©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Integration Signal That Is Composed of Two Transmembrane Segments Is Required to Integrate the Neurospora Plasma Membrane H-ATPase into Microsomes (*)

(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, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Neurospora plasma membrane H-ATPase belongs to a family of cation-motive porters called P-type ATPases. Putative transmembrane segments of these enzymes contain one or more charged residues. Conditions were determined by which a transmembrane segment with charged residues is integrated into its cognate membrane. We constructed fusion proteins flanked by the hydrophilic domains of the amino and carboxyl termini of the H-ATPase that contained either one or two transmembrane segments. Neurospora in vitro translation system supplemented with homologous microsomes was programmed with RNA transcripts of these constructs. When transmembrane segment number one (M1) or number two (M2) of the H-ATPase was engineered into the construct, the resultant protein did not integrate into microsomes. When M1 and M2 were placed in tandem, the resultant protein integrated into microsomes as judged by the criteria of resistance to extraction at pH 11.5 and protection from protease digestion. The integration event depended on ATP and GTP and on microsomal protein(s). We posited that membrane topology of the amino-terminal third of the H-ATPase, and perhaps of other P-type ATPases is achieved by inserting transmembrane segments into membrane in pairs.


INTRODUCTION

The Neurospora plasma membrane electrogenic, proton-translocating ATPase (H-ATPase) (^1)transduces the chemical energy of ATP hydrolysis into a transmembrane proton-motive force (Addison and Scarborough, 1981). The latter functions to drive the uptake and extrusion of various ions and molecules through chemiosmotic coupling devices called porters (Mitchell, 1967). The Neurospora H-ATPase belongs to a family of P-type cation-motive ATPases that includes the Na/K, the H/K, and the Ca motive ATPases from higher eukaryotic cells (Addison, 1986). We know a great deal about the reaction mechanisms and the physiological roles of these cation-motive ATPases. Their primary structure is known mainly because of the methods of gene cloning and DNA sequencing. Yet little is known about the structure-function relationships of these remarkable energy transducers.

Knowledge of the membrane topology of P-type ATPases is essential to gaining insights into their structure-function relationships. Such knowledge, unfortunately, is not easy to obtain. Membrane topological models of P-type ATPases are based almost exclusively on hydropathy analysis of their primary structure. There is confusion about the actual number of transmembrane segments. Most agree these enzymes contain either 8 or 10 segments. Because of these uncertainties, a method is needed to verify if a predicted segment is within the membrane. One method is to study the topogenesis of P-type ATPases. The molecular mechanisms by which polytopic integral membrane proteins achieve their membrane topology are poorly understood, however. Topogenesis of membrane proteins is presumed to occur by sequential insertion of topogenic signals into the endoplasmic reticulum (Blobel, 1980). Transmembrane segments serve as the topogenic signals; odd-numbered segments initiate translocation of the carboxyl-terminal residues across the membrane, while the even-numbered segments stop the translocation process.

Using the Neurospora H-ATPase as a model P-type ATPase, we are investigating the integration of this P-type ATPase into its cognate membrane (Addison, 1990, 1991, 1993; Lin and Addison, 1994). We constructed fusion proteins flanked by hydrophilic amino acids of the amino and carboxyl termini of the H-ATPase containing putative transmembrane segments. RNA transcripts of these constructs were translated in a Neurospora in vitro system supplemented with homologous microsomes (Addison, 1987). The results demonstrate that transmembrane segments of Neurospora H-ATPase, when assayed out of context, have various degrees of effectiveness as a topogenic signal and provide functional evidence for a novel integration signal that is composed of two transmembrane segments. In considering the results obtained, a model is presented of how the amino-terminal third of the H-ATPases achieves its membrane topology.


EXPERIMENTAL PROCEDURES

Materials

The oligonucleotide primers for the polymerase chain reactions (PCR) were synthesized at the Molecular Resource Center of the St. Jude Children's Research Hospital (Memphis, TN). Restriction endonucleases, mung bean nuclease, T4 DNA ligase, Klenow fragment, and N-glycosidase F were from New England Biolabs. pBluescript II KS, T3 RNA polymerase, and RNase Block I were from Stratagene (La Jolla, CA). Proteinase K, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, and soybean trypsin inhibitor were from Worthington (Freehold, NJ). Phenylmethylsulfonyl fluoride and emetine were from Sigma. L-[S]Methionine (1,120 Ci/mmol) was from DuPont NEN. Bio-Gel P-4 (200-400 mesh) was from Bio-Rad (Richmond, CA). The molecular mass markers were from Bio-Rad or Life Technologies, Inc. The other reagents were of the highest grade commercially available.

