©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hydrophobicity and Subunit Interactions of Rod Outer Segment Proteins Investigated Using Triton X-114 Phase Partitioning (*)

(Received for publication, November 14, 1994; and in revised form, April 14, 1995)

John M. Justice (§) James J. Murtagh , Jr. (¶) Joel Moss Martha Vaughan

From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Triton X-114 phase partitioning, a procedure used for purifying integral membrane proteins, was used to study protein components of the mammalian visual transduction cascade. An integral membrane protein, rhodopsin, and two isoprenylated protein complexes, cyclic GMP phosphodiesterase and G, partitioned into the detergent-rich phase. Arrestin, a soluble protein, accumulated in the aqueous phase. G distributed about equally between phases whether GDP (GGDP) or GTP (GGTP) was bound. G increased recovery of GGDP but not GGTP in the detergent phase. Trypsin-treated G, which lacks the fatty acylated amino-terminal 2-kDa region, accumulated to a greater extent in the aqueous phase than did intact G. Trypsinized cGMP phosphodiesterase, which lacks the isoprenyl group, partitioned into the aqueous phase. A carboxyl-terminal truncated mutant (Val-331 stop) of G accumulated more in the aqueous phase then did recombinant full-length G, supporting the role of the carboxyl terminus in increasing its hydrophobicity. N-Myristoylated recombinant G was more hydrophobic than recombinant G without myristate. ADP-ribosylation of G catalyzed by NAD:arginine ADP-ribosyltransferase, but not by pertussis toxin, increased hydrophilicity. Triton X-114 phase partitioning can thus semiquantify the hydrophobic nature of proteins and protein domains. It may aid in evaluating changes associated with post-translational protein modification and protein-protein interactions in a defined system.


INTRODUCTION

Heterotrimeric guanine nucleotide-binding proteins (G proteins),()consisting of , , and subunits, couple cell-surface receptors with effectors and may also participate in signaling involving intracellular organelles(1) . G proteins are activated by GTP, which upon binding to the subunit (G), causes a conformational change (2, 3) and promotes its dissociation from the complex (G). GTP hydrolysis and GTP-GDP exchange, reactions critical for regulation of G activity, are dependent on the presence of receptor, G, and effector(4, 5, 6, 7) .

Domains of G involved in interaction with receptor and G have been mapped using a variety of methods. It was concluded that the amino-terminal region of G is important for its interaction with G. Removal of the first 18 amino acids from the amino terminus of G by trypsin abolished G stimulation of pertussis toxin-catalyzed ADP-ribosylation, although the ADP-ribosylation site itself was intact(8) . Limited proteolysis with staphylococcal V8 protease, which removes 21 amino acids from the amino terminus, inhibited other -dependent reactions including receptor-stimulated GTP hydrolysis, GTP-GDP exchange, and binding of G to rhodopsin(4) . A monoclonal antibody directed against the amino terminus of G interfered with pertussis toxin-catalyzed ADP-ribosylation(9) , as did deletions in the amino terminus of recombinant G(10) . Amino acids 7-10 in recombinant G were deemed critical for interaction with G(11) .

The carboxyl terminus of G subunits is important for receptor interaction. ADP-ribosylation of cysteine 347, four residues from the carboxyl-terminal end of G, functionally uncoupled it from receptor(12, 13, 14) . Peptides possessing the carboxyl-terminal sequence of G inhibited its binding to rhodopsin(15) . The behavior of chimeric G/G recombinant proteins was consistent with the conclusion that the carboxyl terminus of G is critical for coupling the -adrenergic receptor to adenylyl cyclase (16) . The carboxyl terminus is important in the membrane association of G(17, 18) , and the carboxyl terminus of G was postulated to be involved in anchoring the Gphosphodiesterase complex to membranes(19) .

G proteins appear to interact with the inner surface of the plasma membrane. Association of G with membranes is facilitated by farnesylation or geranylgeranylation of a cysteine four amino acids from the carboxyl terminus of the subunit(20, 21, 22, 23) . Membrane association of some G subunits (e.g. G and G) was dependent on the amino terminus as limited digestion with trypsin, which removes an amino-terminal 2-kDa fragment, produced a soluble protein(24) . The amino-terminal glycines of G and G are myristoylated, a modification that enhances membrane association (25, 26, 27, 28) . Membrane association may occur directly via the myristoyl moiety and its neighboring amino acids, as has been postulated for the Src protein(29, 30) , or indirectly if it increases the association of the subunit with the prenylated and membrane-associated G subunits(31, 32) . In support of the latter hypothesis, both -stimulated ADP-ribosylation and the affinity of G for -Sepharose were significantly greater with the myristoylated than with the nonmyristoylated protein(11, 31) . G modified with more hydrophilic myristate analogues was less likely than native G to associate with membranes(25) . In sum, the data are consistent with a role for a myristoylated amino terminus in membrane association, although with the more recent recognition that several G subunits are also palmitoylated, the contribution of the less hydrophobic myristate may need to be reassessed(33) .

