©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Membrane Topology of the Carboxyl-terminal Third of the Neurospora Plasma Membrane H-ATPase (*)

(Received for publication, August 16, 1994; and in revised form, December 22, 1994)

Jialing Lin Randolph Addison

From the Department of Biochemistry, University of Tennessee, Health Science Center, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

To localize transmembrane segments in the carboxyl-terminal third of the Neurospora plasma membrane H-ATPase, we constructed fusion proteins on the cDNA level. These contained DNA fragments encoding hydrophilic residues of the amino and carboxyl termini of the H-ATPase with a DNA fragment encoding the putative transmembrane segment. To report translocation into microsomes, a DNA fragment encoding three consensus N-linked glycosylation sites was engineered carboxyl-terminal to the putative transmembrane segment. Fusion proteins were synthesized in a Neurospora in vitro translation system supplemented with homologous microsomes. By the criteria of glycosylation of fusion proteins by microsomes, sedimentation of products with microsomes after alkaline extraction, and analysis of protected fragments generated from proteinase K digestion of integrated products, we localized six transmembrane segments in the carboxyl-terminal third of the H-ATPase. These results support a 10-segment model of the Neurospora H-ATPase.


INTRODUCTION

The plasma membrane electrogenic, proton-translocating ATPase (H-ATPase) (^1)of Neurospora crassa belongs to a family of cation-motive porters called P-type ATPases. A prerequisite for understanding this family of remarkable cation-motive ATPases is knowledge of their membrane topology. This knowledge will contribute significantly to elucidating their structure-function relationships. A major impediment to achieving this goal is the inability to obtain crystals of cation-motive ATPases suitable for x-ray structural analysis. Accordingly, topological models of these ATPases are based almost exclusively on hydropathy plots of their primary sequence. These plots, unfortunately, are ambiguous for regions spanning the membrane, leading to publication of different models for the identical protein. For instance, there are three models of the Neurospora plasma membrane H-ATPase that have eight- (Hager et al., 1986), 10- (Addison, 1986), or 12-transmembrane segments (Rao et al., 1991). Confusion is caused primarily by the fact that hydropathy profiles of the carboxyl-terminal third of this enzyme as well as of other P-type ATPases are not as clear as those of the amino-terminal third. This has made it difficult to predict the number of transmembrane segments in this region. Nevertheless, reliable topological models of these porters are needed to assist in designing and interpreting mutagenesis studies. Without structural data, these studies can provide insights into functionally significant regions of these ATPases. To provide a model of these ATPases, other experimental approaches will be required to verify predicted transmembrane segments.

Toward this goal, biochemical, immunological, and genetic approaches have been used to localize the amino and carboxyl termini of P-type ATPases to the cytosol (Mandala and Slayman, 1989; Reithmeier and MacLennan, 1981). This implies an even number of segments. The best characterized cation-motive porter is the Ca-ATPase of the sarcoplasmic reticulum. The Ca-ATPase has no large exoplasmic (trans) domain. A loop of 20 amino acids between transmembrane segments number 7 and 8 constitutes about 50% of the mass on the exoplasmic side (Clarke et al., 1990). By site-directed mutagenesis of the Ca-ATPase cDNA and expression of the resultant trancripts in an in vivo system, Ca-binding sites were localized to predicted transmembrane segments (Clarke et al., 1989). A low resolution three-dimensional model of the Ca-ATPase constructed from cryoelectron microscopy and helical image analysis supports a 10-segment model of the Ca-ATPase (Toyoshima et al., 1993). Another P-type ATPase, the Mg-ATPase of Salmonella typhimurium, was predicted also to have 10 segments (Smith et al., 1993). In contrast, immunoelectron microscopy studies of the alpha-subunit of the Na,K-ATPase localized three epitopes to the exoplasmic side (Mohraz et al., 1994). Since this was inconsistent with current models, other models were proposed. Image analysis of three-dimensional crystals of the Na,K-ATPase from pig kidney and the Ca-ATPase of sarcoplasmic reticulum suggest that these enzymes have similar three-dimensional structures (Taylor and Varga, 1994). The fact that there are various topological models of this highly conserved protein family of cation-motive ATPases attests to the absence of reliable methods that can predict and verify if a stretch of hydrophobic residues span the membrane.