Constructing Plasmids

All constructs contained portions of the Neurospora H-ATPase gene cloned between the HindIII and SacII sites of pBluescript II KS (Stratagene). As illustrated in Fig. 1, the control protein (pDeltaM) was encoded from a DNA construct containing 1) a DNA fragment containing 170 bp of 5`-untranslated sequence and 345 bp encoding amino acid residues 1-115 of the H-ATPase; 2) a 12-bp fragment containing PstI-BamHI restriction sites encoding Pro-Ala-Gly-Ser; 3) an 180-bp fragment of the yeast invertase (In) gene encoding residues 73-132 of the invertase precursor, a stretch that contains three N-linked glycosylation sites; 4) a 6-bp fragment containing a XbaI restriction site encoding Ser-Arg; and 5) a 144-bp fragment encoding the last 42 amino acid residues of the H-ATPase followed by a stop codon and 15 bp of 3`-untranslated sequence. For pM1, pM2, pM3, pM4, and pM3M4, additional fragments of the H-ATPase gene, were inserted into(2) , as illustrated in Fig. 1. For pM1M2, the fragment (1) of the DNA construct for pDeltaM was replaced by a 659-bp fragment containing 170-bp of 5`-untranslated sequence and 489 bp encoding amino acid residues 1-163 of the H-ATPase. For pM1M4, the fragments (1) and (2) of the DNA construct for pM4 were replaced by a 599-bp fragment containing 170 bp of 5`-untranslated sequence and 429 bp encoding amino acid residues 1-143 of the H-ATPase.


Figure 1: Constructing fusion proteins. Single-letter code is used for the amino acids. Numbersabove each correspond to its position in the primary sequence of the H-ATPase. Residues marked by an asterisk were introduced with restriction endonuclease recognition sequences of oligonucleotide primers used in PCR. Protein containing transmembrane segment was designated pM. Number following M represents the position of the segment in the H-ATPase. The yeast invertase fragment is represented by the thickerbar with verticallines, putative glycosylation sites, in pDeltaM.



pM1InM2 was made by inserting between the DNA fragments (2) and (3) of the construct for pDeltaM a 69-bp fragment encoding amino acid residues 116-138 (M1) of the H-ATPase and between DNA fragments (3) and (4) a 60-bp fragment encoding amino acid residues 141-160 (M2) of the H-ATPase. pM3InM4 was made by inserting between the DNA fragments (2) and (3) of the construct for pDeltaM a 69-bp fragment encoding amino acid residues 292-314 (M3) of the H-ATPase and between the DNA fragments (3) and (4) a 69-bp fragment encoding amino acid residues 322-354 (M4) of the H-ATPase. The overhang ends of DNA fragments were either filled-in using Klenow fragment or deleted using mung bean nuclease to maintain the correct reading frame of some inserts. All constructions introduced DNA fragment encoding amino acid residues Gly-Ser-Ile at the 3` end of M2 and M4, DNA fragment encoding Ser-Ala-Glu at the 5` end of M2, and DNA fragment encoding Ser-Ala-Ala at the 5` end of M4.

For the energy-dependent studies, we used RNA transcripts transcribed from DNA constructs for pM1M2 and pM3M4 without a stop codon based on the reason described under ``Results.'' These were generated by replacing DNA fragment (5) in the constructs with a 126-bp fragment that codes for the last 42 amino acid residues of the H-ATPase. The recombinant plasmids were restricted with PvuII. The resultant transcripts contained no stop codon and encoded an additional 45 amino acid residues derived from the plasmid.

For all constructs, DNA fragments that encoded for the amino acids indicated in Fig. 1by asterisks were introduced with the oligonucleotide primers for the PCR. The single strand DNA that served as template for the PCR was generated from the recombinant phagemid pBluescript II KS as described by the supplier (Stratagene, La Jolla, CA). The PCR products were generated using the Perkin Elmer's GeneAmp kit and DNA Thermal Cycler 480 according to protocols from supplier (Perkin Elmer). Primers were removed from the PCR mixtures using the PrimeErase Quick push column from Stratagene. The resultant products were digested with restriction endonucleases. The resultant mixtures were resolved on an agarose gel; the product of the expected size was excised and eluted from the gel using Glassmilk or Glassfog (Bio 101, La Jolla, CA). The PCR product was cloned into the plasmid as outlined above. All plasmid constructs were verified by dideoxy sequencing (Sanger et al., 1980).

Translating RNA Transcripts

Purified plasmids were linearized with PvuII and then transcribed by T3 RNA polymerase as described by the supplier (Stratagene). The resultant transcripts were translated in a Neurospora in vitro system in the presence or absence of homologous microsomes (Addison, 1987). Afterward, reactions were placed on ice and then were either treated with proteinase K or fractionated into a soluble fraction and a membrane pellet, as described below.