Integral membrane proteins have been identified and purified based on their phase-partitioning characteristics(34) . Solutions of Triton X-114 separate into detergent-rich and detergent-depleted (aqueous) phases at 30 °C. It is generally agreed upon that the more ``hydrophobic'' a protein or peptide, the more likely it will partition into the detergent-rich phase. This partitioning difference has been the basis for separating integral from peripheral membrane proteins(34) . We investigated this partitioning procedure as a new way of characterizing the hydrophobic and membrane-association domains of native and recombinant G and myristoylated and nonmyristoylated rG as well as possibly assessing protein-protein interactions in a purified defined system.


EXPERIMENTAL PROCEDURES

Materials

Gpp(NH)p, GTP, and GTPS were purchased from Boehringer Mannheim; [P]NAD (30 Ci/mmol), [S]GTPS (40 Ci/mmol), [H]GTP (15 Ci/mmol), [H]myristic acid (16 Ci/mmol), GA/1 antibody, and AS/7 antibody from DuPont NEN; Triton X-114 from Fluka; TPCK-treated trypsin from Worthington; glutathione-agarose, NAD, ATP, and soybean trypsin inhibitor from Sigma; blue Sepharose CL-6B and PD-10 columns from Pharmacia Biochem Inc.; Triton X-100 from Research Products International Corp.; isopropyl--D-thiogalactopyranoside from United States Biochemical Corp.; goat anti-rabbit IgG horseradish peroxidase-coupled antibody from Pierce; goat anti-mouse IgG horseradish peroxidase-coupled antibody from Promega; LB medium from Digene; ampicillin sodium from Amresco; and molecular weight standards from Life Technologies, Inc. Densitometry was performed with a Molecular Dynamics laser densitometer and Image Quant software. SDS-PAGE and Western blotting were done in a Novex Minicell apparatus using 12% or 4-20% gradient Tris/glycine gels.

Methods

Preparation of PDE

PDE was essentially prepared as described previously(35, 36) . Briefly, fresh bovine eyes were obtained from a local abattoir; retinas were dissected, frozen on dry ice, and stored at -70 °C. After thawing, retinas were suspended in buffer A (60 mM KCl, 30 mM NaCl, 1 mM dithiothreitol, 10 mM MOPS, pH 7.5, 1 mM NaN, 2 mM MgCl, 0.1 mM EGTA) containing 45% sucrose, vortexed, and centrifuged (27,750 g, 20 min). After diluting the supernatant with an equal volume of buffer A without sucrose, rod outer segments (ROS) were pelleted by centrifugation (27,750 g, 20 min). Pellets were washed twice by suspending in buffer A and centrifuging (27,750 g, 20 min). Washed ROS were then suspended in buffer B (5 mM Tris-HCl, pH 7.5, 0.5 mM MgCl, 0.05 mM EGTA, 0.5 mM NaN, 1 mM dithiothreitol, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin) and centrifuged (45,000 g, 30 min). This supernatant, which contained mainly PDE (35) and arrestin, was concentrated in a Macrosep 100 concentrator (Filtron Technology Corp.) and used as such or further purified by molecular sizing on an Ultrogel AcA-34 column.

Preparation of Transducin and Subunits

ROS, after extraction of PDE, were washed three additional times by centrifugation in buffer B without soybean trypsin inhibitor or leupeptin (45,000 g, 30 min). Transducin was then eluted with buffer B containing 100 µM GTP with centrifugation (45,000 g, 30 min). G and G subunits were resolved by blue Sepharose chromatography(39) .

To prepare G with radiolabeled GDP or GTP, PDE-depleted ROS were washed three times in buffer A, and G was eluted by two extractions with buffer A containing 50 µM [S]GTPS (2-5 mCi/mmol) or 100 µM [H]GTP (4-10 mCi/mmol)(38) . G was concentrated with a Centriprep-10 (Amicon, Inc.), and free nucleotide was removed using PD-10 columns. Samples were stored at 4 °C and used within 3 days. Bound nucleotide was identified by heating G at 95 °C and analyzing the released nucleotide on polyethyleneimine-cellulose TLC plates developed with 1.6 M LiCl(40) , which verified that G eluted with [S]GTPS retained this nucleotide. [H]GTP bound to G was, however, hydrolyzed to [H]GDP. To obtain G in GDP-bound (GGDP) and GTPS-bound (GGTPS) states, procedures were identical except that hypotonic conditions were used for elution with labeled nucleotide(38) .