As an approach toward finding a solution to this problem, we developed an experimental method based on the idea that transmembrane segments function as topogenic signals in the topogenesis of polytopic integral membrane proteins (Lin and Addison, 1994). Using this method, we verified predicted transmembrane segments in the amino-terminal third of the H-ATPase and identified an integration signal that consists of two transmembrane segments (Lin and Addison, 1995). In this report, we use this method to determine the number of transmembrane segments in the carboxyl-terminal third of the H-ATPase. The results support a 10-segment model of the Neurospora H-ATPase (Addison, 1986).


EXPERIMENTAL PROCEDURES

Materials

The ^14C-labeled molecular mass markers were from Life Technologies, Inc. The other reagents were as described previously (Lin and Addison, 1994; 1995).

Plasmid Construction

All constructs contained portions of the cDNA of the Neurospora H-ATPase cloned between HindIII and SacII sites of pBluescript II KS (Stratagene). Constructs were generated by replacing a DNA fragment that codes for transmembrane segment number four (M4) and replacing amino acid residues 322-354 (Lin and Addison, 1995) with DNA fragments that encode putative transmembrane segments of the carboxyl-terminal third of the H-ATPase as predicted by the 10- and 12-segment models (Addison, 1986; Rao et al., 1991). Constructions were done as described previously (Lin and Addison, 1994, 1995). pM7InM8 contains an additional invertase segment (In) inserted between M7 and M8. We replaced the In segment, a BamHI-XbaI fragment, in construct M7 with a BamHI-PstI DNA fragment from M3InM4, which encodes In and a PstI-XbaI DNA fragment from construct M8, which encodes M8 and an In carboxyl-terminal to M8. Additional amino acid residues were introduced by the restriction sites. These were Gly-Ser between M7 and In and Ser-Ala-Val between M8 and the In segment amino-terminal to M8. DNA fragments were generated by polymerase chain reaction (Scharf, 1990) and verified by dideoxyl sequencing.

Other Methods

Translation of the RNA transcripts in the presence of [S]methionine in the Neurospora in vitro system, fractionation of the products on an alkaline sucrose step gradient, digestion of products by proteinase K and by N-glycosidase F, immunoprecipitation of products, and preparation of samples for SDS-polyacrylamide gel electrophoresis were as described (Lin and Addison, 1994; 1995).


RESULTS

We used the polymerase chain reaction in constructing fusion proteins of the Neurospora H-ATPase. The amino and carboxyl termini of the H-ATPase flanked the fusion proteins (Fig. 1). The primary sequence of the putative transmembrane segments was from the 10- and 12-segment models (Addison, 1986; Rao et al., 1991). Carboxyl-terminal to the putative transmembrane segment was placed a segment of yeast invertase with three consensus N-linked glycosylation sites (Taussig and Carlson, 1983) to serve as a reporter of translocation into microsomes. RNA transcripts were generated from DNA constructs using T3 RNA polymerase and were translated in a Neurospora in vitro translation system supplemented with homologous microsomes (Addison, 1987). Integration and translocation events were monitored by sedimentation of products with microsomes after alkaline extraction, formation of glycosylated products, and protection of products from protease digestion. Products were identified by immunoprecipitation using rabbit polyclonal antibodies directed against carboxyl-terminal residues of the H-ATPase.


Figure 1: Constructing fusion proteins. The single-letter code of the amino acids is used. Numbersabove each correspond to its position in the primary sequence of the H-ATPase. Residues marked by an asterisk were introduced by recognition sequences of restriction endonucleases in the primers of the polymerase chain reaction. The yeast invertase fragment is represented by the thickbar with three verticallines, putative N-linked glycosylation sites. Proteins with transmembrane segment are designated pM. The number after M represents the position of the segment in the 10-segment model, except for M7`, which represents the seventh transmembrane segment of the 12-segment model.



Translation and Glycosylation of the Fusion Proteins with One Transmembrane Segment

There are three topological models of the Neurospora H-ATPase that have 8, 10, or 12 segments. The latter is inaccurate in predicting transmembrane segments in the amino-terminal third of the H-ATPase (Lin and Addison, 1994). Nevertheless, this model predicts six transmembrane segments in the carboxyl-terminal third of the H-ATPase, like the 10-segment model. Discrepancy lies in the assignment of two of the six segments. To rigorously examine every stretch of residues capable of serving as a transmembrane segment, we examined the transmembrane segments as predicted by the 12-segment model by our fusion protein method.

