The KdpF Subunit Is Part of the K+-translocating Kdp Complex of Escherichia coli and Is Responsible for Stabilization of the Complex in Vitro *

The kdpABC operon codes for the high affinity K+-translocating Kdp complex (P-type ATPase) ofEscherichia coli. Upon expression of this operon in minicells, a so far unrecognized small hydrophobic polypeptide, KdpF, could be identified on high resolution SDS-polyacrylamide gels in addition to the subunits KdpA, KdpB, and KdpC. Furthermore, it could be demonstrated that KdpF remains associated with the purified complex. As determined by mass spectrometry, this peptide is present in its formylated form and has a molecular mass of 3100 Da. KdpF is not essential for growth on low K+ (0.1 mm) medium, as shown by deletion analysis of kdpF, but proved to be indispensable for a functional enzyme complex in vitro. In the absence of KdpF, the ATPase activity of the membrane-bound Kdp complex was almost indistinguishable from that of the wild type. In contrast, the purified detergent-solubilized enzyme complex showed a dramatic decrease in enzymatic activity. However, addition of purified KdpF to the KdpABC complex restored the activity up to wild type level. It is interesting to note that the addition of high amounts of E. coli lipids had a similar effect. Although KdpF is not essential for the function of the Kdp complex in vivo, it is part of the complex and functions as a stabilizing element in vitro. The corresponding operon should now be referred to askdpFABC.

and is phosphorylated during the catalytic cycle (12,13). KdpB is homologous to the large subunit of other P-type ATPases and shares the common key structures of this ATPase class (14,15). The 59-kDa KdpA subunit is predicted to span the membrane 10 times and is believed to be the subunit that binds and transports K ϩ (16). The 20-kDa KdpC subunit probably traverses the membrane once close to the N terminus, leaving the C-terminal portion exposed to the cytoplasm, and is important for the assembly of the enzyme complex (17). The adjacent kdpDE operon codes for the two proteins KdpD and KdpE, which regulate the expression of the structural genes (18) and which belong to the family of sensor kinase/response regulator systems (19,20).
An extended region was recognized between the start site of the mRNA transcript of the kdp operon and the ATG start codon of kdpA. Sequence analysis revealed that an additional open reading frame, kdpF, exists upstream of the kdpABC structural genes. Therefore, the corresponding operon should be referred to as kdpFABC. Deduced from the DNA sequence, the putative KdpF protein seems to be another candidate for a diverse group of recently identified small, hydrophobic proteins associated with membrane transport complexes (for details, see "Discussion"). In bacteria, the function of these subunits has still to be elucidated.
In this report it is clearly demonstrated that kdpF is indeed expressed and that KdpF is associated with the purified complex. Although deletion of kdpF revealed that KdpF is not necessary for growth of cells on low K ϩ medium, the ATPase activity of the purified complex is impaired in the absence of KdpF. By addition of the purified KdpF or high amounts of E. coli lipids to the purified, detergent-solubilized KdpABC complex, an K ϩ -stimulated ATPase activity comparable with that of the wild type enzyme could be recovered. In summary, it could be demonstrated that the KdpF protein stabilizes the enzyme complex in vitro, thereby possibly acting as a kind of lipid-like peptide for the holoenzyme.

EXPERIMENTAL PROCEDURES
Materials-All chemicals were of analytical grade. [  Construction of Plasmids and Strains-The methods used for handling recombinant DNA and for transformation of E. coli cells were as described (27). Treatment of DNA with restriction endonucleases, T4 DNA ligase, shrimp alkaline phosphatase, and isolation of plasmids was performed following the protocols of the suppliers.
Plasmid pTM1 was generated by digestion of vector pJLA503 (24) with SalI, treatment with Klenow DNA polymerase and shrimp alkaline phosphatase, and ligation of the FspI fragment from plasmid pTM002 (see below) containing the kdpF gene. The resulting plasmid was digested with EcoRI (to remove sequences from pTM002) and religated to yield pTM1.
