beta -Subunit Assembly Is Essential for the Correct Packing and the Stable Membrane Insertion of the H,K-ATPase alpha -Subunit*

Ahmed T. BeggahDagger , Pascal BéguinDagger , Krister Bamberg§, George Sachs, and Käthi GeeringDagger parallel

From the Dagger  Institut de Pharmacologie et de Toxicologie de l'Université, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland, § Astra Hässle AB, S-43183 Möldahl, Sweden, and  West Los Angeles, Wadsworth Veterans Administration-Medical Center, Los Angeles, California 90073

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

The alpha -subunits of H,K-ATPase (HKAalpha ) and Na,K-ATPase require a beta -subunit for maturation. We investigated the role of the beta -subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha  constructs, M1-3 alpha -proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha -proteins are not resistant to cellular degradation. However, co-expression with beta -subunits increases the membrane insertion of M9 in a M1-9 alpha -protein and completely protects M1-10 alpha -proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha -subunit depend on intramolecular and intermolecular beta -subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha -subunit.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

In eukaryotic cells, membrane proteins are co-translationally inserted into the endoplasmic reticulum membrane, and their final membrane topologies are determined during membrane integration. They are targeted to the endoplasmic reticulum membrane by the signal recognition particle and interaction with the signal recognition particle receptor. A polar, transmembrane channel (the translocon) consisting of several subunits permits the passage of hydrophilic protein domains as well as the integration of hydrophobic membrane segments into the lipid bilayer (for recent reviews, see Refs. 1-3). It has been proposed that membrane proteins integrate into the membrane by a series of alternating signal anchor (SA)1 or stop transfer (ST) sequences. However, increasing evidence suggests that the acquisition of a correct membrane topology of polytopic membrane proteins is often more complicated and involves intermolecular interactions that require specific sequence determinants within or adjacent to the hydrophobic sequences. Furthermore, in oligomeric proteins, subunit assembly may play a role in the correct packing of polytopic proteins.

We studied the in vivo membrane insertion of the polytopic alpha -subunit of H,K-ATPase which, in its mature form, is associated with the beta -subunit, a type II membrane protein. Little is known as to how the assembly of the beta -subunit may influence the membrane topology of transmembrane segments of the alpha -subunit. However, it is clear that association with the beta -subunit is needed for the structural maturation of the alpha -subunit of oligomeric P-type ATPases such as H,K-ATPase (HKAalpha ) and Na,K-ATPase (NKAalpha ) because only beta -subunit-assembled alpha -subunits become trypsin-resistant, are protected from cellular degradation, and become functionally active (4, 5).

There is general agreement that the HKAalpha and NKAalpha have 10 transmembrane segments with the N and the C termini located at the cytoplasmic side in the mature beta -associated enzyme (for reviews and references, see Refs. 6-8). The hydropathy profile of the two enzymes is quite similar but differs significantly in the level of hydrophobicity in M7, M8, M9, and M10. Thus, M7, for example, has a lower hydrophobicity value in HKAalpha than in NKAalpha , and M9 has a higher hydrophobicity value in HKAalpha than in NKAalpha . An in vitro transcription/translation assay showed that the putative transmembrane segments M1, M2, M3, M4, and M9 of the gastric HKAalpha can act both as signal anchor (SA) and stop transfer (ST) sequences, M8 and M10 can act only as ST sequences, and M5, M6, and M7 have no membrane insertion properties (9). Thus, there is a gap of three known membrane-inserted sequences in a sequential folding model determined entirely by the hydrophobicity of the membrane segments. Similarly, in vitro translation assays of M5 and M6 of the Na,K- (10) or endoplasmic reticulum Ca-ATPase alpha -subunits (11) have been unable to show efficient SA and ST properties of these segments. In vivo translation/insertion experiments in Xenopus oocytes showed that M5, M7, and M9 of NKAalpha exhibit partial SA function. In the mature enzyme, the beta -subunit is closely associated with the extracellular loop immediately preceding M8 in both NKAalpha and HKAalpha , and the assembly of the beta -subunit plays an important role in the correct packing of the C-terminal membrane domain in NKAalpha (12).

