Folding a WD Repeat Propeller
1998; Elsevier BV; Volume: 273; Issue: 15 Linguagem: Inglês
10.1074/jbc.273.15.9041
ISSN1083-351X
AutoresIrene García-Higuera, Chrysanthe Gaitatzes, Temple F. Smith, Eva J. Neer,
Tópico(s)Electrospun Nanofibers in Biomedical Applications
ResumoThe β subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of an amino-terminal α-helical segment followed by seven repeating units called WD (Trp-Asp) repeats that occur in about 140 different proteins. The seven WD repeats in Gβ, the only WD repeat protein whose crystal structure is known, form seven antiparallel β sheets making up the blades of a toroidal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell83, 1047–1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369–374). It is likely that all proteins with WD repeats form a propeller structure. Alignment of the sequence of 918 unique WD repeats reveals that 85% of the repeats have an aspartic acid (D) residue (not the D of WD) in the turn connecting β strands b and c of each putative propeller blade. We mutated each of these conserved Asp residues to Gly individually and in pairs in Gβ and in Sec13, a yeast WD repeat protein involved in vesicular traffic, and then analyzed the ability of the mutant proteins to fold in vitro and in COS-7 cells. In vitro, most single mutant Gβ subunits fold into Gβγ dimers more slowly than wild type to a degree that varies with the blade. In contrast, all single mutants form normal amounts of Gβγ in COS-7 cells, although some dimers show subtle local distortions of structure. Most double mutants assemble poorly in both systems. We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the propeller structure. Some may affect the folding pathway or the affinity for chaperonins. Mutations of the conserved Asp in Sec13 affect folding equally in vitro and in COS-7 cells. The repeats that most affected folding were not at the same position in Sec13 and Gβ. Our finding, both in Gβ and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant for the same protein. The β subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of an amino-terminal α-helical segment followed by seven repeating units called WD (Trp-Asp) repeats that occur in about 140 different proteins. The seven WD repeats in Gβ, the only WD repeat protein whose crystal structure is known, form seven antiparallel β sheets making up the blades of a toroidal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell83, 1047–1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369–374). It is likely that all proteins with WD repeats form a propeller structure. Alignment of the sequence of 918 unique WD repeats reveals that 85% of the repeats have an aspartic acid (D) residue (not the D of WD) in the turn connecting β strands b and c of each putative propeller blade. We mutated each of these conserved Asp residues to Gly individually and in pairs in Gβ and in Sec13, a yeast WD repeat protein involved in vesicular traffic, and then analyzed the ability of the mutant proteins to fold in vitro and in COS-7 cells. In vitro, most single mutant Gβ subunits fold into Gβγ dimers more slowly than wild type to a degree that varies with the blade. In contrast, all single mutants form normal amounts of Gβγ in COS-7 cells, although some dimers show subtle local distortions of structure. Most double mutants assemble poorly in both systems. We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the propeller structure. Some may affect the folding pathway or the affinity for chaperonins. Mutations of the conserved Asp in Sec13 affect folding equally in vitro and in COS-7 cells. The repeats that most affected folding were not at the same position in Sec13 and Gβ. Our finding, both in Gβ and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant for the same protein. The β subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of two distinct regions as follows: an amino-terminal α-helical segment, followed by 7 repeating units called WD repeats that occur in about 140 different proteins (reviewed in Refs. 1Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar and 2Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). Members of the family of WD repeat proteins do not have an immediately obvious common function but are involved in diverse cellular pathways such as signal transduction, pre-mRNA splicing, transcriptional regulation, cytoskeletal assembly, and vesicular traffic (2Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). Each WD repeat consists of a conserved core of approximately 40 amino acids (typically bracketed by the dipeptides GH (glycine-histidine) and WD (tryptophan-aspartic acid)) and a variable region of 7–11 amino acids (2Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). Gβ is the only WD repeat protein whose crystal structure is known (3Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1051) Google Scholar, 4Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar, 5Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1014) Google Scholar). The seven WD repeats in Gβ are arranged in a ring to form a propeller structure with seven blades. Each blade of the propeller consists of a four-stranded antiparallel β sheet oriented so that the outer surfaces of the torus are composed of the sheet edges, whereas the turns protrude from the two flat surfaces (see Fig. 1). It is likely that all proteins with WD repeats form a propeller structure, although with varying numbers of blades corresponding to varying numbers of repeating units. WD repeats are not essential to form a propeller. Other families of proteins with no sequence similarity to WD repeat proteins form propellers whose blades are virtually identical to those in Gβ (reviewed in Ref. 6Neer E.J. Smith T.F. Cell. 1996; 84: 175-178Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Nevertheless, within the subset of propellers formed of WD repeats, it is reasonable to suppose that the most highly conserved residues play an important role either in the function or the structure. The WD repeats are not characterized by a rigidly conserved sequence but rather by their fit to a regular expression that allows limited variation at each position (2Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar). However, alignment of the sequences of 918 unique WD repeats in our data set reveals that one residue is the most conserved; an aspartic acid residue (D, not the D in WD) located in the loop connecting β strands b and c of each propeller blade in Gβ (and presumably in all other WD repeat proteins) occurs in 85% of the repeats. In another 9%, the residue is Glu or Asn. This extraordinary conservation suggests that the Asp residue performs an important function that is shared by all WD repeats. Since the WD repeat proteins do not appear to bind to any common molecule, we tested the hypothesis that the conserved Asp plays a role in the folding of the propeller. The occurrence of a conserved residue at an equivalent position in each repeat allowed us to ask a number of questions. Are all the Asp residues equivalent within a protein? Are the consequences of mutating Asp to Gly the same in different proteins? It is not known whether the WD repeat or other propeller proteins fold by a single or multiple pathways. If there is a single pathway, we would expect that mutation of a critical Asp would have a large effect on folding kinetics, whereas if multiple pathways to the final structure exist, a single mutation might have little effect since it would be kinetically less important if an alternative pathway could be followed (7Harrison S.C. Durbin R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4028-4030Crossref PubMed Scopus (177) Google Scholar). To analyze such questions, we mutated the conserved Asp to Gly in two WD repeat proteins, Gβ and Sec13, a yeast protein involved in vesicular traffic (8Pryer N.K. Salama N.R. Schekman R.W. Kaiser C.A. J. Cell Biol. 1993; 120: 865-875Crossref PubMed Scopus (120) Google Scholar). Mutations were inserted one at a time or two at a time. We mutated Asp