Digesting Proteins with Proteinase K

Translation mixture (20 µl) was adjusted to 10 mM CaCl(2), 150 or 300 µg/ml proteinase K and incubated for 1 h at 0 °C. The digestion was terminated by adding phenylmethylsulfonyl fluoride to a final concentration of 25 mM from a 0.5 M stock in Me(2)SO and then adding an equal volume of 25% trichloroacetic acid. Where indicated, Triton X-100 was present at 1% (v/v). Samples were prepared for urea-SDS-polyacrylamide gel electrophoresis (PAGE) as described previously (Burr and Burr, 1983), except that the resolving gel was 15% acrylamide and 0.13% bisacrylamide with a 5% stacking gel that was made from a stock solution of 30% acrylamide, 0.8% bisacrylamide without urea; the sample disaggregation buffer contained 15 mM phenylmethylsulfonyl fluoride.

Other Methods

Methods of N-glycosidase F digestion, immunoprecipitation, and SDS-PAGE and subsequent fluorography of the dried gels were as described previously (Lin and Addison, 1994). Microsomes were digested with trypsin as described by Addison(1993). Five µl of the trypsin- or mock-treated microsomes were added co-translationally to the 20-µl translation mixture. Mock-treated microsomes were obtained by adding premixed trypsin, soybean trypsin inhibitor, and phenylmethylsulfonyl fluoride to microsomes and were incubated similarly as the trypsin-treated microsomes. For fractionation on sucrose step gradient, an equal volume of 0.2 M Na(2)CO(3), pH 11.5, was added to the translation mixture and then fractionated as described (Lin and Addison, 1994). An estimate of the relative quantity of integrated or glycosylated protein was performed by densitometric analysis of autoradiograms by a Bio Image Radioanalytic Imaging System (MilliGen/Biosearch, Bio Image, Ann Arbor, MI).


RESULTS

Topological models of the Neurospora plasma membrane H-ATPase are based mainly on hydropathic plots of the primary sequence that predict either eight (Hager et al., 1986), 10 (Addison, 1986), or 12 (Rao et al., 1991) transmembrane segments. This method predicts transmembrane segments by scanning the primary sequence for stretches of hydrophobic residues long enough to span the lipid bilayer as alpha-helices. The eight- and 10-segment models agree that the amino-terminal third of the H-ATPase contains four transmembrane segments. The relative position of these transmembrane segments is supported by trypsin digestion of the reconstituted Neurospora H-ATPase (Rao et al., 1991). Integration of the H-ATPase and truncated forms of the H-ATPase into microsomes suggests the presence of internal signal sequences (Addison, 1990, 1991). Also, a fusion protein with transmembrane segment number 3 (M3) of the H-ATPase integrated into microsomes as a type II integral membrane protein (Lin and Addison, 1994). The objective of this report is to determine how M1, M2, and M4 of the H-ATPase are integrated into the membrane.

Using the PCR (Scharf, 1990), fusion proteins were constructed containing sequences of putative transmembrane segments. The hydrophilic amino acid residues of the amino and carboxyl termini of the H-ATPase flanked the fusion proteins (Fig. 1). Carboxyl-terminal to the putative transmembrane segment was engineered a segment of invertase that contains three consensus N-linked glycosylation sites (Taussig and Carlson, 1983) to serve as a reporter of translocation. In constructing the fusion proteins, we used primary sequence of transmembrane segments as predicted by the 10-segment model (Addison, 1986). Complementary DNA of fusion proteins was transcribed in vitro by T3 RNA polymerase; the resultant transcripts were translated in a Neurospora in vitro system with or without homologous microsomes. The proteins were identified by immunoprecipitation using rabbit polyclonal antibodies directed against carboxyl-terminal residues of the H-ATPase. Integration of proteins into microsomes was determined by monitoring formation of glycosylated products, cosedimentation of products with microsomes after alkaline treatment, and digestion of products by protease.

Integrating Proteins with One Transmembrane Segment into Microsomes

In the absence of microsomes, RNA transcripts for pDeltaM, pM1, pM2, pM3, or pM4 yielded one product (Fig. 2, lane1). The control protein (pDeltaM) had a M(r) of 34,200 when resolved by SDS-PAGE (Fig. 2, lane1). The relative molecular mass of all fusion proteins was about 4,500 above what was expected based on the number of residues found in each. The exact reason for their aberrant migration in the SDS-polyacrylamide gel is not apparent. In the presence of microsomes, there was no change in the M(r) of pDeltaM (Fig. 2, lane3), nor did pDeltaM cosediment with microsomes (Fig. 2, lane4). These results are consistent with models that predicted no transmembrane segment within the first 115 nor the last 42 amino acid residues of the H-ATPase.


Figure 2: Translation and membrane integration of in vitro synthesized [S]methionine-labeled pDeltaM, pM1, pM2, pM3, and pM4. Transcripts were translated in Neurospora in vitro system with microsomes (nRM) at 3 A units/ml as indicated. Translation mixtures were extracted with 0.1 M Na(2)CO(3), pH 11.5, and fractionated into soluble (S) fraction and membrane pellet (P) as described under ``Experimental Procedures.'' Proteins were resolved on 10% polyacrylamide gel with SDS and visualized by fluorography. Glycosylated protein is indicated by an asterisk.