All other vectors used in this study are derivatives of plasmids pSR4 (25) and pJDkdp (kindly provided by Dr. W. Epstein, University of Chicago). In both plasmids an SspI/EcoRI deletion was introduced to inactivate the posterior of two EcoRI restriction sites enclosing the kdpFABC genes. Each of the resulting plasmids pTM001 and pTM002 gave rise to a series of mutant vectors, designated pTMX01 and pTMX02 (Fig. 1). The difference between vectors of the series pTMX01 and pTMX02 resides in the regulatory region upstream of the kdpFABC operon. In vectors of the series pTMX01 this region resembles that of the wild type sequence including 472 bp 1 upstream of the kdpA start codon. The expression of the kdp genes is under control of the regulatory proteins KdpD and KdpE, which are chromosomally encoded. In vectors of the series pTMX02 the kdpFABC genes are constitutively expressed under control of a promoter, which is still part of the truncated tetracycline resistance gene of the vector. With the latter series of vectors it was possible to express the mutated kdp genes in minicells.
Transfer of in vitro constructed, plasmid-encoded kdpF mutations to the chromosome was carried out using the polA technique based on the method described by Gutterson and Koshland (28). For this purpose strain TKW228505 was used. Only carbenicillin-resistant clones growing well on K10 agar plates were used for further investigations.
Site-directed Mutagenesis-To introduce point mutations the two stage PCR technique was used (29). The outside primers kdp11 (5Ј-C-TACCCGGTGAATGGCAC-3Ј) and kdp12 (5Ј-AAGATCGCCA GCATC-TGC-3Ј) employed in all experiments were complementary to the sequences of bp Ϫ261 to Ϫ244 (kdp11) and bp 896 to 879 (kdp12) (numbering is according to Ref. 30). The mutant primers (20 nucleotides) encompassed the kdpF start codon and differed in the first base of the start codon (position 29). The plasmids pTM301 and pTM302 were constructed with an exchange at position 29 from G to C. For plasmid pTM401 and pTM402, an exchange at the same position from G to A was introduced. The mutant PCR products were digested with EcoRI/StyI, and the resulting fragments were subcloned in the vector pTM001 or pTM002.
The plasmid pTM501 was constructed using the internal primers kdp21 (5Ј-CTCTATAAGCTTGTGTTTTTATTACTGG-3Ј) and kdp22 (5Ј-AGTATTAAGCTTCACAGTGCACCTCCAG-3Ј) corresponding in the 3Ј parts (underlined) to the following sequences in kdp: bp 62-77 (kdp21) and bp 32-16 (kdp22). The 5Ј part of the primers contains a HindIII restriction site and in addition 7 nucleotides to ensure the cleavability of the PCR products by HindIII. With these primers a 24-bp deletion was introduced at the 5Ј end of kdpF and at the same time a new HindIII restriction site suitable for identification of relevant clones was generated. After the first stage of PCR, the resulting fragments were tailored with HindIII, ligated, and after gel purification subjected to a second run. The mutant PCR product was digested with EcoRI/StyI, and the resulting fragment was cloned into vector pTM001. Plasmid pTM201 was generated from the predecessor pTM101, in which a double point mutation was introduced at position 24/25 to create a SalI restriction site right in front of the GTG start codon. Following digestion with SalI/BclI and treatment with Klenow polymerase, and blunt end ligation yielded a 72-bp deletion encompassing the start codon. These manipulations leave 21 bp upstream of the kdpA gene intact including the putative ribosome binding site of kdpA.
Synthesis and Detection of Plasmid-encoded Proteins in Minicells-For the preparation of minicells according to the method of Reeve (31) strain DK6 was used. Cells harboring one of the designated plasmids were grown and harvested as described (23). To perform the labeling reactions 0.1-ml aliquots of minicells were diluted in 1 ml of ice-cold K115 salt solution, pelleted by centrifugation for 3 min at 10,000 ϫ g (4°C) and resuspended in 100 l of K115 medium containing 0.4% glucose and 20 g/ml D-cycloserine. The cells were preincubated for 30 min at 30°C. After addition of 5 l of DIFCO methionine assay medium, the probes were shifted to 37 or 42°C (DK6/pTM1). After 2 min of equilibration, 10 Ci of [ 35 S]methionine were added, and the incubation was continued for 10 min. The reaction was stopped by addition of 10 l of unlabeled methionine (0.1 M) and centrifugation for 2 min at 8,000 ϫ g. Pellets were resuspended in electrophoresis sample buffer (32), heated for 5 min at 100°C, treated for 2 min in a sonifier bath, and subjected to SDS-PAGE (32). For autoradiography, the gels were pretreated with 55% methanol, 2% glycerol to prevent cracking, dried, and exposed for 3 days to an x-ray film (Kodak, X-OMAT AR, BIOMAX MR).
