The vacuolar proton ATPase (vacuolar H-ATPase) is a large enzyme complex that is present in several intracellular membrane compartments (such as endosomes, lysosomes, and the Golgi apparatus) and drives the unidirectional flux of protons from the cytoplasmic to lumenal sides of these organelles(5, 6, 7) . This enzyme complex has also been detected in the plasma membranes of renal epithelial cells (5) and, in some cells, may contribute to the intracellular alkalinization observed during cellular transformation(8) . This proton flux causes a limited acidification, which is critical for cell viability(5, 6, 7, 9) as well as for the proper processing, targeting, and function of cellular proteins in these compartments(10, 11, 12, 13, 14, 15) . The vacuolar H-ATPase is composed of approximately 5-10 different proteins ranging in size from 16-115 kilodaltons, and the membrane proton pore appears to be composed of four of these proteins (116K, 39K, 20K, and 16K)(5, 16, 17, 18) . The 16K protein, which is highly hydrophobic and composed almost entirely of four putative transmembrane domains, is believed to be the main component of the proton pore(16, 19, 20) .

Acidification of endosomes appears to be critical for determining the fate of receptor-ligand complexes following endocytosis(5) , making the pump an important regulator of signal transduction pathways. In addition, acidification of the trans-Golgi apparatus may regulate the transport/processing of proteins that proceed through this compartment, including growth factor receptors. Interestingly, activation and altered processing of epidermal growth factor receptors(21) , as well as activation of PDGF ()receptors(22, 23) , is a characteristic of cell transformation induced by the bovine papillomavirus type-1 E5 oncoprotein. The E5 gene is the main transforming gene of bovine papillomavirus type-1(24) , and it encodes a 44-amino acid oncoprotein that is composed of two distinct domains: a hydrophobic transmembrane region and a hydrophilic dimerization domain (25) . E5 has recently been shown to exist in an in vivo complex with the PDGF receptor(22, 26, 27) , the epidermal growth factor receptor(26) , and the 16K subunit of vacuolar H-ATPase(1, 2, 22, 28) . It appears that E5 may actively recruit 16K into a ternary complex with PDGF receptor, suggesting a role for 16K in signal transduction.

The intent of this investigation was to define the domain(s) of 16K that is responsible for binding E5 and to evaluate the role of 16K in cell transformation. 16K has been cloned and sequenced from several organisms and exhibits strong conservation at the amino acid level. This conservation is especially high in the transmembrane domains, with the carboxyl-terminal fourth transmembrane domain (TM4) showing 100% homology in yeast, nehrops, mouse, and cow. Since previous studies have shown that the yeast 16K can bind efficiently to the E5 oncoprotein(4) , we targeted TM4 for mutagenesis. More specifically, the interaction between the hydrophobic transmembrane domains of 16K and E5 most likely involves the interaction between charged or polar residues since the replacement of the glutamine residue in the transmembrane domain of E5 dramatically inhibits its binding to 16K (as well as its transforming activity)(1, 28) . The 16K TM4 domain is the only transmembrane domain containing a charged amino acid (glutamic acid). This glutamic acid residue is also critical for the biological activity of the vacuolar proton pump, and its conversion to alternative amino acids is lethal for yeast(9) . Our studies indicate that the glutamic acid residue of 16K participates in the binding of E5, that its mutagenic alteration converts the 16K protein into a transforming protein, and that wild-type 16K can suppress the oncogenic activity of E5.

MATERIALS AND METHODS

Plasmid Construction

Plasmids used for transformation assay were generated by a two-step cloning procedure. XhoI-BamHI 16K fragments from pSVL constructs were cloned into the SalI-BamHI site of the vector pIC (a gift of M. Finbow). The pIC-16K plasmids were cleaved with HindIII and ClaI and cloned into the HindIII-ClaI sites of the LNCX expression vector (32) .

DNA Transfection

Cell transformation assays by E5 were performed using the transfection procedure described above. Following the glycerol shock, however, the cells were cultured until confluent, transferred into 75-or 162-mm flasks, and then fed every 3 days thereafter. 2-3 weeks later, the cells were washed once with 1 PBS and then stained with 1% methylene blue in 100% ethanol, and the number of foci were counted.