to Gly because a Gly residue makes the polypeptide chain flexible and is compatible with formation of a turn. Futhermore, the side chain of Asp points into the structure of β and, in some cases, makes contact with other residues within the propeller blade (see “Discussion”). Therefore, we wanted an amino acid that had a small side chain not to confound interpretation by effects produced by the side chain of the amino acid substituted for the aspartic acid residue. Gβ was chosen because its crystal structure is known. Sec13 has 6 repeats and no amino- or carboxyl-terminal extension. We have made and tested a model of Sec13 based on the structure of Gβ (9Saxena K. Gaitatzes C. Walsh M.T. Eck M. Neer E.J. Smith T.F. Biochemistry. 1996; 35: 15215-15221Crossref PubMed Scopus (37) Google Scholar). The model predicts that the conserved Asp are in equivalent positions to Gβ. The Gβ and Sec13 differ in their requirements for folding. Gβ cannot fold completely without Gγ (10Schmidt C.J. Neer E.J. J. Biol. Chem. 1991; 266: 4538-4544Abstract Full Text PDF PubMed Google Scholar) to which it is very tightly bound in the native structure. Furthermore, folding and/or assembly probably requires as yet undefined chaperones (11Mende U. Schmidt C.J. Yi F. Spring D.J. Neer E.J. J. Biol. Chem. 1995; 270: 15892-15898Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In contrast, Sec13 can fold into a globular, trypsin-resistant structure when synthesized in Escherichia coli, wheat germ, rabbit reticulocyte lysate in in vitro translation systems, or in mammalian cells (9Saxena K. Gaitatzes C. Walsh M.T. Eck M. Neer E.J. Smith T.F. Biochemistry. 1996; 35: 15215-15221Crossref PubMed Scopus (37) Google Scholar, 12Garcia-Higuera I. Fenoglio J. Li Y. Lewis C. Panchenko M.P. Reiner O. Smith T.F. Neer E.J. Biochemistry. 1996; 35: 13985-13994Crossref PubMed Scopus (164) Google Scholar). If it requires chaperones at all, it can productively interact with several different ones. We have analyzed the ability of Gβ to fold and assemble with Gγ and of Sec13 to form a compact structure after synthesis in vitro and in COS-7 cells. This comparison allows us to discriminate between mutations that affect the end state and those that affect the rate of folding. COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 1The abbreviations used are: FBS, fetal bovine serum; PBS, phosphate-buffered saline; Hβ1, hexahistidine-tagged β1; HA, hemagglutinin; BMH, 1,6-bismaleimidohexane; PAGE, polyacrylamide gel electrophoresis; wt, wild type. 2 mmglutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin. Transfections were done with LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Typically, cells on 6-well dishes were transfected with 2 μg of total DNA and 15 μg of LipofectAMINE in 1 ml of Opti-MEM (Life Technologies, Inc.) for 5–6 h, after which 1 volume of Opti-MEM supplemented with 8% FBS was added to each well. 18–24 h after the start of transfection, this medium was replaced with complete culture medium (Dulbecco's modified Eagle's medium + 10% FBS), and cells were incubated at 37 °C overnight and then biosynthetically labeled. For labeling, cells were first starved in a methionine/cysteine-deficient RPMI medium containing 5% dialyzed fetal bovine serum for 30–45 min and then labeled with 0.1 mCi of Express Protein Labeling Mix (NEN Life Science Products) per well (1 ml) in the presence of 10% dialyzed FBS. After 2.5–3 h at 37 °C, the medium was removed, and cells were washed twice with PBS and harvested by trypsinization. Mutations in the β1 cDNA were generated using the Altered Sites in vitromutagenesis system (Promega). To construct a hexahistidine-tagged β1 (Hβ1) subunit, the initial methionine was mutated to glutamine, and at the same time, a HindIII and a PstI site were introduced. An annealed double-stranded DNA encoding the first methionine and six histidines was synthesized and ligated between the new HindIII site and theEcoRI site from the pAlter vector. The amino acid sequence of the amino-terminally tagged