In the presence of microsomes, there was no change in the relative molecular mass of pM1, pM2, or pM4 (Fig. 2, lanes1 and 3) as well as no significant increase in the quantity of proteins found in the pellet fraction after alkaline treatment (Fig. 2, lanes2 and 4). In the absence of microsomes, a small percentage of product is found in the pellet fraction (lane2). This is caused most likely by aggregate forms of the products that sediment under these conditions (Addison, 1990). Fractionation of products using sucrose step gradients containing 0.15 M KOAc gave similar results (data not shown), precluding the possibility that pM1, pM2, and pM4 were bound to but not integrated into microsomes. Only pM3 showed a change in its relative molecular mass in the presence of microsomes; one product had a M(r) of 36,100, and the other had a M(r) of 42,700 (Fig. 2, lane4). All of the M(r) 42,700 product and about 60% of the M(r) 36,100 product were found in the pellet fraction after extracting the microsomes with 0.1 M Na(2)CO(3), pH 11.5, and then fractionating the products through a sucrose step gradient containing 0.1 M Na(2)CO(3), pH 11.5 (Fig. 2, lanes3 and 4). Alkaline extraction converts closed microsomes into opened sheets concomitantly extracting proteins from the exposed faces of the sheets (Fujiki et al., 1982). Peripheral membrane and lumenal proteins are found in the supernatant, but integral membrane proteins sediment with the sheets or membrane remnants. When the translational mixture with pM3 was treated with N-glycosidase F, an amidase that cleaves between the innermost GlcNAc of oligosaccharide chains and the asparagine residue, only the M(r) 36,100 product was observed on SDS-PAGE (data not shown). This demonstrated that the increase in the relative molecular mass of pM3 was caused by adding N-linked oligosaccharide side chains to the invertase segment as it was translocated into the lumen of the microsomes.

Integrating Proteins with Two Transmembrane Segments into Microsomes

The sequential insertion model predicts that M1, like M3, should initiate translocation of the carboxyl-terminal residues of the fusion protein. M1 did not, implying that it is not a topogenic signal. Indeed, results with pM1 and pM2 were unexpected, for a truncated H-ATPase containing M1 and M2 was integrated into microsomes in earlier experiments (Addison, 1993). This implied the presence of a topogenic signal. Maybe M1 and M2 do serve as topogenic sequences, but only with native flanking sequences. To evaluate this possibility, we made a protein containing M1 and M2 (Fig. 1).

In the absence of microsomes, the transcripts for pM1M2 yielded one product (Fig. 3, lane1). In the presence of microsomes, approximately 50% of the product cosedimented with microsomes after alkaline extraction (Fig. 3, lanes3 and 4). The presence of pM1M2 in the pellet fraction implies that this protein has topogenic information for integration. Less than 10% of pM1M2 is glycosylated by microsomes. When we engineered about 20 additional native residues carboxyl-terminal to M2 in pM1M2, glycosylation of the resultant protein by microsomes was not observed (data not shown). Clearly, combining M1 and M2 created a topogenic signal. Joining M1 and M2 may have created a stretch of hydrophobic amino acid residues. In the primary sequence of M1 and M2 (Fig. 1), the longest continuous stretch of hydrophobic amino acids is 10, and this is located in M2. As demonstrated, pM2 did not sediment with microsomes after alkaline extraction (Fig. 2). Alternatively, M1 and M2 may have created a structure that served as the topogenic signal. If so, this implies a crucial role for the proximity of the transmembrane segments. To disrupt this putative structure of the transmembrane segments, we inserted the invertase segment (see Fig. 1) between M1 and M2. Integration effectiveness of the resultant protein pM1InM2 was reduced to 20% of that of pM1M2 (Fig. 3, lanes3 and 4), and it did not become glycosylated. This was interpreted to imply that the topogenic information for integration is a function of the proximity of the transmembrane segments.


Figure 3: Translation and membrane integration of in vitro synthesized [S]methionine-labeled pM1M2, pM3M4, pM1InM2, pM3InM4, and pM1M4. Transcripts were translated with microsomes (nRM) as indicated. Mixtures were extracted with 0.1 M Na(2)CO(3), pH 11.5, and fractionated into soluble (S) fraction and membrane pellet (P) as described under ``Experimental Procedures.'' Proteins were resolved on 10% polyacrylamide gel with SDS and visualized by fluorography. Glycosylated protein is indicated by an asterisk.



