INTRODUCTION

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The  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 -helical segment, followed by 7 repeating units called WD repeats that occur in about 140 different proteins (reviewed in Refs. 1 and 2). 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 (2).

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 (2). G is the only WD repeat protein whose crystal structure is known (3-5). The seven WD repeats in G are arranged in a ring to form a propeller structure with seven blades. Each blade of the propeller consists of a four-stranded antiparallel  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 G (reviewed in Ref. 6). 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 (2). 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  strands b and c of each propeller blade in G (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 (7).

To analyze such questions, we mutated the conserved Asp to Gly in two WD repeat proteins, G and Sec13, a yeast protein involved in vesicular traffic (8). 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  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. G 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 G (9). The model predicts that the conserved Asp are in equivalent positions to G. The G and Sec13 differ in their requirements for folding. G cannot fold completely without G (10) to which it is very tightly bound in the native structure. Furthermore, folding and/or assembly probably requires as yet undefined chaperones (11). 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 (9, 12). If it requires chaperones at all, it can productively interact with several different ones.

We have analyzed the ability of G to fold and assemble with G 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.

	EXPERIMENTAL PROCEDURES

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Cell Culture, Transfection, and Biosynthetic Labeling-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS),¹ 2 mM glutamine, 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.

Mutagenesis and Plasmid Construction-- Mutations in the 1 cDNA were generated using the Altered Sites in vitro mutagenesis system (Promega). To construct a hexahistidine-tagged ₁ (H₁) 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 the EcoRI site from the pAlter vector. The amino acid sequence of the amino-terminally tagged ₁ 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 ₁ to create a unique KpnI site. This construct (H₁ in pAlter) was used as a template for creating all mutants. The mutated residues were Asp-76 in repeat 1 (H₁[D1]), Asp-118 in repeat 2 [H₁[D2]), Asp-163 in repeat 3 (H₁[D3]), Asp-205 in repeat 4 (H₁[D4]), Asp-247 in repeat 5 (H₁[D5]), Asp-291 in repeat 6 (H₁[D6]), and Asp-333 in repeat 7 (H₁[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) H₁ cDNA or the mutated forms were transferred to the pcDNA3 vector (Invitrogen). The single mutants H₁[D2] and H₁[D3] were obtained from the double mutant H₁[D2-3] by inserting a HindIII-KpnI fragment containing the [D2] mutation or a KpnI-BamHI fragment with the [D3] mutation into an H₁-pcDNA3 background. Likewise, the double mutants H₁[D1-7] and H₁[D2-7] were generated by inserting the HindIII-KpnI fragment from either H₁[D1] or H₁[D2] into H₁[D7] in pcDNA3. For H₁[D4-7], H₁[D4] in pcDNA3 was cut with NdeI and ligated into H₁[D7].

In Vitro Translation, Immunoprecipitation, and Trypsin Digestion-- 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  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 (13). 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 (14) followed by autoradiography with overnight exposure. Mixtures of independently translated  and  were then made, such that  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.

Immunoprecipitation from COS-7 Cells and ADP-Ribosylation-- 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 mM phenylmethylsulfonyl 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 pertussis toxin. 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 mM NADP, 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 (14), 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.

Trypsin Digestion and Cross-linking of Samples from Transfected Cells-- 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 µM L-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 mM benzamidine. 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% -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.

Nickel Nitriloacetic Acid-Agarose Purification-- After labeling, transfected cells were lysed in buffer A (6 M guanidinium HCl, 0.1 M Na₂HPO₄/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.

	RESULTS

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Mutation of ₁-- Our goal was to determine if the most conserved residue in the WD repeat family of proteins (the Asp in the turn between strands b and c of each blade structure, see Fig. 1) is essential for a WD repeat protein to fold into a -propeller. Mutations of aspartic acid to glycine (Gly) were introduced in individual repeats of the ₁ subunit, 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 ₁ tagged at the amino terminus with six histidine residues (H₁), which was useful because it allowed us to distinguish mutated from wild-type  in the transfection experiments to be described below. There was no difference between H₁ and the wild-type ₁ 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. H₁ [D1], H₁ [D2], etc.