Constructs with putative transmembrane segments were designated pM followed by a number indicating the position of the segment within the H-ATPase. The sequential insertion model of polytopic integral membrane proteins predicts that an odd-numbered transmembrane segment initiates translocation and an even-numbered segment stops the translocation process. Therefore, if correctly identified, an odd-numbered segment engineered into constructs should initiate translocation of the invertase segment into the lumen of the microsomes (see Fig. 2).


Figure 2: Fusion proteins membrane topology. Thin lines with N or C represent the amino or carboxyl-terminal hydrophilic residues of fusion proteins, respectively. Rectangular boxes with M or M represent transmembrane segments. The invertase segment is represented by thickbars with three perpendicularlines, putative N-linked glycosylation sites. Smallcircles attached to these lines indicate addition of oligosaccharide chains. Trans and Cis correspond to exoplasmic (lumenal) and cytosolic sides of the membrane, respectively. Depending on the construct, the fusion protein may integrate into microsomes as a type II bitopic integral membrane protein (I) or a polytopic integral membrane protein with the invertase segment on the cis side (II) or on both sides (III) of the membrane.



pM7` contains the putative seventh transmembrane segment of the H-ATPase as predicted by the 12-segment model. This segment includes amino acid residues 660-681 (Rao et al., 1991). These residues were predicted only by the 12-segment model to span the membrane. RNA transcripts for pM7` yielded a product with a M(r) of 34,100 (Fig. 3, lane1). The relative molecular mass of all fusion proteins was about 4,500 higher than expected based on the number of residues contained in each. The reason for this is not apparent. There was no change in the relative molecular mass of pM7` in the absence or presence of microsomes, nor did it sediment with microsomes after alkaline extraction (lanes1-4), suggesting that the sequence for M7` has no topogenic information.


Figure 3: In vitro synthesis of fusion proteins. Transcripts were translated with microsomes (nRM) as indicated. Mixtures were extracted at pH 11.5 and then fractionated on an alkaline sucrose step gradient into soluble fraction (S) and membrane pellet (P) as described under ``Experimental Procedures.'' Oligosaccharide chains were removed by digesting the total (T) reaction mixture with N-glycosidase F (PNGaseF). Products were precipitated by trichloroacetic acid and resolved on 10% polyacrylamide gel with SDS; radioactive proteins were visualized by fluorography. Glycosylated proteins are indicated by an asterisk.



DNA fragments encoding M5 (amino acid residues 688-713) and M7 (amino acid residues 755-779) were also placed in constructs. The residues of M5 are not predicted to span the membrane by the eight-segment model (Hager et al., 1986). In the 10-segment model, M5 and M7 are putative initiate-translocation signals. RNA transcripts of these yielded one product when translated without microsomes (Fig. 3, lane1). When translated with microsomes, two products were observed (lane4). The increase in the relative molecular mass of the slower migrating band, indicated by an asterisk in Fig. 3, is consistent with addition of three N-linked oligosaccharide side chains to the invertase segment and is corroborated by results obtained after incubating products with N-glycosidase F, an amidase that cleaves between the asparagine residues of the glycosylation site and the innermost GlcNAc of oligosaccharide chains. N-Glycosidase F converted the slower migrating product to that observed without microsomes (compare lanes5 and 1). This substantiated the conjecture that the increase in molecular mass was caused by the addition of oligosaccharide chains to the products. The relative quantity of pM10 after treatment with N-glycosidase F was consistently low (lane5). The reason for this is not apparent. M9 (amino acid residues 827-847), a predicted initiate-translocation signal, did not initiate transfer of the carboxyl residues into microsomes (Fig. 3). In the presence of microsomes, pM9 sedimented with microsomes after alkaline extraction (Fig. 3, compare lanes2 and 4), suggesting that it is integrated into microsomes, albeit with a very low efficiency.

When M6 (amino acid residues 721-738) and M8 (amino acid residues 807-826) (Fig. 1), as predicted by the 10-segment model, were engineered into fusion proteins to make pM6 and pM8, the resultant proteins did not associate with microsomes after alkaline extraction (Fig. 3). The residues 807-826 are predicted to span the membrane only in the 10-segment model. In this model, M6 and M8 function as stop-transfer signals. One predicted stop-transfer signal, M10 (amino acid residues 854-878), in pM10 initiated translocation of the carboxyl-terminal residues into microsomes (Fig. 3, lane4).