pM4 did not sediment with microsomes after alkaline extraction. M4 is a presumed stop-transfer signal. To evaluate this possibility, we constructed pM3M4 (Fig. 1). M4 partially blocked transfer of the invertase segment into microsomes (compare Fig. 3, lane4, with Fig. 2, lane4). To make charge differences in the residues flanking the segments in pM3M4 similar to that of the native protein, hydrophilic residues flanking the segments were engineered into the construct (see Fig. 1). Despite this precaution, about 30% of pM3M4 had an increase in the relative molecular mass of 2,400, suggesting the addition of one N-linked oligosaccharide side chain to the invertase segment. It is not apparent why only one site in the invertase segment was glycosylated. The appearance of glycosylated pM3M4 demonstrated that the four lysines carboxyl-terminal to M4 (Fig. 1) could not completely block transfer of the invertase segment into the lumen of microsomes. When 29 native residues (residues 364-392 of the H-ATPase) were engineered carboxyl-terminal to M4 in pM3M4, the resultant protein was still glycosylated. When native residues 262-283 were engineered amino-terminal to M3 and residues 364-392 carboxyl-terminal to M4, the resultant protein was not glycosylated (data not shown). This was interpreted to suggest that factors other than difference in the charge of residues flanking the transmembrane segments and the presence of positively charged residues dictate orientation of segments in the membrane. These results were unexpected and apparently add an additional level of complexity as to the nature of the factors controlling orientation of transmembrane segments in the membrane.

Although M4 did not function independently, it functioned as a stop-transfer signal when engineered carboxyl-terminal to M3. Did the activity of M4 in pM3M4 depend on its proximity to M3? To evaluate this, we inserted the invertase segment between the segments in pM3M4 to give pM3InM4. In the presence of microsomes, pM3InM4 gave a slower migrating product on SDS-PAGE with a M(r) of 44,200 that sedimented with microsomes after alkaline extraction (Fig. 3, lanes3 and 4). N-glycosidase F converted the slower migrating product to a product with M(r) of 38,100 (data not shown), implying that the invertase segment was glycosylated. To determine if M4 blocked transfer of the carboxyl terminus into the lumen of the microsomes, pM3InM4 was digested by proteinase K in the absence or presence of microsomes (Fig. 4). The protected fragment observed only when microsomes were present in the mixture was not immunoprecipitated by polyclonal antibodies directed against the amino or the carboxyl-terminal residues (Fig. 4, lanes4 and 5). These results demonstrate that pM3InM4 was integrated into microsomes as a polytopic IMP, implying that the decoding of M4 in pM3InM4 is independent of its proximity to M3.


Figure 4: Analysis of pM3InM4 by proteinase K digestion. pM3InM4 was synthesized in the absence or presence of microsomes (nRM) and then digested by proteinase K at 150 µg/ml as described under ``Experimental Procedures.'' Triton X-100 (TX-100) was present at 1% during the proteinase K (lane3). Digested mixtures were either precipitated by trichloroacetic acid (lanes1-3) or immunoprecipitated by polyclonal antibodies (Ab) directed against amino (N, lane4) or carboxyl (C, lane5) terminal residues. Molecular mass markers are hen egg white ovalbumin, bovine carbonic anhydrase, and soybean trypsin inhibitor with M(r) of 42,700, 31,000, and 21,500, respectively. Precipitates were resolved on 15% polyacrylamide gel with SDS; product was visualized by fluorography.



The integration efficiency of pM1M2 was reduced by inserting amino acids between the segments. This suggests that decoding the segments in pM1M2 is a function of the proximity of M1 and M2. Does the putative association of M1 and M2 represent specific interactions between these segments? Can another segment in the proximity of M1 create the structural motif that is decoded as an integration signal? To answer this question, we replaced M2 with M4 to give the resultant protein pM1M4 (Fig. 1).

Without microsomes in the translation mixture, transcripts for pM1M4 yielded one product (Fig. 3, lane1). With microsomes in the translation mixture, there was no change in the relative molecular mass of pM1M4, but about 40% of the product sedimented with microsomes after alkaline treatment (Fig. 3, lanes3 and 4). This implies that M4, like M2, could form with M1 the structural motif that functioned as a topogenic signal, albeit not as effectively.

Protease Protection Analysis

Resistance of the proteins to extraction by 0.1 M Na(2)CO(3), pH 11.5, suggests that these had become integral components of the microsomes. To further test this, proteins were digested with proteinase K in the absence or presence of microsomes. Membrane-integrated transmembrane segments should resist digestion by protease. Indeed, proteinase K digestion of pM1M2 in the presence of microsomes gave a fragment with a relative molecular mass of about 5,000 (Fig. 5, lane2). The number of residues in M1M2 is 45. This should give a fragment of about M(r) 5,700. The size of the protected fragment is close to this expected value. This fragment was not immunoprecipitated by polyclonal antibodies against either the amino- or carboxyl-terminal residues of the H-ATPase (data not shown). This implies that the termini were exposed to the cytosol, suggesting that pM1M2 was integrated into microsomes as a polytopic integral membrane protein. No protected fragment was observed for pM1M2 in the absence of microsomes (Fig. 5, lane1), supporting the conjecture that the observed protected fragment (lane2) contains the transmembrane segments. In the presence of Triton X-100 (a nondenaturing detergent) (lane3), approximately 20% of the fragment resisted digestion by proteinase K. This protection is apparently caused either by phospholipids that are tightly associated with the transmembrane segments or by the formation of transmembrane segments-Triton X-100 complex (Li and Deber, 1993).