The in vitro transcription/translation system may lack factors present in the intact cell, and it is difficult to study the effect of beta -subunit association in the in vitro system. Therefore, we reassessed the SA or ST properties of the transmembrane sequences of HKAalpha after expressing them in Xenopus oocytes in the presence or absence of the beta -subunit. As a test for membrane insertion, we assessed the presence or absence of glycosylation of chimera containing single or multiple transmembrane segments of alpha -subunits and the ectodomain of the beta -subunit that contains several glycosylation sites. We also assessed the stability of the synthesized proteins by pulse-chase experiments. Whereas the three N-terminal membrane segments are efficiently and stably integrated into the membrane during synthesis, the C-terminal membrane segments do not conform to the sequential signal anchor-hydrophilic segment-stop transfer motif. Furthermore, association with the beta -subunit plays a role in the correct packing of the C-terminal membrane domain and the stabilization of HKAalpha , as has also been observed in NKAalpha (12). However, the specific requirements for the proper folding of the C-terminal membrane domain are distinct in the two P-type ATPases.

    MATERIALS AND METHODS

alpha Constructs-- The rabbit gastric HKA chimera containing single or multiple putative transmembrane segments of the alpha -subunit (see Table I) and the 177 C-terminal amino acids of the rabbit HKA beta -subunit were prepared as described previously (9), but all constructs were subcloned into the pSD5 vector containing a modified Xenopus 5'-untranslated region, which improves the translation of foreign cRNA in Xenopus oocytes as described previously (13).

For the preparation of truncated alpha  constructs lacking the HKAbeta ectodomain, we introduced the modified Xenopus 5'-untranslated region into the original pGEM7zf+ chimeric M1 construct (e.g. containing the cDNA sequence for the first 139 amino acids of the alpha -subunit ending in the extracellular loop following M1 and for the ectodomain of the HKA beta -subunit; Ref. 9) by using appropriate restriction sites. By polymerase chain reaction mutagenesis, we then introduced a stop codon at the junction between the alpha  and the beta  sequence upstream of the artificial BglII/HindIII linker. The variable domains containing the putative transmembrane segments were excised from the chimeric constructs by BglII and HindIII digestion and introduced into the BglII/HindIII linker of this new vector. Finally, the constructs were subcloned from the pGEM7zf+ vector to the pSD5 vector using EcoRI restriction sites. All constructs generated by polymerase chain reaction amplification were sequenced by dideoxy sequencing.

Expression of HKA Constructs in Xenopus Oocytes and Immunoprecipitation of alpha - and beta -Subunits-- Stage V-VI oocytes were obtained from Xenopus laevis females (Noordhoek) as described previously (5). In vitro synthesized RNA (cRNA) was prepared as described by Melton et al. (14). Routinely, 8-10 ng of mutant HKAalpha cRNA and/or 2 ng of rabbit gastric HKAbeta cRNA were injected into oocytes. Oocytes were metabolically labeled with 0.6 mCi/ml [35S]methionine (Hartmann Analytic) for 24 h and, in some instances, subjected to a 48-h chase period. Microsomal fractions were prepared, and the pellets were taken up in 0.5% digitonin and subjected to immunoprecipitation under nondenaturating conditions (5) with polyclonal antibodies that recognize the alpha - and beta -subunits of HKA (15). Endoglycosidase H (Endo H) (Calbiochem) treatment was performed as described previously (16). SDS-polyacrylamide gel electrophoresis was performed on the immunoprecipitated proteins without or with Endo H treatment, and the protein pattern was quantified by densitometry with a LKB 2202 Ultrascan.