Rhodopsin was prepared by the method of Hong and Hubbell(41) . Protein concentrations were determined by Coomassie Blue assay (Bio-Rad) using bovine serum albumin as a standard.

PDE Assay

PDE activity was assayed for 10 min at 30 °C in 0.3 ml of 50 mM HEPES, pH 7.5, 0.1 mM EGTA, 8.3 mM MgCl, 1 µM [H]cGMP (20,000 cpm/assay). Reactions were terminated by addition of 100 µl of 11.25 mM cGMP and 5 mM 5`-GMP in 250 mM HCl; neutralized with 100 µl of 250 mM NaOH in 250 mM Tris-HCl, pH 8.0; and then incubated with 5`-nucleotidase (Crotalus atrox venom) at 30 °C for 20 min. The H-labeled nucleoside produced was collected for radioassay(42) .

Triton X-114 Phase Partitioning

In a modification of the method of Bordier(34) , Triton X-114 was added (final concentration of 1%) to protein samples in buffer C (20 mM Tris-HCl, pH 7.5, 0.5 mM MgCl, 0.05 mM EGTA, 0.5 mM NaN, 1 mM dithiothreitol, 150 mM NaCl) in a final volume of 40-75 µl. Mixtures were gently mixed and kept on ice for 3-5 min. After warming to 37 °C for 5 min, the cloudy solution was centrifuged (300 g, 5 min) in a swinging bucket rotor prewarmed to 30-37 °C. The upper aqueous layer was aspirated. Buffer C was added to the detergent phase, which contained no visible particulates, and Triton X-114 was added to the aqueous phase to equalize the compositions and volumes of the two fractions. Equal volumes of each phase were subjected to SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue and quantified by densitometry. In experiments in which G contained radiolabeled nucleotide, samples of the two phases were subjected to PD-10 column chromatography to separate unbound from protein-bound nucleotide, which was quantified by liquid scintillation counting. Nucleotide that became unbound during partitioning accumulated primarily (>85%) in the aqueous phase.

Trypsinization

Transducin (G; 0.5 mg/ml), G (0.2 mg/ml), or PDE (1 mg/ml) in hypotonic buffer was incubated with TPCK-treated trypsin (1.2 µg/ml) at 30 °C for 2-10 min. Reactions were stopped by adding phenylmethylsulfonyl fluoride or soybean trypsin inhibitor (final concentrations of 1 mM and 0.1 mg/ml, respectively).

Preparation of Recombinant G

DNA inserts for G and a carboxyl-truncated form of G were prepared by polymerase chain reaction using G cDNA as template. The 5`-primer for both transducin molecules was 5`-GCAGCAGCATGATCAGGGGCCAGCGCTGAGGAGAAG-3`, which includes a BclI restriction site enabling excision of the polymerase chain reaction product with BclI and cloning into the BamHI site of the pGEX-2T vector (Pharmacia Biotech Inc.) without cleaving the BamHI site within the G coding sequence. The 3`-primers (5`-GCGCCACGGGAATTCTCAGAAGAGCCCGCAGTCTT-3` and 5`-GCGCCAGCGGAATTCTCAAAACTTGACGTTCTGCGTGTC-3`) included an EcoRI site after the natural stop codon or after a stop codon introduced to replace codon 331 (valine) to yield a truncated protein. Following polymerase chain reaction, Taq polymerase (Perkin Elmer) and unincorporated nucleotides were removed using a QIAGEN polymerase chain reaction purification kit. Products were digested with BclI and EcoRI. G inserts were gel-purified with Qiaex resin (QIAGEN) and ligated into the BamHI/EcoRI-restricted pGEX-2T vector using T4 DNA ligase. Escherichia coli DH5 competent cells (Life Technologies, Inc.) were transformed using heat shock and selected on ampicillin-containing LB agar plates. After isopropyl--D-thiogalactopyranoside induction, positive clones were screened for protein production using SDS-PAGE.