Figure 5: Digesting pM1M2 and pM3M4 with proteinase K. RNA transcripts for pM1M2 and pM3M4 were translated in the in vitro system with microsomes (nRM) as indicated. pM1M2 or pM3M4 was digested with 300 or 150 µg/ml of proteinase K, respectively, as outlined under ``Experimental Procedures.'' Triton X-100 (TX-100) was present at 1% during the proteinase digestion (lanes3 and 6). The molecular mass markers are bovine trypsin inhibitor with a M(r) of 6,200 and insulin with a M(r) of 3,000. Proteins were resolved on a 15% polyacrylamide gel containing 8 M urea and 0.1% SDS; radioactive bands were visualized by fluorography.



When digested by proteinase K in the absence or presence of microsomes, pM3M4 gave similar results (Fig. 5, lanes4-6). The protected fragment had a relative molecular mass of 7,000 (Fig. 5, lane5). The number of residues in M3M4 is 63. The expected relative M(r) is about 8,000. The observed relative M(r) of the protected fragment is close to the expected M(r). This protected fragment could not be immunoprecipitated by polyclonal antibodies against amino- or carboxyl-terminal residues (data not shown). As observed in Fig. 3, about 25% of pM3M4 is glycosylated. Therefore, we expected to find a protected fragment of about M(r) 20,000. No protected fragment, however, with this M(r) was observed. The inability to detect a protected fragment of this size in the autoradiograms could be caused by the fact that the quantity of proteinase K protected glycosylated product is beyond the limit of detection by autoradiography.

Integration Is a Function of Microsomal Protein(s) and Chemical Energy

Is integration of the fusion proteins into microsomes dependent on microsomal proteins? Is integration into microsomes spontaneous? Is chemical energy required for integrating transmembrane segments into microsomes? To answer these questions, first microsomes were treated with trypsin and then added to the in vitro system. Integration of pM1M2, pM3M4, and pM3 into trypsin-treated microsomes was blocked by approximately 75% (Fig. 6, compare lanes2 and 4), implying that a trypsin-sensitive component on the cytosolic side of the microsomes is essential for the integration process.


Figure 6: Integrating proteins into trypsin treated microsomes. RNA transcripts for pM1M2, pM3M4, and pM3 were translated in the in vitro system. Mock-treated nRM (nRM) and trypsin treated nRM (nRM) were added co-translationally to the in vitro system, as indicated. Samples were incubated and fractionated as outlined under ``Experimental Procedures.'' The products were immunoprecipitated using antibodies against carboxyl-terminal residues of the H-ATPase. Samples were prepared for and resolved on 10% polyacrylamide gel with SDS and were visualized by fluorography.



Second, we studied integration into microsomes post-translationally to determine if this process is a function of chemical energy. Truncated forms of the H-ATPase are integrated post-translationally into microsomes when they remain associated with ribosomes. The exception to this rule is pM3. This protein is integrated effectively into microsomes with or without its association with ribosomes (data not shown). Accordingly, RNA transcripts without stop codons that encode pM1M2 and pM3M4 were used in these studies. Small molecules were removed by passage of translation mixtures through a size fractionation column. To the desalted mixtures, microsomes were added in the absence (Fig. 7, lanes3 and 4) or presence (lanes7 and 8) of an energy-regenerating system. The data demonstrate that integrating fusion proteins into microsomes depended on ATP and GTP. The amount of pM1M2 that integrates post-translationally is lower than that observed for pM3M4 and for pM3. The exact reason for this is not apparent. This may suggest that the topogenic signal in pM1M2 had become less accessible to the integration machinery. This could be caused by aberrant folding of nascent polypeptide chain of pM1M2 after or during the desalting step.


Figure 7: Integrating proteins into microsomes as a function of chemical energy. pM3 was synthesized in the in vitro system. For pM1M2 and pM3M4, stop codonless RNA transcripts were used. Protein synthesis was stopped with 0.2 mM emetine. Mixtures were desalted on column containing Bio-Gel P-4. Desalted fractions were divided into 20 µl and adjusted to 30 µl. Assay mixture contained 20 mM Hepes, pH 7.5, 150 mM KOAc, 2 mM Mg(OAc)(2), 6% glycerol, 1 mM dithiothritol, 0.2 mM emetine, and 1 µg/ml each of chymostatin, antipain, leupeptin, and pepstatin. Energy-regenerating system (ATP/GTP) contained 400 µM ATP, 100 µM GTP, 16 mM creatine phosphate, and 50 µg/ml creatine phosphate kinase. For samples in lanes5 and 6, 4 units of glycerol kinase (GK) were added to the desalted fraction (40 µl) and 1 unit of GK was added to nRM (10 µl) with 2.5% glycerol. The resultant mixtures were incubated at 20 °C for 20 min. GK-nRM were added to GK-treated desalted fraction (lanes5 and 6). All samples were incubated at 20 °C for 20 min with or without nRM (3 A units/ml) and then fractionated into soluble (S) fraction and membrane pellet (P) as outlined under ``Experimental Procedures.'' Proteins were resolved on 10% polyacrylamide gel in the presence of SDS and visualized by fluorography. Amount of product in each lane is expressed as a percentage of the total material recovered in the pellet and soluble fractions of that reaction.