β1 is MSHHHHHHGSLLQ. In addition, to facilitate the transfer of the mutants to other vectors, a silent mutation corresponding to amino acids 144 and 145 was introduced into β1 to create a unique KpnI site. This construct (Hβ1 in pAlter) was used as a template for creating all mutants. The mutated residues were Asp-76 in repeat 1 (Hβ1[D1]), Asp-118 in repeat 2 [Hβ1[D2]), Asp-163 in repeat 3 (Hβ1[D3]), Asp-205 in repeat 4 (Hβ1[D4]), Asp-247 in repeat 5 (Hβ1[D5]), Asp-291 in repeat 6 (Hβ1[D6]), and Asp-333 in repeat 7 (Hβ1[D7]), and all were changed to glycine using the codon that allowed a single base substitution (GGT or GGC). All mutations were confirmed by double-stranded sequencing. For expression in COS-7 cells, the wild-type (wt) Hβ1 cDNA or the mutated forms were transferred to the pcDNA3 vector (Invitrogen). The single mutants Hβ1[D2] and Hβ1[D3] were obtained from the double mutant Hβ1[D2–3] by inserting a HindIII-KpnI fragment containing the [D2] mutation or a KpnI-BamHI fragment with the [D3] mutation into an Hβ1-pcDNA3 background. Likewise, the double mutants Hβ1[D1–7] and Hβ1[D2–7] were generated by inserting theHindIII-KpnI fragment from either Hβ1[D1] or Hβ1[D2] into Hβ1[D7] in pcDNA3. For Hβ1[D4–7], Hβ1[D4] in pcDNA3 was cut with NdeI and ligated into Hβ1[D7]. The HA-γ2 construct (γ2 tagged at the amino terminus with the hemagglutinin epitope) previously described (11Mende U. Schmidt C.J. Yi F. Spring D.J. Neer E.J. J. Biol. Chem. 1995; 270: 15892-15898Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) was also subcloned into the pcDNA3 vector. αi-2 cDNA was originally provided by Dr. R. R. Reed and was subsequently transferred to pcDNA3. γ3 (kindly provided by Dr. M. I. Simon, Caltech, Pasadena, CA) was amino-terminally tagged with the hemagglutinin epitope by cloning the coding sequence between the StuI and XbaI sites in the HA-tag pBS (R) vector (a hemagglutinin-tagged Bluescript vector provided by Dr. T. Kirchhausen, Harvard Medical School, Boston). The same sites had been previously introduced at the 5′ and 3′ ends of γ3 by polymerase chain reaction. The HA-γ3 sequence was then cloned in pcDNA3. Prior to mutagenesis of Sec13 cDNA (kindly provided by Dr. C. Kaiser, Massachusetts Institute of Technology, Cambridge, MA), an HA epitope was fused to the amino terminus of the protein. For this purpose, a StuI site was introduced by polymerase chain reaction 3′ to the starting methionine, and aStuI-BglII fragment from the polymerase chain reaction product together with a BglII-SacI fragment from Sec13 in pBluescript (described in Ref. 12Garcia-Higuera I. Fenoglio J. Li Y. Lewis C. Panchenko M.P. Reiner O. Smith T.F. Neer E.J. Biochemistry. 1996; 35: 13985-13994Crossref PubMed Scopus (164) Google Scholar) were cloned between the StuI and SacI sites of the hemagglutinin-tagged Bluescript vector (HA-pBS). The coding sequence of HA-Sec13 was then transferred to the pAlterMax vector (Promega), and this construct was used as template to create mutations using the altered sites mammalian mutagenesis system. The mutated residues were Asp-30 (HA-Sec[D1]), Asp-76 (HA-Sec[D2]), Asp-122 (HA-Sec[D3]), Asp-179 (HA-Sec[D4]), Asp-228 (HA-Sec[D5]), and Asp-275 (HA-Sec[D6]). Each was changed to glycine. All proteins were transcribed and translated using the TNT-coupled reticulocyte lysate system (Promega). Typically, 1 μg of plasmid DNA and 20 μCi of [35S]methionine or [35S]cysteine were used in a 50-μl reaction. In all cases, transcription was directed by the T7 promoter either from pAlter or pcDNA3. To increase expression levels, all γ subunits were subcloned into the PAGA-1 vector (provided by Dr. O. Reiner) that contains a poly(A) sequence and the alfalfa mosaic virus leader sequence, which has been previously shown to improve translation efficiency (13Jobling S.A. Gehrke L. Nature. 