For large-scale purification of fusion proteins, E. coli cells were grown in 500-1000 ml of LB/ampicillin medium at 37 °C to an absorbance of 0.4 at 600 nm. Isopropyl--D-thiogalactopyranoside was added (final concentration of 100 µM), and incubation (with shaking) was continued for 3-4 h. After centrifugation, cell pellets were washed once with phosphate-buffered saline, suspended in 10 ml of phosphate-buffered saline, and lysed using nitrogen cavitation. Sarcosine (0.5%) was added to solubilize the fusion proteins, and cell debris was removed by centrifugation (15,000 g, 4 °C, 10 min). Triton X-100 (final concentration of 1%) and glutathione-agarose beads were added to the supernatant, and the mixture was rocked for 2-3 h at 4 °C. Binding of fusion protein to the beads in the presence of sarcosine required Triton X-100. Bound fusion protein (0.25-1.0 mg) was eluted with 3-5 ml of 10 mM reduced glutathione, 200 mM NaCl, 50 mM Tris, pH 8.0, 0.5% Triton X-100 and cleaved with thrombin (2-4 µg/ml). Intact G was quantified by immunoblot using AS/7, a polyclonal antipeptide antibody directed against the carboxyl terminus of G(43) . GA/1, an amino terminus-directed G protein-specific polyclonal antibody, reacted similarly with intact and carboxyl-truncated forms of recombinant G. Intact G was ADP-ribosylated by pertussis toxin; as expected, the truncated form, which lacks the ADP-ribose acceptor site (Cys-347), was not.

Preparation of rG

The coding region of bovine G cDNA was inserted into the pT7/Nde expression vector(44) . This was transfected into E. coli BL21(DE3) cells (Novagen), which had or had not been previously transfected with the pACYC177/ET3d/yNMT vector(45) . Bacteria cotransfected with both plasmids overexpressed the yeast N-myristoyltransferase and G, with the resultant production of myristoylated rG(46) , whereas those transfected only with pT7/Nde containing G sequence overexpressed nonmyristoylated rG. An overnight culture (0.2 ml) of the cotransfected bacteria was added to 2 ml of LB medium containing the appropriate antibiotics, grown at 37 °C for 1 h, and induced with 0.2 mM isopropyl--D-thiogalactopyranoside in the presence of [H]myristic acid (100 µCi, 16 Ci/mmol). Growth was continued for an additional 3 h before centrifugation (15,000 g, 7 min). Cells were then resuspended in phosphate-buffered saline, pH 7.4; lysed by sonification; and centrifuged (15,000 g, 45 min). The supernatant, which contained crude H-labeled myristoylated rG, was used in phase-partitioning experiments. The same procedure was used for the singly transfected cells, which produced nonmyristoylated rG. Following partitioning, proteins were detected by autoradiography in experiments using H-labeled myristoylated rG and with an anti-G antibody (47) after blotting to nitrocellulose for both H-labeled myristoylated rG and nonmyristoylated rG.

Pertussis Toxin-catalyzed ADP-ribosylation

G (1 µg) with or without the indicated amounts of G was incubated at 30 °C for 20 min (total volume of 50 µl), in 50 mM potassium phosphate, pH 7.5, 10 mM thymidine, 10 µM [P]NAD (1 µCi/assay), 40 µM ATP with 0.1 µg of pertussis toxin-activated with 20 mM dithiothreitol immediately before use(13) . [P]ADP-ribosylated proteins were subjected to Triton X-114 phase partitioning, separated by SDS-PAGE, and exposed to Kodak XAR film at -70 °C for 1-4 h.

NAD:Arginine ADP-ribosyltransferase-catalyzed ADP-ribosylation

G (45 µg) was incubated at 30 °C for 45 min with or without the NAD:arginine ADP-ribosyltransferase (4.5 µg), which had been purified from turkey erythrocytes (48) in 200 µl of buffer containing 50 mM KPO, pH 7.5, 200 mM NaCl, 100 µM NAD (10-15 µCi). Following labeling, samples were applied to NAP-5 columns (Pharmacia Biotech Inc.) to separate protein-bound from unbound radioactivity (enabling an estimation of extent of labeling) before use in phase-partitioning experiments.


RESULTS

Phase Partitioning of Native Rod Outer Segment Proteins

Arrestin, a soluble 48-kDa protein that copurified with G, remained primarily in the detergent-depleted phase (Fig. 1A and Table 1), whereas G (which contains farnesylated G) and PDE (the subunit of which is prenylated) (49) were found mainly in the detergent-rich phase (Fig. 1A and Table 1). Rhodopsin, an integral membrane protein, partitioned, as expected, almost exclusively into the detergent-rich phase (Fig. 1B and Table 1).


Figure 1: Phase partitioning of retinal proteins. A, purified G (8 µg), G (4 µg), and purified G (20 µg) in 40 µl of buffer C were subjected to Triton X-114 phase partitioning. C lanes, sample before partitioning; A lanes, aqueous phase; D lanes, detergent phase. The positions of arrestin (Arr; present in G and G preparations), G, G, and G are indicated on the left. B, rhodopsin (24 µg) in phosphatidylcholine vesicles underwent phase partitioning and SDS-PAGE. The position of rhodopsin (R) is indicated on the left. Lanes are labeled as described for A. Molecular weight markers (10) are indicated on the right. After exposure to Triton X-114, rhodopsin appeared as multiple aggregated species on SDS-PAGE.