The alkaline extraction method, used for fractionating the products, converts closed microsomes into opened sheets. Concomitantly, proteins are extracted from the exposed faces (Fujiki et al., 1982). Peripheral membrane and lumenal proteins are found in the supernatant fraction, while integral membrane proteins are found in the membrane pellet. Taken together, this implies that M5, M7, and M10 served not only as signals that initiated translocation of the carboxyl-terminal residues, but also as signals that anchored the proteins in the membrane.

Membrane Topology of the Fusion Proteins

In the presence of microsomes, pM5, pM7, and pM10 were glycosylated. To examine the membrane topology, the products were digested by proteinase K. In the absence of microsomes, no protected fragment was observed (Fig. 4, lane1). In the presence of microsomes, a protected fragment with a relative molecular mass of 27,000 was observed for all constructs (lane2). When Triton X-100 was added to dissolve the protective membrane barrier, no fragment was observed (lane3). The protected fragment could be immunoprecipitated by polyclonal antibodies directed against the carboxyl-terminal residues of the H-ATPase (lane5) but not by antibodies directed against the amino-terminal residues (lane4). This demonstrates that the fusion proteins had assumed the orientation of a type II bitopic integral membrane protein in the microsomes (Singer, 1990) with carboxyl-terminal residues on the exoplasmic (trans) side and the amino-terminal residues on the cytoplasmic (cis) side of the microsomes (see Fig. 2).


Figure 4: Digesting products by proteinase K. RNA transcripts for pM5 (panelA), pM7 (panelB), and pM10 (panelC) were translated in the Neurospora in vitro system supplemented with microsomes (nRM), as indicated. The resultant mixtures were treated with 100 µg/ml of proteinase K. Triton X-100 (TX-100) was present at 1% during the digestion of products for lane3. Samples for lanes1-3 were trichloroacetic acid-precipitated; others were immunoprecipitated using polyclonal antiserum (Ab) against the amino terminus (N) (lane4) or against the carboxyl terminus (C) (lane5) of the H-ATPase. Precipitates were resolved on a 12.5% polyacrylamide gel with SDS; radioactive fragments were visualized by fluorography. Exposure time of data represented in lanes4 and 5 is 3 times longer than that of the data in lanes1-3. Molecular mass markers are ovalbumin, carbonic anhydrase, and beta-lactoglobulin with molecular masses of 43,000, 29,000, and 18,400 Da, respectively.



The smaller fragments observed in Fig. 4lane5 for pM10 were generated during the immunoprecipitation step. pM10 was more susceptible to apparent proteolytic digestion.

Translation of Fusion Proteins with Two Putative Transmembrane Segments

pM7` did not associate with microsomes after alkaline extraction, suggesting that these residues are not embedded in the membrane. It is possible, however, that M7` functions as a topogenic signal with another transmembrane segment. We demonstrated that certain constructs containing a transmembrane segment will not associate with microsomes unless combined with another transmembrane segment (Lin and Addison, 1995). To determine if pM7` had a similar requirement, we constructed pM7`M5. M5 is a presumed initiate-transfer signal in the 10-segment model and represents the eighth transmembrane segment in the 12-segment model. In the latter, these residues function as a stop-transfer signal. Therefore, pM7`M5 should be integrated into the membrane as a polytopic integral membrane protein with the termini and the invertase segment on the cis side of the microsomes (see Fig. 2). When RNA transcripts of this construct were translated with microsomes, this protein was glycosylated (Fig. 5, lane4). The topmost glycosylated product, indicated by an asterisk, had an increase in relative molecular mass of about 6,000, suggesting the addition of three N-linked oligosaccharide chains to the protein. Since pM7`M5 was glycosylated, this suggests that M7` does not span the membrane as suggested by the eight- and 10-segment models. The quantity of glycosylated products in pM7`M5 is less than that observed in pM5 (Fig. 3), suggesting that M7` apparently affected the decoding of M5 in this construct. When M6, a presumed stop-transfer signal in the 10-segment model, was combined with M5 to give pM5M6, this protein was integrated into microsomes as judged by its resistance to alkaline extraction, but it was not glycosylated (lanes3 and 4). Unlike the combination of M7` and M5, these segments functioned as predicted based on their positions in the 10-segment model. When M9 and M10 were combined, these also functioned as predicted based on their positions in the 10-segment model and gave results similar to those observed with pM5M6 (Fig. 5, lanes1-4). When M8 was engineered downstream of M7, glycosylation of the resultant product was reduced but not completely blocked (compare lane4 in Fig. 3with lane4 in Fig. 5). M8 impaired the translocation process initiated by M7, implying that M8 served as a stop-transfer signal. Protease K digestion of membrane integrated forms of pM5M6, pM7M8, and of pM9M10 yielded no protected fragment that could be immunoprecipitated by polyclonal antisera directed against the amino- or the carboxyl-terminal residues (data not shown). This suggests that these proteins were integrated into microsomes with both termini on the cis side of the microsomes.