1987; 325: 622-625Crossref PubMed Scopus (198) Google Scholar). Synthesis of the desired product was routinely verified by running 2–5 μl of the translation mixture in a small 11 or 13% polyacrylamide gel (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) followed by autoradiography with overnight exposure. Mixtures of independently translated β and γ were then made, such that γ was in excess and the subunits were incubated together at 37 °C for 90 min to dimerize. After dimerization, the samples were either subjected to immunoprecipitation or to trypsin digestion. For immunoprecipitation, 10–20 μl of the mixture was diluted in 500 μl of RIPA buffer (25 mm Tris-Cl, pH 7.6, 150 mm NaCl, 4 mm EDTA, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40), precleared with 40 μl of protein A-Sepharose slurry (50% v/v in PBS) for 45 min at 4 °C, and incubated for 60–90 min with 2 μl of 12CA5 monoclonal antibody (Babco) directed against the HA epitope present in the Gγ subunit used. Protein A-Sepharose (50 μl) was then added, and the mixture was rocked for 45–60 min at 4 °C, washed three times in RIPA buffer, and once in 50 mm Tris-HCl, pH 7.5. Immunobeads were then boiled in Laemmli sample buffer (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) and the proteins resolved by SDS-PAGE on 11% polyacrylamide gels followed by autoradiography. Trypsin digestion of in vitro translated proteins was performed as described previously (15Garcia-Higuera I. Thomas T.C. Yi F. Neer E.J. J. Biol. Chem. 1996; 271: 528-535Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Seven μl of βγ mixture or Sec13 translation reaction were treated with 1 μl of 20 μml-1-tosylamido-2-phenylethylchloromethyl ketone-treated trypsin (Cooper Biomed) and incubated at 30 °C for 10 min, after which 2 μl of 100 mm benzamidine were added to stop the reaction. The samples were then boiled for 5 min in Laemmli sample buffer and analyzed electrophoretically. Labeled cells were lysed in TNE buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA) plus 1% Triton X-100 and 0.25% deoxycholic acid supplemented with protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 3 mm benzamidine, and 1 μg/ml each of soy and lima bean trypsin inhibitor). After lysis, all further steps were performed at 4 °C. The lysates (1 ml/well transfected cells) were first precleared with 50 μl of protein A-Sepharose slurry (50% v/v in PBS) for 45–60 min and then incubated for 2 h or overnight with 4 μl of 12CA5 monoclonal antibody (anti-HA epitope). At this point, each sample was usually split in 2 aliquots (500 μl each), and 40 μl of protein A-Sepharose slurry (1:1) was added to each one. After 45–60 min, samples were washed three or four times with the lysis buffer and once with 50 mm Tris-HCl, pH 8, 2 mm MgCl2, 1 mm EDTA (ADP-ribosylation buffer). One aliquot of each sample was then boiled in Laemmli sample buffer, and the other aliquot was subjected to ADP-ribosylation with Bordetella pertussistoxin. The reaction was carried out in a 30-μl volume containing 50 mm Tris-HCl, pH 8, 2 mm MgCl2, 1 mm EDTA, 10 mm dithiothreitol, 10 mm thymidine, 10 μm NAD, 1 mmNADP, 100 μm GTP, 1 mm ATP, 0.5 μCi of [32P]NAD, and 10 μg/ml activated pertussis toxin. After 30–60 min at 37 °C, the samples were washed with 1 ml of ice-cold ADP-ribosylation buffer, boiled in Laemmli sample buffer (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar), and analyzed on 11% SDS-PAGE. For exposure of [32P] signal without contribution from [35S], a black film was placed between the gel and the film to be exposed. Lysis and immunoprecipitation were performed essentially as described above, except that deoxycholic acid was not included in the lysis buffer. Washes in the lysis buffer were followed by two additional washes in 50 mm Tris-HCl, pH 7.5, and the final pellet of protein A-Sepharose beads was resuspended in 30 μl of this same buffer. For trypsin digestion, 1 μl of 20 μml-1-tosylamido-2-phenylethylchloromethyl ketone-treated trypsin (Cooper Biomed) was then added to one of the aliquots of each sample (see above), and all samples were incubated at 30 °C for 10–15 min. The reaction was stopped with 2 μl of 100 mmbenzamidine. For cross-linking, 1 aliquot of each sample