G, in the absence of G, partitioned about equally between the detergent-rich and detergent-depleted phases (Fig. 1A and Table 1). On repeated partitioning of G from either the aqueous or detergent phase, distribution between detergent and aqueous phases was similar regardless of the phase from which G originated (data not shown). In the presence of G, G was recovered to a greater extent in the detergent-rich phase. Recovery of G in the detergent-rich phase increased with increasing amounts of G (Fig. 2, A and B). In contrast, G appeared not to affect the partitioning of G (Table 1).


Figure 2: Effect of G on partitioning of G. A, G (1.0 µg) was incubated with the indicated amounts of G at 23 °C for 30 min before partitioning. Lanes are labeled as described for Fig. 1A. B, mean ± S.E. (n = 4) of percentage of G in the detergent phase as a function of the amount of G added.



After addition of Gpp(NH)p in the presence of G and rhodopsin, G was recovered to a greater extent in the aqueous phase presumably as a result of dissociation of GGpp(NH)p from G (Fig. 3). In the absence of rhodopsin, Gpp(NH)p had no effect on the partitioning of G (data not shown). Rhodopsin, in the absence of G, did not significantly influence the phase partitioning of G (data not shown), consistent with other evidence that G is important for functional association of G and receptor(5) .


Figure 3: Effect of Gpp(NH)p on partitioning of G and rhodopsin. G (20 µg) and rhodopsin (12 µg) were incubated at 30 °C for 30 min with or without 100 µM Gpp(NH)p (GN) as indicated before phase separation. Lanes are labeled as described for Fig. 1A. Similar results were obtained with 100 µM GTPS (data not shown). The experiment was replicated twice with similar results. Arr, arrestin.



To evaluate directly the role of bound nucleotide in apparent hydrophobicity, samples of GGDP, GGTPS, GGDP, and GGTPS underwent phase partitioning. PD-10 columns were used to separate protein-bound from unbound nucleotide. Ratios of protein-bound nucleotide in aqueous and detergent phases concurred with ratios of total protein on Coomassie Blue-stained SDS-polyacrylamide gels quantified by densitometry (Table 2), indicating that the total protein reflected the fraction containing bound nucleotide, i.e. G retained nucleotide during partitioning. Whether GTPS or GDP was bound made no difference to the overall hydrophobicity of G in the absence of G (Fig. 4A and Table 2). In the presence of G, however, GGDP was recovered to a much greater extent in the detergent phase, likely as a result of its tight association with the more hydrophobic G complex containing the farnesylated subunit. In contrast, GGTPS partitioning was independent of G (Fig. 4B and Table 2).




Figure 4: Effect of bound GDP or GTPS on partitioning of G with and without G. A, G[H]GDP (8 µg) and G[S]GTPS (8 µg) were subjected to partitioning. B, G[H]GDP (30 µg) and G[S]GTPS (30 µg) were subjected to phase partitioning. The positions of G and G are indicated on the left. Nucleotide bound is indicated at the bottom; A and D indicate aqueous and detergent phases, respectively.



Interaction between the PDE complex and GGTPS was studied by partitioning the proteins separately and together, which yielded similar results whether the amounts of G and PDE were equimolar or one or the other was in excess (data not shown). To verify that the proteins were not denatured during partitioning, PDE activity and GGTPS-activated PDE activity were determined. The distribution of PDE activity in detergent and aqueous phases was similar whether or not GGTPS was present during partitioning (Fig. 5) and also when PDE partitioned in the absence of GGTPS was assayed in its presence (data not shown). The partitioning of GGTPS was not affected by PDE whether measured by SDS-PAGE and densitometry or by quantifying protein-bound radioactive nucleotide (data not shown).


Figure 5: Effect of GGTPS on phase partitioning of PDE activity. PDE (0.1 µg) was subjected to phase partitioning with or without GGTPS (2.0 µg). Triton X-114 and buffer A were then added back to equalize detergent and buffer concentrations in the two phases, and an additional 100 µl of buffer A were added to each. Samples (100 µl) of these mixtures were assayed for PDE activity. Emptybars represent activity recovered in the aqueous phase, and hatchedbars in the detergent phase. Errorbars represent mean ± S.E. (n = 3). Bars 1 and 2, PDE partitioned alone; bars3 and 4, PDE partitioned with GGTPS.