Figure 5: In vitro synthesis of fusion proteins with two putative transmembrane segments. RNA transcripts were translated in a Neurospora in vitro system supplemented with microsomes (nRM), as indicated. Samples were extracted at pH 11.5 and then fractionated on a sucrose step gradient under alkaline (pH 11.5) conditions into soluble fraction (S) and membrane pellet (P). trichloroacetic acid-precipitated products were resolved on a 10% polyacrylamide gel with SDS; radioactive bands were visualized by fluorography. See text for more details.



To further substantiate the observation that M8 functioned as a stop-transfer signal, we inserted between M7 and M8 the invertase segment with three consensus N-linked glycosylation sites. pM7InM8 has two invertase segments. If M8 functions as a stop-transfer signal, pM7InM8 will be glycosylated three times; if not, it will be glycosylated six times (see Fig. 2). When synthesized with microsomes, pM7InM8 gave four products that sedimented with microsomes after alkaline extraction (Fig. 5, lane 4): The topmost product, indicated by an opendiamond in Fig. 5, had an increase in relative molecular mass of about 15,000 Da, suggesting the addition of six N-linked oligosaccharides chains; the bottommost product is unmodified protein that sediments with microsomes. A second modified product, indicated by an asterisk in Fig. 5, had an increase in relative molecular mass of about 6,000 Da. The third modified product had an increase of relative molecular mass of 4,000 Da. These products could represent partially glycosylated pM7InM8 that had both invertase segments in the lumen of microsomes. Alternatively, this profile of modified proteins would reflect a mixture of combinations. For example, products with relative increase of molecular mass of 6,000 and 4,000 Da could represent pM7InM8 that had one invertase segment in the lumen of microsomes, and the product that had a relative increase in molecular mass of about 15,000 Da could represent pM7InM8 with both invertase segments in lumen of microsomes. To distinguish between these possibilities, the mixture was digested by proteinase K. Results are depicted in Fig. 6. In the absence of microsomes, no protected fragment was observed (lane1); in the presence of microsomes, two major and one minor protected fragments were observed (lane2). Only the protected fragment with a molecular mass of about 40,000 Da was immunoprecipitated by polyclonal antibodies against carboxyl-terminal residues of the H-ATPase (lane5). The other fragments were not immunoprecipitated by antibodies against either the amino- or the carboxyl-terminal residues of the H-ATPase (lanes4 and 5). This implies that pM7InM8 had two orientations in the membrane. One product had the orientation of a type II bitopic integral membrane protein; the other had that of a polytopic integral membrane protein. This demonstrates that M8 functioned as a weak stop-transfer signal.


Figure 6: Digesting pM7InM8 by proteinase K. pM7InM8 was synthesized in the in vitro system supplemented with microsomes (nRM), as indicated. The resultant mixtures were digested with 100 µg/ml of proteinase K. For sample in lane3, Triton X-100 (TX-100) was present at 1% during the digestion. Samples of lanes1-3 were trichloroacetic acid-precipitated; others were immunoprecipitated with polyclonal antiserum (Ab) against the amino terminus (N) (lane4) or the carboxyl terminus (C) (lane5) of the H-ATPase. Precipitates were resolved on 12.5% polyacrylamide with SDS; radioactive bands were visualized by fluorography. Exposure time of data in lanes 4 and 5 is 5 times that of the data in lanes1-3. Molecular mass markers are identical to those of Fig. 4.