Immunoprecipitation

The procedure described above was modified for a double immunoprecipitation. Following 1.5 h of incubation at 4 °C with the first antibody, the immune complex was washed once in extraction buffer and then treated with 200 µl of SOL buffer (50 mM triethylamine HCl, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.4% SDS, and 2 mM -mercaptoethanol), heated for 2 min at 100 °C, and then cooled on ice. 4 µl of 0.5 M iodoacetamide was added, and the suspension was centrifuged. The supernatant was collected, and 50 µl of 10% Triton X-100 was added. The second immunoprecipitation was performed using 5 µl of the AU1 antibody plus 50 µl of protein A-Sepharose CL-4B beads. Following 1.5 h of incubation at 4 °C, the immune complex was washed once with PBS. Precipitated proteins were separated on a 14% SDS-polyacrylamide gel.

Generation of Stable NIH 3T3 Cell Lines

Anchorage-independent Growth Assay

Southern Blot Analysis

RESULTS

To identify the E5 binding site on the 16K protein and to study the role of 16K in transformation, six 16K constructs were made using PCR techniques: two wild-type 16K constructs (AU1 16K and HA1 16K), a construct expressing helices 1 and 2 (-1,2 16K), a construct expressing helices 3 and 4 (-3,4 16K), a truncated 16K lacking the fourth transmembrane domain, and a single amino acid substitution (glutamic acid to arginine) in the fourth transmembrane domain (Arg-143 16K) (Fig. 1). Due to the lack of antibodies against native 16K, the AU1 or HA1 epitopes were tagged to the amino terminus of 16K proteins to facilitate immunoprecipitation(29, 30) . All constructs were cloned into and expressed by the pSVL vector.

Figure 1: Epitope-tagged forms of wild-type and mutant 16K. PCR techniques were used to append the HA1 and AU1 epitopes to the amino terminus of the indicated 16K mutants. These mutants were then expressed by the SV40 late promoter (pSVL vector) for transient expression in COS cells or by the CMV promoter (LNCX vector) for stable expression in cell lines. Each box represents a transmembrane domain, and the soliddot indicates the substitution of glutamic acid with arginine at position 143.

The 16K Binding Site for E5 Maps to Helices 3 and 4

Figure 2: The main binding site for E5 on 16K maps to helices 3 and 4. COS cells were cotransfected with 1 µg of the HA1 E5 and 1 µg of either the -1,2 16K (lanes1-4) or the -3,4 16K (lanes5-8). Double immunoprecipitation was performed using 5 µl of either 12CA5 (lanes1 and 5) or pre-absorbed 12CA5 (lanes2 and 6) as the first antibody, followed by a second immunoprecipitation with 5 µl of AU1. To verify expression of the E5 and 16K, a single immunoprecipitation was performed using 5 µl of either 12CA5 for E5 expression (lanes3 and 7) or AU1 antibody for 16K expression (lanes4 and 8).

The Binding Site for E5 Maps Predominantly to the Fourth Transmembrane Domain of 16K and Is Modulated by Charge Interactions

Figure 3: Specific amino acids within 16K and E5 transmembrane domains mediate their interaction. COS cells were transfected with the DNAs indicated at the top of each gel and then metabolically labeled with [S]methionine, lysed in radioimmunoprecipitation assay buffer, and immunoprecipitated with either AU1 or 12CA5 antibody. Immunoprecipitated proteins were separated on SDS-polyacrylamide gels and visualized by fluorography. Positions of molecular mass standards (in kilodaltons) are indicated to the left of each gel. A, expression of two mutant forms of 16K. COS cells were transfected with pSVL (lane1), wild-type 16K (lane2), truncated 16K (lane3), or Arg-143 16K (lane4) and immunoprecipitated with 5 µl of AU1. B, mutations in the fourth transmembrane domain of 16K affect binding to the E5 oncoprotein. Coprecipitations using 5 µl of 12CA5 were performed on COS cells cotransfected with the DNAs indicated at the top. Since 16K constructs express more efficiently than E5 constructs, 0.1 µg of wild-type AU1 16K and 0.25 µg of the truncated and Arg-143 16K DNAs were used to cotransfect COS cells with 1 µg of E5 DNA. C, E5/16K binding is governed by charge interactions. COS cells were cotransfected with 1 µg of either of the two E5 mutants (glutamic acid and aspartic acid substitutions) and 1 µg of the Arg-143 16K, as indicated at the top. Labeled cell extracts of cotransfected cells were immunoprecipitated with 5 µl of 12CA5 antibody. D, binding efficiency between E5 and wild-type 16K is reduced by substitution of glutamine 17 with glutamic or aspartic acid. COS cells were transfected with 1 µg of the indicated E5 constructs. Labeled cell extracts were then immunoprecipitated with 5 µl of the monoclonal antibody 12CA5. Positions of molecular mass standards (in kilodaltons) are indicated to the left of each gel.