    RESULTS

Comparison of the Topographic Properties of Transmembrane Segments of the alpha -Subunit of Gastric H,K-ATPase Synthesized in Intact Cells and in Vitro-- To follow the topogenic properties of membrane segments of HKAalpha synthesized in vivo, we used the reporter glycosylation scanning approach. This method has been used successfully for topological mapping of several polytopic membrane proteins (6, 17, 18). The presence or absence of glycosylation of a C-terminal, topographically neutral reporter sequence indicates whether a translated sequence ends on the luminal or the cytoplasmic side of the membrane, if one assumes that all membrane-inserted protein is glycosylated. Glycosylation of putative membrane-spanning sequences containing the last 177 amino acids of the HKA beta -subunit at their C terminus including five glycosylation sites was assessed after their expression in Xenopus oocytes by cRNA injection, radioactive labeling, and immunoprecipitation. Endo H that specifically cleaves N-linked high mannose core sugars was used to distinguish glycosylated protein species from nonglycosylated protein species to confirm the origin of the shift in the protein migration on SDS polyacrylamide gels.

Fig. 1 shows the Endo H sensitivity of several membrane-spanning sequences of HKAalpha (summarized in Table I) expressed in Xenopus oocytes after cRNA injection and labeled for 24 h with [35S]methionine. M1 (lanes 1 and 2), M2 (lanes 3 and 4), M3 (lanes 7 and 8), M4 (lanes 9 and 10), and M9 (lanes 11 and 12), which were synthesized in vivo from cRNAs that are preceded by the first 101 amino acids (cytoplasmic N terminus) of rabbit gastric HKAalpha , were all efficient SA sequences that were Endo H-sensitive due to the glycosylation of the total protein population. In the oocyte, as in vitro (9), glycosylation of M2 was prevented when it was preceded by M1 (M1/2, lanes 5 and 6), indicating that in this construct, M2 acts as a ST sequence, and the first membrane loop is firmly anchored in the membrane.


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Fig. 1.   Membrane insertion of individual membrane segments of H,K-ATPase alpha -subunits expressed in Xenopus oocytes. Oocytes injected with 8 ng of chimeric cRNA coding for individual or combined transmembrane segments M1-M10 of the alpha -subunit of rabbit gastric H,K-ATPase (alpha  HKA) and the ectodomain of the HKA beta -subunit were labeled with [35S]methionine for 24 h. Microsomes were prepared, and immunoprecipitations were performed under nondenaturing conditions with an antiserum that recognizes HKAalpha and HKAbeta (15). Immunoprecipitates were subjected or not subjected to Endo H treatment, which reduces the size of glycosylated signal anchor sequences but not that of nonglycosylated stop transfer sequences. Autoradiograms of immunoprecipitates subjected to SDS-polyacrylamide gel electrophoresis are shown. Some artifactual bands (dots) are observed in lanes 7-10, 13, and 14 that migrate in front of the antibody heavy chains and probably represent actin. In lane 13, the coreglycosylated, Endo H-sensitive M10 is indicated by an asterisk. Right, the position of marker proteins is indicated. ng, nonglycosylated; cg, core glycosylated. One of three similar experiments is shown.

                              
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Table I
Description of alpha  constructs and summary of results

A set of larger constructs (Table I) was used to test whether and how naturally preceding membrane-spanning segments influence the orientation of C-terminal membrane sequences. The total population of a M1-3 translation product was Endo H-sensitive (Fig. 2B, lanes 1 and 2), showing that M3 also acts as an efficient SA sequence when preceded by the membrane-integrated M1/2 pair. M4, which acts as an efficient SA sequence when synthesized alone (Fig. 1, lanes 9 and 10), became partially but not entirely protected from glycosylation when preceded by M1-3. Also, the M1-4 protein showed aberrant migration on SDS-polyacrylamide gels with a lower molecular mass than the M1-3 protein (Fig. 2A, lanes 3 and 4), as noted previously in vitro (9).