Preparations-The Kdp complex was prepared from E. coli strain TKW3205 harboring one of the different plasmids using the two column purification protocol described previously (9). For purification of the KdpF peptide, the protocol for the isolation of subunit c of the ATP synthase using C/M extraction, and diethyl ether precipitations were applied (33-35) starting with 10 g of cells of strains TKW3205/pSR4 or TKW3205/pTM501 resuspended in 12 ml of H 2 O. The sample, obtained from C/M extraction and two successive diethyl ether precipitations is called "KdpF extract" throughout the manuscript. In parallel, the enriched KdpF fraction (without diethyl ether precipitations) was applied to ion exchange chromatography on CM 23-cellulose using C/M/H 2 O mixtures as solvent (35,36). The KdpF peptide eluted from the column in the presence of C/M/H 2 O in a ratio of 5:5:1. The transfer of KdpF from organic solvents to detergent solutions was performed in the following way. A KdpF-containing sample (100 l) was mixed end-over-end with 45 l of chloroform and 25 l of H 2 O (10 times), and after phase separation the upper methanol/H 2 O phase was sucked off. After doubling the volume of the samples with chloroform and traces of methanol to keep the solution clear, the samples were dried in a speed-vac at room temperature, and subsequently the pellet was dissolved in 20 l of 50 mM Tris/HCl, pH 7.5, 2 mM MgCl 2 , 0.2% aminoxide WS 35 by shaking for 1 h at room temperature. The same procedure was used for the application of E. coli lipids (Avanti). For KdpABC/KdpF interaction studies detergent-dissolved KdpF was mixed with the purified KdpABC complex present in the same buffer in a ratio of 5:1 (v/v) and incubated for 30 min at room temperature with shaking.
Mass Spectrometry-After chromatography on CM 23-cellulose the sample was mixed with four parts of a 1% 2,5-dihydroxy benzoic acid solution and treated with an ion exchange matrix (AG 50W-X8, Bio-Rad). The matrix-assisted laser desorption/ionization mass spectrometry measurements were performed with a VISION 2000 (Finigan, Bremen, Germany) endowed with a 2,5-dihydroxy benzoic acid matrix. The total impact energy was 20 keV, and the laser was calibrated at 337 nm. The accuracy of the measurements was between 0.5 and 1.5 Da.
Quantification of the Kdp Complex-Quantification of the Kdp complex was carried out with [ 125 I]protein A. For this purpose samples were subjected to SDS-PAGE according to Laemmli (37) and blotted onto nitrocellulose. Detection of the subunits was carried out as described by Siebers and Altendorf (38) using a polyclonal antiserum raised against the Kdp complex (dilution: 1:10,000) and [ 125 I]protein A (dilution: 1:5000, incubated at room temperature for 3 h). The radioactivity was quantified with a PhosphorImager (Molecular Dynamics). In a second approach the radioactively labeled immunoblot was subsequently incubated with the second antibody (goat anti-rabbit IgG conjugated with alkaline phosphatase), and the visualization was performed according to Blake et al. (39). For quantification the blot was dried, and bands were cut out and directly counted in a ␥-counter. For calibration a preparation of homogeneous Kdp complex was used.
Assays-Protein was determined by the method of Hartree (40). High resolution SDS-PAGE was performed according to Schä gger and von Jagow (32) using 16.5% T and 3% C. To obtain strongly focussed bands, urea was replaced by glycerol (10%). Proteins were visualized by silver staining (41). The phospholipid content in samples obtained from C/M extraction or chromatography on CM 23-cellulose or from E. coli lipid solutions (purchased from Avanti) was determined according to Ames (42). The ATPase activity of the Kdp complex was determined as described by Altendorf et al. (11).

RESULTS
The kdpABC operon contains an unusually large stretch of 116 bp between the kdp regulatory sites and the start codon of the kdpA gene. Analysis of this sequence revealed that an additional open reading frame, kdpF, exists starting at position 29 with a GTG and ending with a stop codon, which overlaps by one nucleotide with the start codon of the kdpA gene. The protein sequence deduced from the DNA sequence calls for a small putative protein consisting of 29 amino acids with a calculated molecular mass of 3072 Da (Fig. 2). A hydrophobicity plot according to the algorithm of Kyte and Doolittle (44) predicts that this protein is very hydrophobic in nature and thus very likely to be membrane-embedded (data not shown).