was treated with 1.6 μl of freshly prepared 50 mm BMH (1,6-bismaleimidohexane, Pierce) in Me2SO, and the other aliquot received only Me2SO. After 20 min on ice, Laemmli sample buffer containing 15% β-mercaptoethanol was added, and the samples were boiled for 5 min. The final products of both reactions were resolved by SDS-PAGE on 11% polyacrylamide gels followed by autoradiography. After labeling, transfected cells were lysed in buffer A (6 mguanidinium HCl, 0.1 mNa2HPO4/NaH2PO4, pH 8, 10 mm imidazole), and the lysate was then mixed with 50 μl of nickel nitriloacetic acid-agarose slurry (50% v/v in buffer A) (Qiagen) on a nutator for 3–5 h at room temperature. The beads were washed 5 times with buffer A and twice with 25 mm Tris-HCl, pH 6.8, 20 mm imidazole. Purified proteins were eluted by boiling the beads in Laemmli sample buffer supplemented with 200 mm imidazole and analyzed by SDS-PAGE followed by autoradiography. Our goal was to determine if the most conserved residue in the WD repeat family of proteins (the Asp in the turn between β strands b and c of each blade structure, see Fig. 1) is essential for a WD repeat protein to fold into a β-propeller. Mutations of aspartic acid to glycine (Gly) were introduced in individual repeats of the β1subunit, as well as in adjacent repeats (2, 3; 4, 5; 6, 7), or pairwise in separate repeats (1, 7; 2, 7; 4, 7). All mutants were made in β1 tagged at the amino terminus with six histidine residues (Hβ1), which was useful because it allowed us to distinguish mutated from wild-type β in the transfection experiments to be described below. There was no difference between Hβ1 and the wild-type β1 in any of the assays used in this study (data not shown). The mutants are designated by the number of the repeat in which the mutation is placed,e.g. Hβ1 [D1], Hβ1 [D2], etc. The β subunit does not fold into a compact structure without Gγ but, instead, aggregates with itself and/or other proteins (10Schmidt C.J. Neer E.J. J. Biol. Chem. 1991; 266: 4538-4544Abstract Full Text PDF PubMed Google Scholar, 12Garcia-Higuera I. Fenoglio J. Li Y. Lewis C. Panchenko M.P. Reiner O. Smith T.F. Neer E.J. Biochemistry. 1996; 35: 13985-13994Crossref PubMed Scopus (164) Google Scholar). βγ dimers can be synthesized and assembled in vitro using β and γ subunits synthesized in a rabbit reticulocyte lysate. Such dimers are indistinguishable in their physical properties from βγ dimers purified from bovine brain (10Schmidt C.J. Neer E.J. J. Biol. Chem. 1991; 266: 4538-4544Abstract Full Text PDF PubMed Google Scholar). Indeed, we were able to estimate the distance between the cysteine residues at the interface between β and α by chemical cross-linking of in vitrosynthesized βγ to purified α. Our estimate was within 2 Å of that subsequently found in the crystal structure (3Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1051) Google Scholar, 5Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1014) Google Scholar, 15Garcia-Higuera I. Thomas T.C. Yi F. Neer E.J. J. Biol. Chem. 1996; 271: 528-535Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Mutant β1 subunits were synthesized in a rabbit reticulocyte lysate and incubated with independently synthesized γ2 subunits tagged at the amino terminus with a hemagglutinin epitope (HA-γ2). The formation of dimers was assessed by immunoprecipitation of the β1 subunit through the HA epitope in γ2. Mutation of Asp to Gly in all repeats, except repeat 2 (Hβ1[D2]), diminishes the amount of βγ subunit immunoprecipitated (Fig. 2and Table I). Mutations in repeats 6 and 7 (Hβ1[D6] and Hβ1[D7]) produce the most severe phenotypes. None of the β1 subunits with two mutated Asp (either in adjacent repeats or in separate repeats) were able to form significant levels of dimers in vitro. All β1 subunits were translated with the same efficiency, so that the starting amount of β and γ was equal in all samples (data not shown). Therefore, the quantitative differences in the co-immunoprecipitation of Hβ1 reflect the relative ability of each