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Fig. 2.   Membrane insertion of combined membrane segments of H,K-ATPase alpha -subunits and topogenic effects of beta -subunit assembly. Oocyte injection, metabolic labeling, sample preparation, and immunoprecipitation were performed as described in the Fig. 1 legend. A, membrane insertion of combined membrane segments of chimeric HKAalpha . Similar to results obtained in vitro (9), M1-4 consistently showed aberrant migration on SDS-polyacrylamide gel electrophoresis. B, effect of H,K-ATPase beta -subunit (beta  HKA) co-expression on membrane insertion of C-terminal membrane segments of HKAalpha . The position of the alpha - and the beta -subunits is indicated. Oocytes were co-injected with HKAalpha cRNAs and 2 ng of HKAbeta cRNA. cg, core glycosylated HKAbeta species; ng, nonglycosylated HKAbeta species. One of three similar experiments is shown.

The observation that M5, M6, and M7 following the large cytoplasmic loop had neither SA nor ST function in an in vitro translation system led to the conclusion that M5-M7 must be inserted into the membrane by a different mechanism than the other seven transmembrane segments (9). One possibility that was considered was the insertion of this region in an already folded state into a membrane domain that included M1-M4 and M9-M10. However, when synthesized in Xenopus oocytes, about 40-45% of the total HKA M1-5 population was glycosylated (Fig. 2A, lanes 5 and 6), suggesting that HKA M5 has some intrinsic SA function in vivo. The addition of M6 (M1-6) completely prevented glycosylation (lanes 7 and 8), indicating that M6 can form a pair with M5 that may be membrane-bound or retained in the cytoplasm.

In combined HKAalpha constructs, M9 of gastric HKAalpha displays SA function and M7 does not display SA function when synthesized in vitro and in vivo in intact cells. When synthesized in the oocyte, HKA M1-7 and M1-8 proteins were Endo H-resistant and thus were not glycosylated (lanes 9 and 10), whereas 40-45% of the total M1-9 population was Endo H-sensitive and glycosylated (lanes 13 and 14).

Individual M10, which was devoid of SA activity in vitro (9), became partially glycosylated when expressed in the oocyte (Fig. 1, lanes 13 and 14). Thus, in vivo, M10 can adopt a Nout-Ccyt as well as a Ncyt-Cout orientation. Although M10 added to M1 could not act as a ST sequence in intact cells (Fig. 1, lanes 15 and 16), it completely prevented glycosylation when added to M1-9 (Fig. 2A, lanes 15 and 16, M1-10), indicating that M10 can form a pair with M9 that is either retained in the cytoplasm or membrane-bound. The results shown in Fig. 2 are also summarized in Table I.

The differences in the membrane insertion efficiency of certain membrane segments of HKAalpha synthesized in vitro or in intact cells cannot be easily explained. It is possible that due to the longer synthesis time allowed in intact cells compared with that used in in vitro translations, a steady-state level of folding and membrane insertion of certain membrane segments can only be achieved in the former and not in the latter translation system. In this context, however, it is interesting to note that P-glycoprotein synthesized in two different in vitro translation systems adopts a different topography, possibly due to the presence or absence of certain soluble cytoplasmic factors (19). Whatever might be responsible for the subtle differences between the in vivo and in vitro systems does not change the main conclusions that can be drawn from the reporter glycosylation scanning assays. The results obtained in both systems indicate that the formation of N-terminal and C-terminal membrane pairs in HKAalpha is governed by different mechanisms: the former is governed by sequential signal anchor and stop transfer mechanisms, and the latter is governed by more complicated interactions. We tested the role of beta -subunit interactions on the correct packing of the C-terminal membrane domain of HKAalpha .

Effect of beta -Subunit Assembly on the Topography of H,K-ATPase alpha -Subunit-- A domain that interacts with the beta -subunit has been identified in the extracellular loop just before M8 in the alpha -subunit of both H,K-ATPase (20, 21) and Na,K-ATPase (22). The effect of beta -subunit assembly on the membrane insertion of HKAalpha was determined by the co-expression of HKAbeta with HKA M1-7-M1-10 alpha -proteins.