Detection of the kdpF Gene Product and Verification of the Start Codon of kdpF by Site-directed Mutagenesis-The expres-  (30,43). In addition, the overlap between the stop codon of kdpF and the start codon of kdpA is indicated. sion of the kdpF gene by plasmid pTM1 was investigated using [ 35 S]methionine-labeled minicells derived from strain DK6 (Fig. 3). In a second approach the presence of the KdpF peptide could be verified in the purified Kdp complex by SDS-PAGE visualizing the proteins by silver staining (Fig. 4). As can be taken from SDS-PAGE (Figs. 3 and 4), KdpF exhibits a molecular mass of 4000 -6000 Da, considerably higher than expected from the amino acid composition. Such a behavior has also been observed for other proteins of this class. This difference seems to be dependent on the protein itself and/or on the lipid content of the sample (compare Figs. 4 and 9).
To substantiate further that the GTG at position 29 represents the start codon for kdpF, the plasmid series pTMX02 was used, in which the genes of the kdpFABC operon were expressed constitutively from a promoter belonging to the regulatory sites of the tetracycline resistance gene (Fig. 1). Replacing the GTG by a CTG codon (pTM302), gene expression in minicells revealed that kdpF was not detectable any more (Fig.  3). This was also the case for pTM202, which carries a deletion of kdpF encompassing the GTG start codon, leaving the putative ribosomal binding site for kdpA intact. In contrast, changing the GTG to an ATG (pTM402), the expression of kdpF was enhanced as expected. Taken together, these data provide strong evidence that the GTG codon at position 29 represents the translational start site for kdpF.
Effect of Mutations within kdpF on Cell Growth under K ϩlimiting Conditions-To address the question whether mutations in kdpF give rise to a detectable phenotype, strain TK2205 (⌬kdpFABC5) was transformed with plasmids of the series pTMX01. In this case the expression of kdpFABC genes was under control of the chromosomally encoded regulatory proteins KdpD and KdpE. When cells of TK2205/pTM301 (start codon: CTG) or TK2205/pTM401 (start codon: ATG) were grown in K0.1 minimal medium, where the limiting K ϩ concentrations served as the environmental stimulus for the expression of the plasmid-encoded kdp genes, no significant change in growth could be observed compared with TK2205/ pTM001 (wild type start codon: GTG) (data not shown). To avoid overproduction from the altered kdpF gene, the mutations were transferred to the chromosome using strain TKW228505 (⌬kdpFABC5 polA). In comparison with the control (TKT2285-001, rate of growth () ϭ 0.60⅐h Ϫ1 ), the growth of strains TKT2285-301 ( ϭ 0.62⅐h Ϫ1 ) and TKT2285-401 ( ϭ 0.62⅐h Ϫ1 ) was hardly affected, whereas the growth rate of TKT2285-201 ( ϭ 0.56⅐h Ϫ1 ) was significantly impaired (Fig.  5). Because in the case of strain DK6/pTM201 the expression of the kdpABC genes was also affected by the deletion of kdpF (Fig. 3), probably produced by polarity effects, a smaller kdpF deletion of only 24 bp was generated (pTM501), leaving the GTG start codon and most of the kdpF gene intact. A transfer of this smaller deletion to the chromosome revealed that the growth rate of TKT2285-501 ( ϭ 0.60⅐h Ϫ1 ) was identical to that of the control (Fig. 5). These data clearly demonstrate that the inactivation of kdpF (pTM501), which does not affect the expression of the kdpABC operon, still leads to a functional KdpABC complex in vivo.
Effect of the Mutations within kdpF on the Kdp Complex in Vitro-To study the influence of the mutations in kdpF at the level of the Kdp complex, the enzyme complex was purified, subjected to SDS-PAGE, and probed with polyclonal antibodies raised against the Kdp complex. The yield of Kdp complexes derived from strains TKW3205/pTM301 and TKW3205/pTM501 was four to eight times lower as compared with the wild type, whereas in the case of TKW3205/pTM401 the yield obtained was comparable with that of TKW3205/pTM001 (wild type). Hardly any complex was obtained from strain TKW3205/ pTM201 (data not shown), indicating a strong polarity effect based on the extent of the deletion within kdpF.