Phase Partitioning of Tryptic Fragments of Gand PDE

To define better the structural domains responsible for partitioning, G, G, and G were subjected to limited proteolysis with trypsin. Cleavage products of G were recovered in the detergent-rich phase to a greater extent than were those from G (Fig. 6). In the presence of G, the fraction of intact 39-kDa G recovered in that phase was greater than that of the 37-kDa fragment lacking the amino terminus (Fig. 6). Since the amino terminus of G is involved in interaction with G, it was unclear whether the difference in behavior of intact G and the 37-kDa protein resulted from disruption of the G-binding site with loss of ability of G to associate with and be influenced by G or whether the amino terminus itself contributed significantly to the hydrophobicity of G. To evaluate this, G was trypsinized without G, and in some experiments, after stopping trypsinolysis with phenylmethylsulfonyl fluoride or soybean trypsin inhibitor, intact G was added back before partitioning to ensure that sufficient intact G would be recovered for densitometric measurement. Even in the absence of G, intact G was recovered to a greater extent in the detergent-rich phase than was the 37-kDa fragment (and other major tryptic products), although amounts of both were less than when G was present (Fig. 7; data not shown), consistent with the conclusion that the amino terminus is primarily responsible for the hydrophobic nature of G and that this hydrophobicity is not a consequence solely of interaction with G. After trypsinolysis, PDE accumulated mostly in the detergent-depleted phase (Table 1).


Figure 6: Phase partitioning of tryptic fragments of transducin. G (20 µg) in hypotonic buffer was subjected to limited proteolysis with trypsin (Try) before partitioning. The positions of protein standards, G, G, G, and arrestin (Arr) are indicated on the left, and those of major proteolysis fragments on the right. Lanes are labeled as described for Fig. 1A. 33% of the intact 39-kDa G was aqueous, whereas 70% of the 37-kDa fragment was aqueous. The experiment was repeated four times with similar results.




Figure 7: Phase partitioning of tryptic fragments of G. G (8 µg) in hypotonic buffer was subjected to limited proteolysis with trypsin before partitioning. The positions of arrestin (Arr), intact G and the 37-kDa fragment are indicated on the right. Lanes are labeled as described for Fig. 1A. Of intact G, 56 ± 2.3% was in the aqueous phase versus 88.3 ± 9.0% of the 37-kDa product. The mean ± S.E. was derived from four separate assays.



Effect of Carboxyl-terminal Truncation of G

Recombinant G and G lacking 20 amino acids at the carboxyl terminus were prepared as described under ``Methods.'' When partitioned in the same tube, carboxyl-terminal truncated G was consistently recovered to a greater extent in the detergent-depleted phase than was intact recombinant G (Fig. 8).


Figure 8: Phase partitioning of intact recombinant and carboxyl-terminal truncated G. Intact (4 µg) and carboxyl-truncated (3 µg; Val-331 stop) G were added to the same microcentrifuge tube for partitioning. The positions of glutathione S-transferase (G S-T), intact recombinant G (rG), and carboxyl-truncated G (Val 331 stop) are indicated on the right. Molecular weight standards are indicated on the left. Lanes are labeled as described for Fig. 1A. The experiment was repeated four times with different preparations of intact and carboxyl-truncated G. Consistently, a higher percentage of the truncated than the intact form was recovered in the aqueous phase. The mean ± S.E. of differences in the percentage of G and truncated G recovered in the aqueous phase was 18.7 ± 5.2% (n = 4).



Effect of Myristoylation of the Amino Terminus of rG

When subjected to phase partitioning, nonmyristoylated rG was recovered primarily in the aqueous phase, whereas myristoylated rG was totally recovered in the detergent-rich phase (Fig. 9, A and B).


Figure 9: Phase partitioning of myristoylated recombinant and nonmyristoylated G. Crude supernatants of bacterially expressed myristoylated and nonmyristoylated G, prepared as described under ``Methods,'' were subjected to phase partitioning and SDS-PAGE (12% acrylamide). Detection of H-labeled myristoylated rG was after treatment of the gel with Promote (Integrated Separation Systems) and exposure to Kodak X-Omat film at -70 °C for 1-4 days. Detection of nonmyristoylated rG was with an anti-G polyclonal antibody (47) after transfer of proteins from the gel to nitrocellulose. The nonmyristoylated form was recovered to a greater extent in the aqueous phase (58.8 ± 7.9%, n = 5), while myristoylated rG was nearly 100% recovered in the detergent phase (n = 6). A, Western blot analysis of phase-partitioned nonmyristoylated rG. A lane, aqueous phase; D lane, detergent phase. B, H-labeled myristoylated rG. Lanes are labeled as described for A.