Endo H sensitivity and thus the apparent topography of HKA M1-7, M1-8, or M1-10 alpha -proteins were similar when they were expressed alone or together with beta -subunits (compare Fig. 2B, lanes 1-4, 7, and 8 with Fig. 2A, lanes 9-12, 15, and 16) showing an absence of glycosylation. In contrast, the M1-9 protein consistently showed a change in the ratio between glycosylated, Endo H-sensitive and nonglycosylated, Endo H-resistant species when it was expressed with beta -subunits. The percentage of glycosylated species increased from 40-45% in M1-9 expressed alone to about 80-85% in M1-9 co-expressed with beta -subunits (compare Fig. 2B, lanes 5 and 6 with Fig. 2A, lanes 13 and 14; Table I). This result suggests that assembly with beta -subunits increases the efficiency of membrane integration of M9 in the HKA M1-9 alpha -protein. To further analyze the role of beta -subunit assembly in the membrane integration of the HKAalpha , we determined the resistance against cellular degradation of various HKAalpha constructs synthesized in the absence or presence of beta -subunits, an indicator of correct folding of polytopic integral membrane proteins.

Effect of beta -Subunit Assembly on the Stability of H,K-ATPase alpha -Subunits-- To test the stability of HKAalpha proteins synthesized in the presence or absence of beta -subunits, we prepared truncated HKAalpha constructs lacking the glycosylation reporter sequence because we have previously observed that the glycosylation reporter sequence itself can destabilize chimeric NKAalpha constructs when it is exposed to the cytoplasm (12), leading to cellular degradation. Fig. 3A shows pulse-chase experiments performed in oocytes expressing truncated HKA M1-3-M1-10 alpha -proteins in the absence of beta -subunits. M1-3 proteins were completely resistant to cellular degradation (Fig. 3A, lanes 1 and 2), consistent with the efficient membrane insertion of this construct observed in the reporter glycosylation scanning assay. M1-4 constructs produced two different protein populations. One of the proteins had a lower molecular mass than the M1-3 protein and likely corresponds to the aberrantly migrating M1-4 protein that shows an inefficient ST activity in the reporter glycosylation scanning assay (Fig. 2A, lanes 3 and 4). This protein population was significantly degraded during a 48-h chase period (Fig. 3A, lanes 3 and 4). The second protein population synthesized from the M1-4 construct exhibited the expected molecular mass, which was greater than that of the M1-3 protein, and it was relatively resistant to cellular degradation (lanes 3 and 4). This result indicates that the particular M1-4 construct used in this study is indeed able to form an alpha -protein population in vivo that corresponds to a stably inserted M1-4 membrane domain. It is possible that the removal of the hydrophilic glycosylation reporter sequence favors the ST function of M4 in this particular construct.


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Fig. 3.   Cellular degradation of truncated H,K-ATPase alpha -subunits and effect of beta -subunit assembly. Oocytes were injected with 10 ng of HKAalpha cRNA coding for combined transmembrane segments M1-M10 in the absence (A) or presence (B) of 1 ng of HKAbeta cRNA, labeled with [35S]methionine for 24 h (odd-numbered lanes), and subjected to a 48-h chase (even-numbered lanes). Microsomes were prepared, and immunoprecipitations were performed as described in the Fig. 1 legend. The position of the alpha - and the beta -subunits and the molecular mass of marker proteins are indicated. HKAbeta is present in its core-glycosylated (cg) form during the pulse and the chase period and partially in its fully glycosylated (fg) form during the chase period due to the endoplasmic reticulum exit of unassembled HKAbeta (26). The band indicated by a dot and visible in lanes 1, 2, and 4 corresponds to endogenous oocyte NKAalpha , which is co-immunoprecipitated and assembled with HKAbeta . One of three similar experiments is shown.

All other truncated HKAalpha proteins comprising C-terminal membrane segments up to M10 (Fig. 3A, lanes 5-16) synthesized in the absence of beta -subunits were almost completely degraded during a 48-h chase, similar to the full-length alpha -subunit (lanes 17 and 18). These results are consistent with the inefficient membrane insertion of M1-5-M1-10.