Furthermore, the kdpF mutations had severe effects on the enzymatic activity of the Kdp complex. Enzyme preparation from TKW3205/pTM301 showed a reduced K ϩ -stimulated ATPase activity (18%) in comparison with enzyme preparations derived from strain TKW3205/pTM001 or TKW3205/pSR4 (100%, wild type). For the Kdp complex isolated from TKW3205/ pTM501, only very low ATPase activity (6%) could be detected (Table II). The difference in the activities may be due to a low level expression of kdpF resulting from CTG as the start codon (similar effect as in Ref. 45) in strain TKW3205/pTM301, whereas kdpF is inactivated in strain TKW3205/pTM501. Only the purified Kdp complex from TKW3205/pTM401 exhibited an ATPase activity (100%) comparable with that of the control (TKW3205/pTM001).
Recovery of K ϩ -stimulated ATPase Activity of the Kdp Complex-To analyze the effect of KdpF on the ATPase activity of the KdpABC complex, the peptide was extracted with C/M followed by two diethyl ether precipitations to reduce the amount of lipids in the sample (Fig. 6). The extract was dried, dissolved in the same buffer as the Kdp complex containing 0.2% aminoxide WS35, and mixed with the KdpABC complex purified from TKW3205/pTM501. Determination of the specific K ϩ -stimulated ATPase activity (see Fig. 8A), based on the amount of KdpABC determined with [ 125 I]protein A labeling, revealed that the activity of the KdpABC complex could be recovered by addition of KdpF extract containing the KdpF peptide and lipids. Interestingly, the ATPase activity could also be recovered with high amounts (Ͼ1.2 mM) of E. coli lipids alone (Fig. 7).
To differentiate between an effect caused by KdpF and that caused by lipids, the recovery of the ATPase activity was related to the amount of phospholipids added. As shown in Fig. 7, 20 times less phospholipids have to be added in the presence of the KdpF peptide in comparison with E. coli phospholipids obtained commercially (Avanti) to achieve half maximal ATPase activity. To verify that no KdpF peptide was present in the E. coli lipids used as control, the lipids were purified on CM 23-cellulose as the KdpF extract. Although a number of proteins could be detected by SDS-PAGE and silver staining, no KdpF peptide was detectable (data not shown). Furthermore, a lipid extract prepared from strain TKW3205/pTM501 (⌬kdpF) also allowed a partial recovery of the K ϩ -stimulated ATPase activity (data not shown). These results clearly demonstrate that the effect caused by the KdpF extract is mainly due to the presence of KdpF and not due to the presence of phospholipids. In addition, the K ϩ -stimulated ATPase activity of the KdpABC complex decreases upon solubilization reaching only 6% of the intact KdpFABC complex after the column steps, probably because of the loss of the natural lipid environment. Addition of KdpF extract brings this residual activity back to the level of the intact KdpFABC complex (Table II). Because the ATPase activity is stimulated by K ϩ (Fig. 8A) and completely inhibited by ortho-vanadate (Fig. 8B), the native conformation of the Kdp  Purification of C/M-extracted KdpF by Ion Exchange Chromatography-To differentiate between the effect caused by KdpF itself on the one hand and by the lipids on the other in more detail, KdpF obtained by C/M extraction was further purified by chromatography on CM 23-cellulose. In accord with the deduced amino acid composition of the KdpF peptide, no absorption could be detected at 280 nm (Fig. 9, A and C). Therefore, each fraction containing proteins of the appropriate size (Fig. 9, B and D) was tested for the ability to recover the K ϩ -stimulated ATPase activity of the purified KdpABC complex. Fig. 9 clearly demonstrates that only fractions 18 -28 (with the maximum at fraction 25) derived from strain TKW3205/pSR4 (kdpFABC), in contrast to that of strain TKW3205/pTM501 (kdpABC), were capable to recover the K ϩstimulated ATPase activity. A determination of the phospholipid content within those fractions revealed that it was below the detection limit. These results provide evidence that the recovery of the ATPase activity is due to the addition of the KdpF peptide.
To verify that the purified peptide is indeed KdpF, a matrixassisted laser desorption/ionization mass spectrometry analysis was carried out of fraction 25 derived from strain TKW3205/ pSR4 (Fig. 9B). With this approach we were able to detect a mass at 3100,7 Da, which corresponds well with the molecular mass of the protonated formyl-KdpF. DISCUSSION A number of membrane-bound complexes that mediate transport of cations across biological membranes in different organisms and tissues have been shown to contain small hydrophobic subunits, like the ␥ subunit of the Na ϩ ,K ϩ -ATPase (46), sarcolipin (47,48), plasma membrane proteolipids 1 and 2 (49), and phospholamban (50). None of these polypeptides, however, is essential for the transport function of the enzyme complexes in vivo. In this study, we report on the smallest protein found to date, KdpF from E. coli, which also belongs to this diverse class of polypeptides. Comparable putative peptides can be found in kdp sequences of other organisms, like Clostridium acetobutylicum (51), Synecchocystis sp. PCC6803 (genome project), or Mycobacterium tuberculosis (genome project).