The ability of the two mutated 16K proteins to form a complex with E5 protein was evaluated by coprecipitation (Fig. 3B). Plasmids encoding the 16K proteins and the HA1 E5 protein were cotransfected into COS cells and, 48 h later, immunoprecipitated with a monoclonal antibody (12CA5) against the HA1 epitope on E5. The amounts of immunoprecipitated E5 protein and coprecipitated 16K protein were quantitated by film densitometry, and the ratio of 16K/E5 was calculated from three different experiments (Table 1A). The deleted form of 16K protein bound 8% (Fig. 3B, lane2), and the arginine mutant bound 28% of wild-type levels of E5 protein (Fig. 3B, lane3). These results demonstrate the importance of TM4 and, more specifically, the glutamic acid in TM4 for E5 binding and indicate that E5 targets an amino acid critical for the function of the proton pump(9) .

Previous studies have implicated glutamine 17 in the hydrophobic domain of E5 as the main binding site for 16K(1, 28) . Our current results indicate that 16K/E5 binding is mediated by polar glutamine 17 (partial positive charge) in E5 and the negatively charged glutamic acid 143 in 16K. To demonstrate the apparent charge dependence of these interactions, coprecipitation experiments were performed in COS cells using Arg-143 16K, which binds with reduced efficiency to wild-type HA1 E5. The COS cells were also transfected with E5 constructs containing either glutamic acid or aspartic acid at position 17 (31) to provide complementary charge interactions with the arginine residue in 16K. These E5 mutants exhibited an anticipated decreased binding to the endogenous wild-type 16K (Fig. 3D, lanes3 and 4, and Table 1C) when compared with wild-type E5 (lane 2). On the other hand, when coprecipitation was performed using the E5 mutants and Arg-143 16K (Fig. 3C, lanes2 and 3), they bound more 16K than wild-type HA1 E5 (Fig. 3C, lane1 and Table 1B). These results demonstrated that the binding of 16K/E5 was regulated by charged intramembrane amino acids and that the interaction was optimal when the 16K and E5 amino acids were of opposite charge.

The striking mobility shift of the E5 mutants (Fig. 3, C and D) was unexpected since only one amino acid was mutated in the transmembrane domain of E5. Conformational changes in the mutated E5 or different interactions between the E5 TM and the detergent might account for this aberrant mobility.

Mutant 16K Proteins Retain Normal Binding to the Wild-type 16K

Figure 4: Mutant 16K proteins retain normal binding to wild-type 16K protein. A, Double immunoprecipitations were performed on [S]methionine-labeled COS cells, which had been cotransfected with 1 µg of HA1 16K and 1 µg of the AU1 16K, truncated 16K, or Arg-143 16K as indicated at the top of each lane. Cotransfected COS cells were labeled, extracted in lysis buffer, and immunoprecipitated first with 5 µl of 12CA5. Immune complexes were dissociated as described under ``Materials and Methods'' and reprecipitated with 5 µl of AU1. The final immune complex was dissociated in SDS sample buffer and separated on 14% polyacrylamide gels. B, coprecipitation of AU1 16K proteins is dependent upon immunoprecipitation of the HA1 16K. Verification of the specificity of the double immunoprecipitation method was performed using a 12CA5 antibody pre-absorbed with an HA1 peptide as the first antibody on COS cells cotransfected with HA1 16K and AU1 16K constructs as described in A. Precipitated proteins were electrophoretically separated on 14% SDS-polyacrylamide gels. Positions of molecular mass standards (in kilodaltons) are indicated to the left of each gel.

Mutant 16K Proteins Transform Mouse Cells

Figure 5: Mutant 16K proteins exhibit cell transformation activity. NIH 3T3 cell lines generated with AU1 16K constructs were evaluated for anchorage-independent growth by plating the cell lines into soft agar and allowing them to grow for 4 weeks. Panela, cell line generated with the vector alone; panelb, cell line generated with wild-type 16K; panelc, cell line generated with truncated 16K; paneld, cell line generated with Arg-143 16K.