Co-expression with the HKAbeta had no effect on the stability of the HKA M1-7 alpha -proteins (Fig. 3B, lanes 1 and 2) but partially protected the M1-8 (lanes 3 and 4) and M1-9 proteins (lanes 5 and 6) from cellular degradation. Furthermore, HKA M1-10 proteins (lanes 7 and 8) were completely stabilized after co-expression with beta -subunits, similar to the full-length HKAalpha (lanes 9 and 10). These results confirm that association with the beta -subunit is an important factor for correct membrane insertion and stabilization of the C-terminal membrane domain of the HKAalpha .

    DISCUSSION

Topological Properties of HKAalpha Membrane Segments-- The present study shows a membrane insertion process of HKAalpha that is characterized by major differences in the mechanisms that govern membrane integration of the four N-terminal and the six C-terminal membrane segments. The former segments act as alternate SA and ST sequences and are sequentially inserted into the membrane to form stable membrane pairs, whereas the latter segments need both intramolecular interactions and assembly with beta -subunits for correct membrane packing and protection against cellular degradation.

Full SA activity of HKAalpha M1 and M3 and an efficient ST activity of M2 were observed in vivo (this study) and in vitro (9), which provide the basis for stable membrane integration of a domain comprising a M1/M2 pair and M3. This was also confirmed by the observation that M1-3 alpha -proteins are completely resistant to cellular degradation. On the other hand, the particular sequence used in this study for HKA M4 produced aberrant migration on SDS-polyacrylamide gels and degradation of part of the M1-4 protein population, and it had inefficient ST activity in vivo. This contrasts with the efficient ST activity observed in vitro (9). This result may indicate that pair formation of M3 and M4 in vivo is necessary but is not sufficient to determine the four-membrane segment topology of the HKAalpha N terminus in contrast to recent results obtained for NKAalpha synthesized in Xenopus oocytes (12). Alternatively, it is more likely that M4 in the HKA M1-4 protein that ends at Leu-357 may be too short to assure its membrane retention as an efficient ST sequence. According to a current model (Ref. 8; see Table I), HKA M4 would end at Ala-359; consequently, the M1-4 protein lacks two positively charged residues that follow the transmembrane domain and may be important for the ST properties of M4 in the HKA M1-4 protein synthesized in vivo. Positive charges following a membrane-inserted domain have been shown to be important for ST function (6). Nevertheless, part of the HKA M1-4 alpha -protein population was able to adopt a relatively stable membrane-inserted conformation with a correct molecular mass, supporting the hypothesis that the N-terminal membrane domain of HKAalpha is formed by sequential insertion of the two membrane pairs M1/M2 and M3/M4, as in NKAalpha (12).

M5, M7, and M9, which should act as SA sequences according to the 10-transmembrane segment model, produced a mixture of glycosylated and nonglycosylated species in HKA M1-5 and M1-9 alpha -proteins and only nonglycosylated species in the M1-7 alpha -protein synthesized in vivo in the absence of beta -subunits. Similar observations were made with NKAalpha , but the insertion efficiency of NKA M5, M7, and M9 differed from that of HKA membrane segments. The NKA M1-5 alpha -proteins produced only about 5% glycosylated species, but M1-7 and M1-9 produced about 50% glycosylated species (12), perhaps due to the greater hydrophobicity of M7 in NKAalpha .

Consistent with the poor membrane insertion properties, all alpha -proteins containing C-terminal membrane segments of both HKAalpha and NKAalpha (23) were rapidly degraded during a chase period. Because we have recently shown that major domains of NKAalpha are not exposed to the lumen of the endoplasmic reticulum during synthesis (12), it is likely that unassembled alpha -subunits of P-type ATPases are degraded from the cytoplasmic side, as recently suggested for misfolded polytopic membrane proteins (24). The degradation of the alpha -subunit, which is likely to start in the C-terminal domain and/or the central cytoplasmic loop, also destabilizes the intrinsically stable N-terminal domain and renders it susceptible to degradation because a proteolytic fragment corresponding to the N-terminal domain is never observed.