Even in the absence of the energy-regenerating system, a small percentage of products sedimented with microsomes after alkaline treatment (Fig. 7, compare lanes3 and 4 with lanes1 and 2). This suggests that either the fusion proteins can integrate spontaneously into microsomes or the post-translational assay that contained microsomes and desalted translation mixture were not depleted of nucleotides. To distinguish between these possibilities, the desalted translation mixtures and microsomes were incubated with glycerol kinase to remove endogenous nucleotides. This reduced the amount of product found in the pellet after alkaline extraction (compare lanes5 and 6 with lanes3 and 4). This was interpreted to imply that even without the energy regenerating system, integration into microsomes is a function of nucleotides. The nucleotides may have been added to the assay by microsomes or they could represent nucleotides that were refractory to the desalting step. The combination of ATP and GTP was essential for optimal integration. This suggests that integrating fusion proteins into microsomes requires an ATPase(s) and a GTPase(s).


DISCUSSION

We investigated conditions required for integrating transmembrane segments from the amino-terminal third of the Neurospora H-ATPase into homologous microsomes. The results allowed us to confirm the number of segments in the amino-terminal third of the H-ATPase and to gain insights into how transmembrane segments containing charged amino acids and proline residues are integrated into membranes.

M3 initiated transfer of carboxyl-terminal residues of pM3 into the lumen of microsomes. Constructs containing either M1, M2, or M4 were not integrated into microsomes as judged by the fractionation of alkaline treated mixtures. When M4 was engineered downstream of M3 to give pM3M4, M4 blocked the glycosylation of the invertase segment. pM3M4 was integrated into microsomes as a polytopic integral membrane protein. When M2 was placed carboxyl-terminal to M1, the resultant protein pM1M2 was also integrated into microsomes as a polytopic integral membrane protein.

Using chimeric polytopic integral membrane proteins constructed by tandemly repeating the transmembrane segment of a bitopic integral membrane protein, researchers demonstrated that each transmembrane segment functioned independently and was inserted sequentially into microsomes (Wessels and Spiess, 1988; Rothman et al., 1988; Lipp et al., 1989). These chimeric polytopic membrane proteins, however, do not resemble P-type ATPases. P-type ATPases contain few, if any, residues on the exoplasmic side of the membrane. The studies presented herein suggest that transmembrane segments may function in pairs.

In vitro studies on the biogenesis of the multidrug resistance protein (MDR1) suggest that cooperative interaction between the first two transmembrane segments is a prerequisite for integrating MDR1 into microsomes. When these segments were assayed independently, each could direct protein translocation, but not integration, into microsomes (Skach and Lingappa, 1993). In contrast, the first two transmembrane segments of the H-ATPase did not function independently. In studies on the biogenesis of lac permease using a gene fusion method, transmembrane segment number nine (M9) gave a low export efficiency of the fused alkaline phosphatase. This was interpreted to imply that Arg-302 in M9 caused the low export efficiency. The authors suggested that M9 is stabilized in the membrane by forming an ion pair with Glu-325, which is found in M10 (Calamia and Manoil, 1992). Forming a salt bridge between M1 and M2 of the H-ATPase cannot explain the behavior of these segments in pM1M2, for M1 and M2 each contain a negatively charged residue, but no positively charged residue is present in either.

Most transmembrane segments of P-type ATPases contain hydrophilic residues that are presumed to serve as specific ligand for cations (Clarke et al., 1989). Proline residues are found in some transmembrane segments of P-type ATPases also. When hydrophilic and proline residues are introduced into transmembrane segments, the resultant segments have a reduced hydrophobicity and alpha helical character (Chou and Fasman, 1978). The introduction also of these residues by site-directed mutagenesis into the hydrophobic core of signal sequences of presecretory proteins blocks or greatly impairs the translocation of the resultant proteins across translocation competent membrane (Emr et al., 1980; Emr and Silhavy, 1983). And as demonstrated herein, the presence of hydrophilic residues in transmembrane segments of H-ATPase impaired their ability to function individually as topogenic signals when assayed out of context. Apparently, pairing of transmembrane segments is essential for masking charged residues and for stabilizing the integrated segments. This conjecture is supported by the following observations. M3 contains no charged residue and functioned independently as a topogenic signal. M1, M2, and M4 each contain a negatively charged residue, and these functioned as a topogenic signal only when combined.