Anchorage-independent cell lines were also evaluated for expression of the mutant 16K proteins. In Fig. 6A, cell lines generated with the LNCX vector, wild-type AU1 16K, truncated 16K, or Arg-143 16K were immunoprecipitated with AU1 antibody. Both the wild-type and arginine point mutant cell lines synthesized detectable levels of 16K protein (Fig. 6A), whereas the deletion mutant cell line did not. Since it was not possible to detect any protein expressed by the truncated 16K, the cause of cell transformation observed with this mutant remains uncertain. The lack of detectable truncated protein might reflect protein stability since this protein appeared less stable in COS cells than wild-type or arginine E5 proteins (Fig. 3A). Southern blot analysis clearly demonstrated that the transformed cell line contained the deleted 16K gene (Fig. 6B, lane3). Therefore, the activity of the 16K truncated protein might be sufficiently high to induce cell transformation in the presence of very low protein levels, a situation that is analogous to a potent Fos recombinant protein(37, 38) .

Figure 6: Evaluation of 16K expression in wild-type and mutant 16K cell lines. A, NIH 3T3 cell lines generated with the AU1 16K constructs were plated onto 100-mm plates, metabolically labeled with [S]methionine, extracted, and immunoprecipitated with 5 µl of AU1 antibody. Lane1, vector alone; lane2, AU1 16K; lane3, Arg-143 16K; lane4, cell line generated with deleted 16K. Proteins were resolved on 14% polyacrylamide gels. AU1 16K (lane2) and Arg-143 16K (lane3), but not the truncated 16K (lane4), expressed detectable levels of 16K proteins. Positions of molecular mass standards (in kilodaltons) are indicated to the left. B, Southern blot analysis of cell lines generated with the wild-type and deleted 16K. High molecular weight DNA from the cell lines generated with the LNCX vector, AU1 16K, and truncated 16K were isolated. DNA was cut with BamHI, separated on a 1% agarose gel, blotted onto GeneScreen Plus membrane, and probed with DNA labeled with [P]ATP harboring the 16K cDNA and the CMV promoter. Lane1, cell line generated with vector alone; lane2, cell line generated with the AU1 16K construct; lane3, cell line generated with deleted 16K.

Cotransfection of Wild-type 16K with E5 Significantly Inhibits the Ability of E5 to Transform Murine Fibroblasts

To evaluate these possibilities, the mouse fibroblast cell lines NIH 3T3 and C127 were plated at 50% confluence on 100-mm plates and cotransfected with 9 µg of wild-type AU1 16K, Arg-143 16K, or truncated 16K expressed from the LNCX vector and 1 µg of wild-type E5. After the plates had reached 100% confluence, they were split into either 75- or 162-cm flasks and allowed to form foci. 2-3 weeks later, the number of foci formed with E5 alone (Fig. 7, panelA) was compared with the number of foci formed when the 16K genes were cotransfected with E5 (Fig. 7, panelsB-D). These studies ( Table 3and Fig. 7) revealed a 75% inhibition of E5-mediated transformation by wild-type 16K (Fig. 7, panelB), 58% inhibition by the Arg-143 16K (Fig. 7, panelC), and only 25% inhibition by the truncated 16K (Fig. 7, panelD), demonstrating that the ability of 16K to inhibit E5 transformation correlated directly with its ability to bind E5.

Figure 7: Inhibition of E5 transformation by coexpression of 16K. Mouse fibroblast cell lines, plated at 50% confluence on 100-mm plates, were cotransfected with 1 µg of wild-type E5 and 9 µg of pUC (panelA), 1 µg of wild-type E5 and 9 µg of AU1 16K (panelB), 1 µg of wild-type E5 and 9 µg of Arg-143 16K (panelC), or 1 µg of wild-type E5 and 9 µg of truncated 16K (panelD). Cells were grown for 3 weeks and stained with 1% Trypan blue; the number of foci were then counted.