Deletion of the kdpF gene did not give rise to a phenotype. However, a purified KdpABC complex (lacking KdpF) did not exhibit ATPase activity any more. Addition of purified KdpF to the KdpABC complex restored that activity back to wild type level. These results point to a stabilizing function of KdpF in vitro, which also could by mimicked by high concentrations of lipids. In summary, KdpF seems to function as a specific, stabilizing lipid-like peptide in the Kdp complex.
A comparable stabilizing function has been shown for the ⑀ subunit (55 amino acids) of the methymalonyl-CoA decarboxylase from Veillonella parvula (52). As in the case of KdpF, this subunit is not essential in vivo but stabilizes the enzyme complex in vitro, especially the interaction of the membrane-bound part with the soluble ␣ subunit. The affinity for substrate and inhibitors of a complex missing subunit ⑀ is not impaired (53). Interestingly, the methylmalonyl-CoA-decarboxylase from Propionigenium modestum lacks the ⑀ subunit, leading to a relatively unstable enzyme complex during purification (54). A comparison with the other members of this diverse class revealed that none of the listed peptides functions like KdpF. For subunit IV of the cytochrome c oxidase from Paracoccus denitrificans (55), it has been suggested (i) to act as an assembly factor, (ii) to be required for cofactor insertion, or (iii) to stabilize the complex. Until now, however, none of these functions could be verified (56). Furthermore, this subunit is not involved  from the ion exchange chromatography (corresponding to A and C) visualized by silver staining and the K ϩstimulated activity (activity*) of the KdpABC complex after addition of an aliquot of the corresponding fraction. As standard, the Mark12 (Novex) was used, and the molecular masses of the marker proteins are indicated. The samples used for the recovery of the K ϩ -stimulated ATPase activity were prepared as described under "Experimental Procedures," and the KdpABC complex was purified from strain TKW3205/ pTM501. The ATPase activity was determined in the presence of 1 mM KCl. 100% ATPase activity is correlated to a turnover of 8.9 mol P i ⅐min Ϫ1 ⅐mg Ϫ1 . n.d., not determined. in or altered during electron or proton transfer (57), and it has been proposed that subunit IV is an evolutionary remnant (56). Another group of peptides that influence the activity/affinity of their corresponding eukaryotic enzyme complexes share with KdpF one membrane-spanning domain followed or flanked by hydrophilic regions. However, these similarities are confined to the structural level and could not be extended to the level of the amino acid sequence. The ␥ subunit (58 -61 amino acids) of the Na ϩ ,K ϩ -ATPase was ultimately proven to be part of the complex by Mercer et al. (58). After dissociation of the ␥ subunit the purified complex is still active, and maximal turnover of the Na ϩ ,K ϩ -ATPase is not affected (59). This subunit, which is tissue specifically expressed, seems to affect the external cation-binding site of the transport complex (46). A more drastic effect could be observed for the plasma membrane H ϩ -ATPase of Saccharomyces cerevisiae, where two highly homologous proteolipids (plasma membrane proteolipids 1 and 2; 43 amino acids) modulate the ATPase activity. Deletion of both genes, coding for the two peptides, results in a membrane-bound enzyme complex exhibiting only 50% of the normal ATPase activity (49). Sarcolipin (31 amino acids) (47) and phospholamban (52 amino acids) (60) seem to be regulatory components of the sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA) (48). In both cases the synthesis of these peptides is tissue-specific, and both peptides influence the affinity of the corresponding Ca 2ϩ -ATPase (sarcolipin/SERCA class 1 and phopholamban/SERCA class 2a). Furthermore, phopholamban can be phosphorylated at three different sites and forms pentamers after phosphorylation (for review see Ref. 61). Although sarcolipin and KdpF are similar in size and embedded in the membrane by one membrane-spanning domain, sarcolipin is involved in regulatory control of the SERCA class 1, whereas no regulatory effects have been observed so far for KdpF. In summary, the peptides associated with eukaryotic transporters appear to function as activity/affinity-modulating subunits, whereas the peptides from the prokaryotic transporters, like KdpF, act as stabilizing elements.