View larger version (84K): [in this window] [in a new window]   Fig. 1.   Structure of the G subunit. A, in this space-filling model, the G subunit is shown in yellow, and the G subunit is shown in red. The conserved Asp residues in the hairpin turn between the b and c strands are shown in blue. B, a ribbon representation of blade three of G is shown in yellow. The position of the conserved Asp residue is shown in blue. The figures were generated using coordinates provided by Dr. S. Sprang, University of Texas Southwestern, Dallas (see Ref. 5).

Analysis of ₁ Mutants Synthesized in Vitro-- The  subunit does not fold into a compact structure without G but, instead, aggregates with itself and/or other proteins (10, 12). dimers can be synthesized and assembled in vitro using  and  subunits synthesized in a rabbit reticulocyte lysate. Such dimers are indistinguishable in their physical properties from dimers purified from bovine brain (10). Indeed, we were able to estimate the distance between the cysteine residues at the interface between  and  by chemical cross-linking of in vitro synthesized to purified . Our estimate was within 2 Å of that subsequently found in the crystal structure (3, 5, 15).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Immunoprecipitation of in vitro translated H1 Asp mutants through HA-2. Equal amounts of wild-type or mutated H1 subunits translated in vitro in a rabbit reticulocyte lysate were incubated with an excess of independently translated HA-2 to allow dimerization. The dimers were immunoprecipitated with an anti-HA antibody. Proteins were separated on 11% SDS-PAGE and visualized by autoradiography. The position of H1 is indicated by an arrow. The numbers under each lane indicate the repeat(s) in which the Asp was mutated. Shown on top (A) is an autoradiogram of a representative experiment. The bar graph (B) shows the average result (± S.E.) of 3 independent experiments. Where there are no error bars, the error was too small to plot. The immunoprecipitated H1 band was quantitated by densitometry, and the values obtained for the mutants are given as percent of the value for the wild-type subunit.

View this table: [in this window] [in a new window]   Table I Summary of properties of the D to G mutants in vitro and in cells The numbers given were calculated as indicated in the legend to figures. All are expressed as percentage of the values obtained for wild-type H1. They represent the mean ± S.E. of 3-8 independent experiments. The mutants are named according to the WD repeat(s) in which the Asp was mutated to Gly. The results in vitro were obtained using proteins translated in a rabbit reticulocyte lysate, as described under "Experimental Procedures." The results in cells come from experiments with transiently transfected COS-7 cells, as described under "Experimental Procedures."

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Trypsin digestion of in vitro translated H1 Asp mutants dimerized with 2. In vitro translated wild-type or mutant H1 subunits were incubated with G2 and treated with (+) or without () trypsin. The control is wild-type H1 incubated without G2. The fragments were resolved on 11% SDS-PAGE and visualized by autoradiography. The 1st lane of each pair corresponds to undigested samples (trypsin). The bands corresponding to undigested H1 and to the carboxyl-terminal proteolytic fragment are indicated by arrows. The number at the top of each pair of lanes indicates the repeat in which the Asp was mutated. The radioactive bands below the H1 band in the lanes without trypsin correspond to incomplete G proteins generated during the in vitro translation as a result of either a premature termination or an internal start. These truncated proteins do not dimerize with the G subunit and therefore do not interfere with our experimental procedures. Note that the (trypsin) lanes contain both dimerized and undimerized H1, but the tryptic product arises only from the dimerized component which is about 20-30% of the total.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Time course of dimerization of wild-type H1 and H1D1. Wild-type H1 and the D1 mutant were translated in vitro and incubated with HA-2 for the indicated periods. The dimerization reaction was stopped by dilution with ice-cold RIPA buffer. The dimers were immunoprecipitated using an anti-HA antibody and resolved by SDS-PAGE. Both dimerization mixtures contained the same amount of H1 protein (either wt or mutated) and an excess of HA-2. A shows the autoradiogram of a representative experiment. The radioactive band corresponding to H1 was then quantitated by densitometry and the results plotted (B). Similar results were obtained in two other experiments.