The poor efficiency of membrane insertion and the associated instability of the C-terminal membrane domain likely reflect a distinct, low level of hydrophobicity of HKAalpha and NKAalpha M5, M7, and M9 that reduces interactions with nonpolar surfaces. In NKAalpha , the presence of proline, polar, and/or charged residues in NKA M5, M7, and M9 was shown to be responsible for the partial membrane insertion of these membrane segments (12). As a consequence of their moderate hydrophobicity, membrane insertion of M5, M7, and M9 of HKAalpha and NKAalpha must be mediated by other factors, for instance, interaction between membrane peptide segments in the translocon during translation. Such peptide-peptide interaction is consistent with the recently proposed two-dimensional crystal structure of the Ca-ATPase (25) that would place two or three membrane segments in a structure where their surfaces interact largely with other transmembrane segments, whereas the other segments face the hydrophobic core of the membrane. Furthermore, we show in this study and in previous studies of NKAalpha (12, 23) that in oligomeric P-type ATPases, association with the beta -subunit plays an important role in the stabilization of the moderately hydrophobic membrane pairs in the membrane.

Effect of beta -Subunit Assembly on the Membrane Topography and Stabilization of HKAalpha -- Co-expression of HKAalpha proteins with beta -subunits had two major effects. On the one hand, it increased the efficiency of membrane insertion of M9 in a M1-9 alpha -protein, and on the other hand, it partially protected M1-8 and M1-9 alpha -proteins and completely protected M1-10 proteins against cellular degradation. Together with the observation that the interaction of the beta -subunit with the region of the extracytoplasmic loop between M7 and M8 participates in the stable membrane insertion of the M7/M8 pair in the NKAalpha (12), this result indicates that in hetero-oligomeric P-type ATPases, not only intramolecular but also intermolecular interactions between subunits participate in the correct packing of the C-terminal membrane domain of the alpha -subunits.

A Model for Stable Membrane Integration of the C-terminal Membrane Domains of HKA and NKA alpha -Subunits-- Our data indicate that the precise role of beta -subunit assembly and intramolecular interactions in the stable membrane insertion of HKAalpha and NKAalpha may vary, possibly due to the differences in the intrinsic SA and ST function of M7 and M10, respectively, in the two proteins. According to the reporter glycosylation scanning assay, HKA M7 in a HKA M1-7 alpha -protein has no visible SA function (Ref. 9 and this study), but NKA M7 in a NKA M1-7 alpha -protein has partial SA function and produces 50% glycosylated forms (12). In both alpha -subunits, the addition of M8 produces only nonglycosylated forms. These observations suggest that in NKA M1-8 alpha -proteins, an equilibrium exists between membrane-inserted and cytoplasmic forms of the NKA M7/M8 pair, whereas in HKA M1-8 proteins, the equilibrium may be significantly shifted in favor of the cytoplasmic forms of the HKA M7/M8 pair, as illustrated in the model in Fig. 4. In consequence, the probability of beta -subunit assembly with the extracellular loop between M7 and M8 in M1-8 proteins is smaller in HKAalpha than in NKAalpha . This may be reflected by the partial stabilization as compared with the complete stabilization of the HKA M1-8 and NKA M1-10 alpha -proteins, respectively, when they are co-expressed with beta -subunits. The difference in the stabilizing effect of the beta -subunit on HKA and NKA M1-8 alpha -proteins also indicates that M9/M10 pair formation has a distinct role in the correct folding of the HKAalpha and NKAalpha . In HKAalpha , co-expression with beta -subunits favors membrane insertion of M9, but only M1-10 proteins are stabilized (Fig. 4). This infers that M9/M10 pair formation and probably its interaction with the M7/M8 pair are necessary for an efficient membrane insertion of HKA M7/M8 and consequently for an efficient beta -assembly and stabilization of HKAalpha . On the other hand, in NKAalpha , beta -assembly with the M7/M8 extracellular loop is sufficient to stabilize the entire alpha -subunit, although it restrains M9 membrane insertion (Fig. 4). In NKAalpha , the formation of the M9/M10 pair may only be possible due to the better ST function of NKA M10 as compared with that of HKA M10.