DISCUSSION

We have shown that the fourth transmembrane domain of 16K is critical for its interaction with the E5 oncoprotein. More specifically, the glutamic acid residue within this domain, which is essential for the activity of the proton pump, regulates the binding of E5. Mutation of glutamic acid 143 reduced the binding to 28% of wild-type, which suggested that other residues within the domain were also involved in the interaction. Accordingly, the truncation of the fourth transmembrane domain virtually abolished binding, suggesting that the predominant binding site for E5 may be the fourth transmembrane domain. The importance of charged amino acids for the interaction between E5 and 16K was supported by the demonstration that binding between the two proteins can be restored by reversing the charges in TM4 of 16K and E5 ( Fig. 3and Table 1). These results strongly indicated that the interaction between E5 and 16K was governed by charge interaction. The targeting of such a critical domain of 16K by E5 suggests that modulation of proton pump activity may represent one of the mechanisms by which E5 contributes to cellular transformation.

Even more suggestive of the relevance of TM4 for E5 action is the finding that mutation of the glutamic acid residue converts 16K into a transforming protein. This might indicate that disrupting the function of the critical fourth transmembrane domain either by mutation or by binding E5 will cause abnormalities in the function of the pump, thereby leading to cell transformation. However, although the Arg-143 16K mutant is transforming, it does not exactly mimic the activity of E5. For example, while both E5 and the Arg-143 16K induce anchorage-independent growth, only E5 induces efficient focus formation on immortalized mouse cells. ()This suggests that the 16K mutant is only able to partially mimic E5 transformation. While mutant 16K might only be able to interfere with the function of the proton pump, E5 may also activate tyrosine kinase growth factor receptors, a function that has not yet been shown for the mutated 16K. These combined functions carried out by the E5 protein may be necessary to attain full transformation. However, it is also possible that the observed biologic differences between mutant 16K and E5 might reflect variations in the level of protein expression since different vectors and promoters have been used to express 16K and E5 proteins in mouse cells.

The simplest hypothesis for the mechanism by which E5 and 16K might mediate cell transformation involves their direct interaction with growth factor receptors. Thus, the dimeric form of E5 (39) might facilitate cross-linking of the PDGF receptor. This cross-linking would then initiate receptor transphosphorylation and the consequent hierarchy of signaling events leading to a mitogenic response. In this scheme, 16K, via its ability to bind E5 protein and receptors(22) , would facilitate the cross-linking of receptors or their activation. Finally, there is also the possibility that E5 transforms cells by mediating both receptor activation and altered receptor processing (proton pump activity)(3) .

It is presumed that the 16K mutant proteins transform cells by interfering with the proton pump, although direct evidence for this hypothesis has not been obtained. Mutant 16K proteins retain their ability to interact with wild-type forms of 16K and could potentially interfere with the acidification of intracellular compartments such as endosomes and consequently inhibit the degradation of internalized, activated receptors. Three reports have demonstrated that the E5 proteins of both the bovine and human papillomaviruses inhibit the degradation of internalized growth factor receptors(21, 36, 40) .

The high specificity and the nature of the binding site on 16K strongly suggest that E5-16K interaction is important for cell transformation. Verification of a direct interaction between E5 and 16K within cells was also inferred from the ability of wild-type 16K, but not non-binding mutants, to efficiently inhibit the transforming activity of E5 ( Fig. 7and Table 3). These results suggest that providing the target cell with excess wild-type 16K might supply enough 16K to restore normal proton pump activity. An alternative explanation for these results might be that excess 16K sequesters E5 away from other cellular targets, such as the PDGF receptor.

Our experiments with reciprocal charge exchange have demonstrated that E5 and 16K interact via hydrophilic, charged residues within their transmembrane domains. Not only do these experiments suggest new mechanisms for conferring specific protein interactions, they also supply the first evidence that E5 binds directly to the 16K cellular protein target. In contrast, it has not been possible to show that E5 binds directly to the PDGF receptor, although they are present within the same immunoprecipitate. Future experiments will be necessary to define the complex formed between E5, 16K, and PDGF receptor in transformed cells.

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.

§

To whom correspondence should be addressed: Dept. of Pathology, Georgetown University Medical School, 3900 Reservoir Rd., NW, Washington, D. C., 20007.

()

The abbreviations used are: PDGF, platelet-derived growth factor; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TM, transmembrane; MOPS, 4-morpholinepropanesulfonic acid.

()

T. Andresson, and R. Schlegel, unpublished results.

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