Effect of ADP-ribosylation of Gon Phase Partitioning

G and ADP-ribosylated G were evaluated in the same assay by protein staining and autoradiography. Pertussis toxin-catalyzed ADP-ribosylation did not significantly alter the partitioning of G alone. Of radiolabeled ADP-ribosylated G, 70.0 ± 11.6% (n = 3) was recovered in the detergent phase compared with 63.3 ± 3.3% (n = 3) of G that had been incubated with pertussis toxin without NAD (see also Table 1). There was a tendency for ADP-ribosylated G to associate more than unmodified G with G since when it was partitioned with equimolar G, 89 ± 5.1% (n = 3) of the ADP-ribosylated G was recovered in the detergent phase, whereas only 77.3 ± 1.8% (n = 3) of the protein was recovered in the detergent phase when no NAD was included (see also Table 1). NAD: arginine ADP-ribosyltransferase modified G to the extent of 5 mol of ADP-ribose/mol of G. Comparing this G with G that had been processed in an identical fashion without transferase, recovery of ADP-ribosylated G in the aqueous phase was greater, i.e. 57.0 ± 2.9% (n = 4) versus 46 ± 2.4% (n = 4) as shown in Fig. 10. When equal amounts of labeled and unlabeled G were mixed and partitioned, more of the unlabeled G was aqueous (55.7 ± 2.5%, n = 3), whereas the partitioning of the labeled G was unchanged (56.5 ± 1.7%, n = 3) (Fig. 10). When partitioned in the presence of excess native G, the partitioning of ADP-ribosylated G was not significantly affected as 58.7 ± 5.9% (n = 3) was recovered in the aqueous phase, whereas native G remained 46.0 ± 3.6% (n = 3) aqueous.


Figure 10: Effect of NAD:arginine ADP-ribosyltransferase-catalyzed ADP-ribosylation of G on phase partitioning. G was ADP-ribosylated and subjected to phase partitioning as described under ``Methods.'' SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue, and proteins were quantitated by densitometry; or in the case of P-labeled ADP-ribosylated G, quantitation was also done by phosphoimaging on a Molecular Dynamics PhosphorImager. Coomassie Blue-stained gel of 2 µg of phased-partitioned arginine-ADP-ribosylated G (G*), native G (G), or both (G/G*). The positions of G* and G are indicated on the right. A and D represent aqueous and detergent phases, respectively.




DISCUSSION

Triton X-114 phase partitioning initially received attention as a way of purifying integral membrane proteins, which are typically retained completely within the detergent phase(34) . It also has been used for the separation of more hydrophobic proteins from those that are less so, as well as for evaluating the hydrophobicity of proteins (50, 51, 52, 53, 54, 55, 56) . Reasons for hydrophobic behavior in a detergent environment include either the post- or cotranslational addition of hydrophobic moieties (myristoyl, palmitoyl, isoprenyl, etc.) or intrinsic features of protein secondary structure.

On Triton X-114 phase partitioning, the integral membrane receptor rhodopsin partitioned completely into the detergent-rich phase, whereas soluble arrestin was mainly aqueous. The farnesylated G complex was recovered mostly in the detergent-rich phase. The PDE complex, which was intact based on its enzymatic activity, initially partitioned as a highly hydrophobic complex. After trypsinization, 88% was recovered in the aqueous phase, consistent with previous findings that trypsin removes a prenyl moiety from the PDE subunit, releasing the PDE complex from membranes(57) . Similarly, addition of a hydrophobic myristate group to a recombinant heterotrimeric G protein subunit, G, shifted its recovery to the detergent-rich phase, whereas the nonmyristoylated form was mostly aqueous. Therefore, in certain instances, the presence of a hydrophobic group may affect the overall hydrophobicity measured in this assay.

G protein subunits are localized at the cytosolic face of the plasma membrane(1) . Membrane attachment of at least two of these proteins, G and G, is facilitated by their myristoylated amino termini, although myristoylation alone may not be sufficient for membrane attachment(58) . G, modified at its amino terminus in a heterogeneous manner by lauroyl (C), myristoyl (C), (cis-5)-tetradecaenoyl (C), or (cis,cis-5,8)-tetradecadienoyl (C) fatty acylation(59, 60) , is mainly a soluble protein and, unlike other subunits, does not require detergents for purification. It is released from ROS by addition of MgGTP. After binding GTP, G undergoes a conformational change, documented in recent crystallographic studies(2, 3) , although how this change affects overall hydrophobicity has not been well defined.

The partitioning of G was consistent with other evidence that it does not behave as either a typical integral membrane protein or a soluble protein. In the absence of G, G partitioned approximately equally between the detergent-rich and detergent-depleted phases. On sequential phase partitioning, G derived from either the detergent-rich or detergent-depleted phase partitioned in much the same way as it did initially. Thus, there was no evidence of heterogeneity of preparations isolated in the same way. There was, however, a small difference between the apparent hydrophobicity of G preparations isolated differently. The G subunits in the experiments of Table 2were isolated from ROS using a nearly isotonic wash, which selectively eluted G, leaving behind G, whereas the apparently more hydrophobic G used in Table 1was eluted from ROS as a G complex using hypotonic conditions and subsequently separated from G using blue Sepharose.