Because M1 and M2 acted concertedly as a topogenic signal for integration, we will refer to this topogenic signal as an integration signal. Although we can not say with certainty what type of structure is formed by M1 and M2, a reasonable structure would be an antiparallel arrangement of the transmembrane segments held together by hydrophobic interaction to minimize interaction with water. This structure most likely would resemble a hairpin with the transmembrane segments serving as the arms. This arrangement would be favored by the short stretch of residues between the segments that acts as a physical constraint, analogous to disulfide bonds, and by the optimal arrangement of the helix dipoles of the segments.

Integration effectiveness of M1M2 was almost abolished by inserting the invertase segment between the segments. This was interpreted to imply that effective decoding of the integration signal depended on the proximity of or of a crucial number of residues between the segments. In contrast, transmembrane segments in pM3InM4 functioned individually and as predicted by the sequential insertion model. Nevertheless, in pM3M4 as well as in the H-ATPase, M3 and M4 are separated by seven residues. In this close association will M3 and M4 act concertedly or independently? Because M4 formed an integration signal with M1, this suggests that the putative integration machinery recognizes some general structural feature of the paired segments. In pM1M4, only eight residues lie between the transmembrane segments (see Fig. 1). It is likely that M3 and M4 in pM3M4 and in the H-ATPase form a structural motif similar to that formed by M1 and M2 in pM1M2. If this is true, then integrating the amino-terminal third of the H-ATPase into microsomes uses just one type of topogenic signal and not multiple topogenic signals.

Additional evidence supporting this conjecture is obtained from the following. Recognition of the signal sequence by the signal recognition particle (SRP) varies with the length of the nascent polypeptide chain. With some nascent presecretory proteins, effective binding of SRP with signal sequences requires that approximately 70-140 residues be synthesized. Depending on the length of the signal sequence, this suggests that 20-60 residues carboxyl-terminal to the signal sequence be in the cytosol (Siegel and Walter, 1988). Integration of the Ca motive ATPase, a P-type ATPase, from rabbit sacrcoplasmic reticulum into heterologous microsomes is SRP-dependent (Anderson et al., 1983). It is likely that SRP acts as a general adapter in the targeting of most nascent secretory proteins to the endoplasmic reticulum. If so, targeting of the Neurospora H-ATPase to the endoplamic reticulum could be SRP-mediated also. If this is true, and taking into consideration SRP recognization of signal sequences, this would imply that in the nascent H-ATPase when M1 and M3 are in the cytosol the 20-60 residues carboxyl-terminal to these segments would contain M2 or M4, respectively. Once in the cytosol, these hydrophobic segments will interact to minimize their interaction with water (Engelman and Steitz, 1981). This implies that closely juxtaposed transmembrane segments in nascent polypeptide chain must interact in vivo. Assuming that this is true, juxtaposed segments would form hairpin-like structures before the decoding process begins. Alternatively, the looped segments could be disrupted by an effector exposing the individual segments, which are then decoded. This interpretation of the data is not consistent with results obtained with pM1 or pM2. Also, this process would require the use of energy to disrupt a thermodynamically stable structure. Furthermore, experimental observations suggest that the hydrophobic core of signal sequences, which consists of eight or 12 amino acids, exists in a looped structure resembling a hairpin (Nouwen et al., 1994; Rizo et al., 1993). If this is true, it is possible the SRP recognizes topogenic sequences in a hairpin-like configuration. Therefore, the hairpin-like structure of paired transmembrane segments may actually represents a common structural motif of topogenic signals.

From the above considerations, we propose that membrane topology of the amino-terminal third of the H-ATPase is achieved by inserting into the membrane-paired transmembrane segments in a receptor-mediated, energy-dependent event. Paired transmembrane segments would be used as an integration signal only by polytopic integral membrane proteins that do not have large exoplasm domains. This model implies that for a region containing four transmembrane segments, there would be two integration events. This conjecture is consistent with the results of Homareda et al.(1989). This group studied integration of truncated forms of the alpha-subunit of the Na/K ATPase into heterologous microsomes; the results imply that for the first four transmembrane segments there are two topogenic signals causing two insertion events. Clearly, the structural analysis of transmembrane segments in an aqueous milieu will provide valuable knowledge about the nature of integration signals for polytopic integral membrane proteins.

In conclusion, the results presented herein provide functional evidence for a novel type of topogenic signal for integrating a polytopic integral membrane protein into membrane. This signal consists of two transmembrane segments. The data also provide evidence that transmembrane segments, when assayed out of context, have various degrees of effectiveness as topogenic signals. This method can serve as a paradigm for identifying transmembrane segments functioning as integration signal.


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 abbreviations used are: H-ATPase, plasma membrane electrogenic, proton-translocating ATPase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; SRP, signal recognition particle; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. J. Foster for the method of sequencing double-stranded DNA.


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