mutant to fold into a βγ dimer.Table ISummary of properties of the D to G mutants in vitro and in cellsMutantHβ1Sec 13In vitroIn cellsIn vitroIn cellsDimer formationDimer formationTrypsin resistanceCross-link to γ3Interaction with αCorrect Stokes radiusTrypsin resistanceTrypsin resistance% wt% wt% wt% wt[D1]30 ± 885 ± 691 ± 4+99 ± 1+++[D2]87 ± 691 ± 392 ± 8+63 ± 8+−−[D3]32 ± 170 ± 95 ± 1+60 ± 10+−−[D4]48 ± 283 ± 691 ± 9+78 ± 8+−−[D5]52 ± 377 ± 667 ± 20+76 ± 12+−−[D6]9 ± 266 ± 993 ± 7+99 ± 1+++[D7]2 ± 176 ± 545 ± 5+99 ± 1[D2–3]17 ± 473 ± 61-2001Tryptic cleavage of Hβ1[D2–3] and Hβ1[D2–7] gave little or no stable carboxyl-terminal tryptic product.24 ± 3[D4–5]8 ± 526 ± 3[D6–7]2 ± 116 ± 3[D1–7]2 ± 117 ± 2[D2–7]1 ± 160 ± 101-2001Tryptic cleavage of Hβ1[D2–3] and Hβ1[D2–7] gave little or no stable carboxyl-terminal tryptic product.63 ± 3[D4–7]2 ± 137 ± 9[D1–6]++1-2001 Tryptic cleavage of Hβ1[D2–3] and Hβ1[D2–7] gave little or no stable carboxyl-terminal tryptic product. Open table in a new tab To determine whether the dimers that did form from some of the single mutants were properly folded, we used a well established tryptic cleavage assay. When Gβ is associated with Gγ, only one of its 32 potential tryptic cleavage sites is accessible to trypsin. Trypsin generates two fragments, an amino-terminal 14-kDa fragment, which is sometimes unstable and difficult to detect on SDS-PAGE, and a stable carboxyl-terminal 24-kDa fragment. The latter is a sensitive indicator of the formation of a properly folded βγ dimer (for example, see Ref. 15Garcia-Higuera I. Thomas T.C. Yi F. Neer E.J. J. Biol. Chem. 1996; 271: 528-535Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Except for Hβ1[D3], the amount of protected carboxyl-terminal fragment (see Fig. 3), produced from each mutant, correlates well with the ability of that β1 mutant to be co-immunoprecipitated through the γ subunit. For this assay, dimerized Gβ is not separated from undimerized aggregated Gβ. The stable tryptic fragment can, therefore, only arise from the approximately 20% of total Gβ that dimerizes, so the yield appears low. The additional bands in the uncleaved lanes are internal starts or premature terminations of Gβ. This experiment suggests that even though most of the single mutants form a substantially reduced amount of dimers compared with Hβ1, the dimers that do form retain the native conformation. The exception is repeat 3 (Hβ1[D3]). Mutating that Asp leads to dimers that are not resistant to tryptic cleavage. The abundance of potential tryptic cleavage sites in the β sequence makes this assay extremely sensitive, since it only takes a small variation in the final structure of the protein to make some of these sites accessible to the enzyme. Therefore, changes in the three-dimensional structure of the Hβ1[D3] mutant could be very subtle. Fig. 4 shows the rate of formation of wild-type Hβ1γ2 dimers compared with Hβ1[D1], a moderately affected mutant shown in Fig. 2. Assembly of wild-type and mutant βγ is only linear for about 30 min. In this initially linear interval, Hβ1[D1]γ2 forms 28% as fast as Hβ1γ2. After 60 min, no more βγ is formed, even though only 20–30% of wild-type Hβ1 has dimerized. Hβ1[D1] dimerizes more slowly, but plateaus at the same time as wild type. The dimerization rate of three other mutants (Hβ1[D3], Hβ1[D4], and Hβ1[D5]) was also slowed to a degree consistent with the yield of βγ dimers, and all plateaued at 30 min (data not shown). The observation that both wild-type and mutant β stop dimerizing before available monomers are depleted suggests that a necessary component in the reticulocyte lysate is depleted or degraded. Adding fresh lysate increases the yield of mutant and wild-type βγ proportionately (about 2-fold) but does not overcome the difference. Adding fresh lysate did not cause synthesis of β or γ to resume (data not shown). The inefficiency of dimerization of Gβ and Gγ in a rabbit reticulocyte lysate suggests that the missing or labile comp
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