, Y., A. L. Hubbard, S. Fowler, and Lazarow, P. B. (1982) J. Cell Biol. 93, 97-102 [Abstract/Free Full Text]
20. Garoff, H., Kondor-Koch, C., Petterson, R., and Burke, B. (1983) J. Cell Biol. 97, 652-658 [Abstract/Free Full Text]
21. Heim, R., Iwata, T., Zvaritch, E., Adamo, H. P., Rutishauser, B., Strehler, E. E., Guerini, D., and Carafoli, E. (1992) J. Biol. Chem. 267, 24476-24484 [Abstract/Free Full Text]
22. Hofmann, F., James, P., Vorherr, T., and Carafoli, E. (1993) J. Biol. Chem. 268, 10252-10259 [Abstract/Free Full Text]
23. Humphrey, J. S., Peters P. J., Yuan, L. C., and Bonifacino, J. S. (1993) J. Cell Biol. 120, 1123-1135 [Abstract/Free Full Text]
24. Inesi, G., and Kirtley, M. R. (1992) J. Bioenerg. Biomembr. 24, 271-283 [Medline] [Order article via Infotrieve]
25. James, P., Maeda, R., Fischer, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J Biol. Chem. 263, 2905-2910 [Abstract/Free Full Text]
26. James, P., Pruschy, M., Vorherr, T., Penniston, J. T., and Carafoli, E. (1989a) Biochemistry 28, 4253-4258 [CrossRef][Medline] [Order article via Infotrieve]
27. James, P., Vorherr, T., Krebs, J., Morelli, A., Castello, G., McCormick, D. J., Penniston, J. T., De Flora, A., and Carafoli, E. (1989b) J. Biol. Chem. 264, 8289-8296 [Abstract/Free Full Text]
28. Klausner, R. D., and Sitia, R. (1990) Cell 62, 611-614 [CrossRef][Medline] [Order article via Infotrieve]
29. Laemmli, U. K. (1970) Nature 227, 680-685 [CrossRef][Medline] [Order article via Infotrieve]
30. Lederkremer, G. Z., and Lodish, H. F. (1991) J. Biol. Chem. 266, 1237-1244 [Abstract/Free Full Text]
31. Munro, S. (1991) EMBO J. 10, 3577-3588 [Medline] [Order article via Infotrieve]
32. Murre, C., Reiss, C. S., Bernabeu, C., Chen, L. B., Burakoff, S. J., and Seidman, J. G. (1984) Nature 307, 432-436 [CrossRef][Medline] [Order article via Infotrieve]
33. Nilsson, T., Lucocq, J. M., MacKay, D., and Warren, G. (1991) EMBO J. 10, 3567-3575 [Medline] [Order article via Infotrieve]
34. Pedersen. P. L., and Carafoli, E. (1987a) Trends Biochem. Sci. 12, 146-150 [CrossRef]
35. Pedersen. P. L., and Carafoli, E. (1987b) Trends Biochem. Sci. 12, 186-189 [CrossRef]
36. Puddington, L., Machamer, C. E., and Rose, J. K. (1986) J. Cell Biol. 102, 2147-2157 [Abstract/Free Full Text]
37. Rechsteiner, M. (1988) Adv. Enzyme Regul. 27, 135-151 [CrossRef][Medline] [Order article via Infotrieve]
38. Renaud, K. J., Inman, E. M., and Fambrough, D. M. (1991) J. Biol. Chem. 266, 20491-20497 [Abstract/Free Full Text]
39. Sanger, F., Nicklen, S., Coulson, and A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract/Free Full Text]
40. Sarkadi, B., Enyedi, A., Földes-Papp, Z., and Gardos, B. (1986) J. Biol. Chem. 261, 9552-9557 [Abstract/Free Full Text]
41. Stafford, F. J., and Bonifacino, J. S. (1991) J. Cell Biol. 115, 1225-1236 [Abstract/Free Full Text]
42. Strehler, E. E. (1991) J. Membr. Biol. 120, 1-15 [CrossRef][Medline] [Order article via Infotrieve]
43. Summers, M. D., and Smith, G. E. (1987) Tex. Agric. Exp. Stn. Bull. 1555
44. Swift, A. M., and Machamer, C. E. (1991) J. Cell Biol. 115, 19-30 [Abstract/Free Full Text]
45. Tsao, Yu. Sh., Ivessa, N. E., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell. Biol. 116, 57-67 [Abstract/Free Full Text]
46. Teasdale, R. D., D'Agostaro, G., and Gleeson, P. A. (1992) J. Biol. Chem. 267, 4084-4096 [Abstract/Free Full Text]
47. Wang, K. W., Villalobo, A., and Roufogalis, B. D. (1989) Biochem. J. 262, 693-706 [Medline] [Order article via Infotrieve]
48. Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085 [Abstract/Free Full Text]
49. Wikström, L., and Lodish, H. F. (1992) J. Biol. Chem. 267, 5-8 [Abstract/Free Full Text]
50. Wong, S. H., Low, S. H., and Hong, W. (1992) J . Cell Biol. 117, 245-258 [Abstract/Free Full Text]
51. Zimmermann, J., Voss, H., Schwager, C., Stegemann, J., Erfle, H., Stucky, K., Kristensen, T., and Ansorge, W. (1990) Nucleic Acids Res. 18, 1067 [Free Full Text]
52. Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N., and Carafoli, E. (1990) Biochemistry 29, 8070-8076 [CrossRef][Medline] [Order article via Infotrieve]
53. Zvaritch, E., Guerini, D., and Carafoli, E. (1992) 8th International Symposium on Calcium Binding Proteins , August 23-27, 1992, Switzerland, Davos, Abstr. B49

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