DISCUSSION

The results described herein support the transmembrane segments as assigned by the 10-segment model (Addison, 1986). A membrane topological model of the H-ATPase is depicted in Fig. 7. Numbers within boxes represent the putative boundaries of the transmembrane segments. The amino acid residues 688-713 were not predicted to span the membrane in the eight-segment model (Hager et al., 1986). The fact that pM5 formed a type II integral membrane protein demonstrated that these residues can serve as a transmembrane segment. pM7` did not interact with microsomes, nor did M7`, when combined with M5, form an integration signal. In light of these results, M7` is not a transmembrane segment.


Figure 7: Model of the Neurospora plasma membrane H-ATPase. This model is based on data presented herein and on previously published data as mentioned in the text. Rectangularboxes represent transmembrane segments (M). Boundaries of transmembrane segments are based on the amino acid sequences used in the fusion proteins. Trans and Cis correspond to exoplasmic and cytoplasmic sides of the membrane, respectively. Length of lines is not proportional to the number of amino acids predicted to lie therein.



M7 functioned as the strongest of the sequences that initiated translocation of carboxyl-terminal residues of the fusion protein into microsomes. The fact that M8 impaired translocation initiated by M7 suggests that M8 functioned as a stop-transfer signal. M8 (amino acid residues 807-826) is predicted to span the membrane only in the 10-segment model. No protected fragments were observed after digesting membrane integrated forms of pM5M6, pM7M8, and of pM9M10 by proteinase K that could be immunoprecipitated by antibodies directed against the amino- or carboxyl-terminal residues. This is possible only if the proteins were integrated into microsomes as polytopic integral membrane proteins (see Fig. 2).

The notion that M8 is a transmembrane segment can also be inferred from experimental observations on the Ca-ATPase of sarcoplasmic reticulum. First, site-directed mutagenesis studies on the Ca-ATPase identified Glu-908, within M8 of the Ca-ATPase, as a ligand-binding site (Clarke et al., 1989). This residue is conserved among the cation-motive ATPases. In the Neurospora H-ATPase, this residue is Glu-805. In the 10-segment model, the amino-terminal boundary of M8 is two residues carboxyl-terminal to Glu-805. If this residue has a function similar to that of Glu-908 in the Ca-ATPase, the boundary of M8 must extend beyond what was originally proposed. pM7M8 was engineered to contain Glu-805 and all native residues of the H-ATPase between M7 and M8 (see Fig. 1). In constructing pM7InM8, we used, however, amino acid residues 807-826 for M8. These residues may represent only a portion of the actual M8. If so, this would explain why M8 in pM7InM8 functioned as a weak stop-transfer signal. The fusion protein method can not resolve boundaries of transmembrane segments. Therefore, another approach will be required before it can be stated with certainty which amino acids form the boundaries of M8.

Second, the largest exoplasm domain in the Ca-ATPase exists between M7 and M8. Only in the 10-segment model of the Neurospora H-ATPase will a similar loop exist. Taken together, this supports the conjecture that M8 is a transmembrane segment.

Of the transmembrane segments predicted as stop-transfer signals, only M10 initiated translocation. This is not caused simply by hydrophobicity, for hydropathy plots generated by the algorithm of Eisenberg et al.(1984) predict M4 as the most hydrophobic of the presumed stop-transfer signals; pM4 did not associate with microsomes after alkaline extraction (Lin and Addison, 1995). After surveying the literature of this field, we found no satisfactory explanation as to why a predicted stop-transfer signal functions to initiate translocation. It is clear that as yet we have no insight into the intrinsic property of a stretch of hydrophobic residues making it function as a stop-transfer or a start-transfer signal.

Any putative transmembrane segment containing a negatively charged amino acid did not function when engineered in the fusion proteins. These are M1, M2, M4, M6, and M9 (Lin and Addison(1995) and this report). M5 contains a negatively and a positively charged residue seven residues apart. This segment initiated translocation of the invertase segment into microsomes. It is possible that the position of these charged residues allowed for formation of a salt bridge that masked their charges, thus creating a functional topogenic signal. M10 and M8 each contains a positively charged residue. M10 initiated translocation of the carboxyl-terminal residues into microsomes; M8 did not. M8 also contains two proline residues. Apparently, the combination of charged and proline residues impaired the ability of M8 to function individually in the construct (Emr and Silhavy, 1983). Pairing of transmembrane segments in the H-ATPase will juxtapose only two pair of segments with opposite charged residues. A simple, planar model of these segments positioned the charged residues near the ends of each pair. For example, in the pair M9 and M10, the negatively charged residue in M9 is positioned near the cis side of the membrane, while the positively charged residue in M10 is near the trans side. Clearly, the ion-pairing of segments cannot be the basis of the driving force to forming the putative paired segments.