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Fig. 4.   Comparison of membrane insertion properties and stability of HKAalpha and NKAalpha and effect of beta -subunit assembly. The membrane retention and cellular degradation of HKA (this study) and NKA (12, 25) M1-8, M1-9, and M1-10 alpha -proteins synthesized in the absence or presence of beta -subunits are shown. In HKAalpha and NKAalpha , the four N-terminal membrane segments are stably inserted into the membrane due to sequential SA and ST mechanisms independent of the beta -subunit. On the other hand, insertion of the six C-terminal membrane segments is mediated by interactions between membrane segments and by beta -subunit assembly with the extracytoplasmic loop (el) between M7 and M8. Our results are compatible with a model in which, in the absence of the beta -subunit, the C-terminal membrane pairs of M1-8, M1-9, and M1-10 exist as membrane-inserted and cytoplasmic forms in a dynamic equilibrium (shown in the diagrams and indicated by an arrow). The membrane insertion of the NKAalpha and HKAalpha M7/M8 pair in the M1-8 protein cannot be directly quantitated, but due to the poor SA function of HKA M7, it can be predicted that the equilibrium of the HKAalpha M7/M8 pairs is significantly shifted toward the cytoplasmic forms. On the other hand, the NKAalpha M7/M8 pairs are evenly distributed between the membrane and the cytoplasm. From these data, it may be inferred that beta -subunit interaction with the extracellular loop between M7 and M8 (el) is more likely to occur in NKA M1-8 alpha -proteins than in HKA M1-8 alpha -proteins. This is supported by the observation that HKA M1-8 is only partially protected, but NKA M1-8 is completely protected against cellular degradation after co-expression with beta -subunits. According to the reporter glycosylation scanning assay, HKA and NKA M9 in M1-9 alpha -proteins have a partial SA function. beta -Subunit assembly favors HKA M9 membrane retention and impedes that of NKA M9. Nevertheless, beta -subunit assembly only partially protects HKA M1-9 alpha -proteins but fully protects NKA M1-9 alpha  proteins from degradation similar to HKA M1-8 and NKA M1-8, respectively. Finally, membrane retention of the M9/M10 pairs in HKA and NKA M1-10 alpha -proteins in the absence of beta -subunit assembly cannot be predicted, but it is fully achieved in the presence of beta -subunit assembly, because NKA M1-10 as well as HKA M1-10 become completely protected from cellular degradation. Together with the observation that HKA M1-5 and M1-6 alpha -proteins are completely degraded independent of the presence of beta -subunits, these results also imply that after assembly with beta -subunits, the HKA M5/M6 pair is correctly membrane-inserted in the HKA M1-10 alpha -protein. For further discussion, see text.

In conclusion, in both HKAalpha and NKAalpha , the correct membrane integration of the N-terminal domain appears to be determined by the properties of the individual segments, whereas the insertion and stabilization of the C-terminal membrane domain depend on interactions with other membrane segments as well as on intermolecular association with the beta -subunit. However, subtle differences exist between the two P-type ATPases in the fine control of the insertion process.

    ACKNOWLEDGEMENTS

We thank Dirk Claeys for providing the HKAalpha and HKAbeta antibody. We also acknowledge the technical assistance of Sylvie Girardet, Sophie Roy, and Danièle Schaer.

    FOOTNOTES

* This work was supported by Swiss National Fund for Scientific Research Grants 31-42954-95 and 31-53721.98 (to K. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed. Tel.: 41-21-692-54-10; Fax: 41-21-692-53-55; E-mail: kaethi.geering{at}ipharm.unil.ch.

    ABBREVIATIONS

The abbreviations used are: SA, signal anchor; ST, stop transfer; HKA, H,K-ATPase; NKA, Na,K-ATPase; HKAalpha , HKA alpha -subunit; NKAalpha , NKA alpha -subunit; HKAbeta , HKA beta -subunit; Endo H, endoglycosidase H.

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TOP
ABSTRACT
INTRODUCTION
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