It appears that both the amino and carboxyl termini contribute to the hydophobicity of G. The phase-partitioning data reported here are consonant with other observations on the trypsin-cleaved protein (61, 62) that implicate the amino terminus as the major hydrophobic segment of G. Unlike G, G, and G, G is not N-terminally myristoylated. The carboxyl terminus of G has been implicated in its ability to associate with membranes(17, 18) . To determine whether the carboxyl terminus of G similarly imparts hydrophobicity, recombinant G and a carboxyl-truncated mutant were synthesized. Recombinant G did not bind labeled nucleotide in the presence of G and rhodopsin, which may have been secondary to its initially insoluble state and the consequent use of detergent for solubilization. Lack of N-terminal fatty acylation, resulting in lower affinity of G for G, may have been a factor as well. Although G did not stimulate the reaction, recombinant wild-type G was ADP-ribosylated by pertussis toxin, from which retention of at least some degree of native structure in the carboxyl terminus was inferred. ADP-ribosylation did not occur with G that had been heat-denatured or, as expected, with the carboxyl-terminal truncated variant (data not shown). Nearly 20% (18.7 ± 5.2%, n = 4) more of the full-length than the truncated G was recovered in the detergent-rich phase on partitioning, from which it was inferred that the carboxyl terminus contributes to its hydrophobicity, as it does in G(17, 18) , although to a lesser extent than the amino terminus.

It has been suggested that the amino and carboxyl termini of G are in close proximity and that the amino terminus of G is critical for G binding. The apparent hydrophobicity of G was enhanced in a concentration-dependent manner by G, but only when G was in the inactive GDP-bound form, i.e. the conformational change induced by GTP binding did not affect the overall hydrophobicity of G, but rather influenced its partitioning indirectly by altering G interaction with the more hydrophobic G. If Triton X-114 does interfere with the GGDP-G interaction, sufficient interaction apparently persists to permit observation of an effect. The failure of GGTPS or PDE to alter the partitioning of the other protein could mean that Triton X-114 interferes with this protein-protein interaction. (The increased activity recovered upon partitioning PDE in the presence of GGTPS suggests, but does prove, that interaction occurred during partitioning.) Formation of a ``tight complex'' between PDE and GGTPS that would partition differently from the individual proteins may require an additional protein or environment supplied by ROS but lacking in this simple system, although it is not clear that a tight complex is required for activation of PDE. In studies reported to demonstrate a tight complex of PDE and G, either ROS were the supporting membrane surface (63, 64, 65) or the fraction of proteins in the complex was so small (<4%) (66, 67) that it would not have been detected in the partitioning assay. In one study, phospholipid vesicles were used as a support for cross-linking PDE and G(67) . This, however, requires only an association close enough to enable cross-linking, and the existence of a stable complex in the artificial membrane was not established(67) .

Pertussis toxin-catalyzed ADP-ribosylation of a cysteine four amino acids from the carboxyl terminus of G did not alter its partitioning in the absence of G. However, ADP-ribosylated G appeared to have enhanced affinity for G, as when partitioned with G, more ADP-ribosylated G than non-ADP-ribosylated G was recovered in the detergent phase. There was, however, a clear difference in the partitioning behavior of arginine-ADP-ribosylated G, a difference likely explained by the greater extent of ADP-ribosylation. At least 5 mol of ADP-ribose/mol of G were incorporated using NAD:arginine ADP-ribosyltransferase as catalyst, whereas pertussis toxin modifies only a single cysteine(12) . It is also possible that the specific site ADP-ribosylated by pertussis toxin (Cys-347) is relatively inaccessible to the external milieu and therefore does not influence partitioning; the ADP-ribosylated arginines may be more exposed to the medium. In equimolar mixtures of arginine-ADP-ribosylated G and native G, the latter behaved similarly to the modified G, i.e. its recovery in the aqueous phase was increased. This might be interpreted as a reflection of dimerization of G, as has been suggested on the basis of other kinds of studies(37, 68) .


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: NIH, 10 Center Dr. MSC 1434, Bldg. 10, Rm. 5N/307, Bethesda, MD 20892. Tel.: 301-496-4554; Fax: 301-402-1610.

Present address: Pulmonary and Critical Care Medicine, Decatur VA Medical Center, Emory University, VAMC Rm. 1253, 1670 Clairmont Rd., Decatur, GA 30033.

The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; G, subunit of transducin; G, subunit of transducin; rG, recombinant G; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; GTPS, guanosine 5`-O-(3-thiotriphosphate); TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; PDE, retinal cGMP phosphodiesterase; MOPS, 4-morpholinepropanesulfonic acid; ROS, rod outer segments.


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