A report on the topological analysis of the H,K-ATPase (Bamberg and Sachs, 1994) used a method similar to the one developed in this laboratory to identify transmembrane segments of this P-type ATPase. The results were consistent with a 10-segment model. In this ATPase, certain transmembrane segments did not function as predicted by the sequential insertion model of polytopic integral membrane proteins. For example, M5 of the H,K-ATPase, a presumed initiate-transfer signal, did not initiate translocation of the carboxyl-terminal residues of the fusion protein into the lumen of microsomes. Similar observations were reported for other polytopic integral membrane proteins (Audigier et al., 1987; Ebele et al., 1987; Sengstag et al., 1990). It is assumed, without any experimental data, that in the topogenesis of P-type ATPases and perhaps of other porters, transmembrane segments are decoded individually. Therefore, segments not behaving as predicted by the sequential insertion model are posited to be integrated into membrane by another mechanism. If this notion is correct, a particular polytopic integral membrane protein would use multiple integration mechanisms and perhaps multiple integration machineries to obtain its membrane topology. While this explanation may be correct, it should be kept in mind that it assumes that each transmembrane segment is decoded individually. This assumption is not valid if transmembrane segments acted concertedly as they are integrated into the membrane. For instance, when assayed individually, M9 and M10 did not function as predicted by the sequential insertion model. When these segments were combined, pM9M10 was integrated into microsomes as a polytopic integral membrane protein, suggesting that M9 and M10 acted concertedly in the integration event. Since transmembrane segments of the Neurospora H-ATPase are separated by a few residues, it is possible that closely juxtaposed segments are decoded as a unit and are then integrated into the membrane (Lin and Addison, 1995).

A combination of M5, M6, and M7 from the carboxyl-terminal third of the H,K-ATPase could not integrate co-translationally into microsomes (Bamberg and Sachs, 1994). This phenomenon was not observed with segments from the Neurospora H-ATPase. Fusion proteins of the H-ATPase with various combinations of transmembrane segments were all integrated co-translationally into microsomes (data not shown). This apparent discrepancy between the systems may be explained on the basis of differences in the in vitro systems. We investigated integration of transmembrane segments from the Neurospora H-ATPase into their cognate membrane. In the analysis of the H,K-ATPase, an in vitro system composed of heterologous components was used. Also, we engineered carboxyl-terminal to the putative transmembrane segment a segment of invertase, a secretory protein, with the amino and carboxyl termini of the H-ATPase flanking the fusion proteins. In constructing the fusion proteins of the H,K-ATPase, the carboxyl terminus of the fusion proteins is a portion of the beta-subunit of the H,K-ATPase engineered carboxyl-terminal to the putative transmembrane segments. The function of the beta-subunit is unknown. It may participant in the topogenesis of the alpha-subunit. If so, it is possible the beta-subunit has specific interactions with certain transmembrane segments of the alpha-subunit. This is another potential factor that could explain the differences between the systems. Clearly, in constructing fusion proteins, a reporter sequence should be chosen that has no potential role in the topogenesis of the protein under study. The combined effects of a heterologous in vitro system and the use of beta-subunit in the fusion proteins can probably account for the apparent differences in the behavior of the transmem-brane segments of the P-type ATPases observed in the two laboratories.

The major assumption for the basis of the experiments described herein is that transmembrane segments in fusion proteins mimic their behavior in the native protein. In view of the similarities of the Neurospora H-ATPase to the other P-type ATPases, it can be anticipated that what is learned about the integration mechanism of the Neurospora H-ATPase will contribute to our understanding of the integration mechanism of P-type ATPases in general.


FOOTNOTES

*
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.

(^1)
The abbreviation used is: H-ATPase, plasma membrane electrogenic, proton-translocating ATPase.


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