Analysis of the ₁ Mutants Transiently Expressed in COS-7 Cells-- The inefficiency of dimerization of G and G in a rabbit reticulocyte lysate suggests that the missing or labile components could be provided by a living cell. Therefore, we analyzed the folding of mutant ₁ subunits in COS-7 cells. The function of transfected wild-type or mutated H₁ was again assessed by immunoprecipitation of  by co-transfected HA-2. Because all  constructs were tagged with 6 histidines, we could distinguish transfected  from endogenous  by the difference in size (see Fig. 5). Therefore, we could always measure how much of the  subunit brought down by HA-2 represented the transfected mutant protein. Fig. 5C and Table I summarize the results of such experiments. Fig. 5 also shows the association of with  that is discussed below. We quantitated the intensity of the precipitated H₁ band for each mutant and expressed it as a percentage of wild-type H₁. In contrast to the results in the reticulocyte lysate, there was no large difference in the amount of dimers precipitated between wild type and any single mutant. Control experiments verified that antibody was in excess in these experiments (not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Immunoprecipitation of H1 Asp mutants in transiently transfected COS-7 cells. COS-7 cells were transfected with pcDNA3 vector alone (V), HA-2 alone (), HA-2 plus i-2 ( + ) or HA-2, i-2, and either wt H1 or the Asp mutants. dimers and the associated  subunit were immunoprecipitated from 35S-labeled cell lysates using a monoclonal antibody against the HA epitope in the  subunit. In some experiments, the samples were split in two, and one aliquot was [32P]ADP-ribosylated by pertussis toxin after immunoprecipitation as described under "Experimental Procedures." Proteins were separated on 11% SDS-PAGE and visualized by autoradiography. A and B show two autoradiograms of the same experiment, one labeled with 35S and the other in which the 35S is shielded to show only 32P. The positions of endogenous , H1, and i-2 are indicated by arrows in A. In B, only the  subunit is visible. The numbers below each lane correspond to the repeat(s) in which the Asp was mutated. The band seen in the lane marked V is a nonspecific background product of the same apparent molecular weight as H1 that is seen in some but not all experiments. Where it appears it is subtracted from all the lanes. C, H1 band was quantitated by densitometry and the values obtained for the mutants referred as percent of the values for the wild type. Data from 5 experiments performed as described in A and B plus 3 additional experiments in which no i-2 was transfected were pooled (total: n = 8, ± S.E.) since there was no difference in the relative amount of dimers formed in the two protocols. D, the amount of 35S i-2 was quantitated by densitometry and normalized taking into account the amount of H1 immunoprecipitated in each case. The value obtained for the wt H1 was taken to be 100 and the result for each Asp mutant was then expressed as percent of the wt. The bar graph summarizes the mean (± S.E.) of 5 independent experiments. Where no error bar is shown (6, 7), the error was too small to plot.

Interaction of the Mutant Dimers with the  Subunit in Transfected COS-7 Cells-- COS-7 cells were transfected with wild-type or mutated H₁, HA-₂, and _i2. After labeling COS cells with [³⁵S]methionine, the H₁ mutants together with any associated  subunit were immunoprecipitated through the HA epitope on ₂. In each experiment, we measured the amount of  precipitated through the endogenous  that associated with HA-₂ when no H₁ was transfected. Therefore, we could subtract the amount of  that could be accounted for by the small amount of endogenous  precipitated in each experimental situation. This was always a very small correction (see Fig. 5A). To verify that the strong 39-kDa band that co-immunoprecipitated with was indeed , the immunoprecipitate was treated with pertussis toxin and [³²P]NAD (Fig. 5B). In every experiment, the intensity of the ³⁵S-labeled  band was quantitated and related to the amount of H₁ present in the immunoprecipitate (see Fig. 5D and Table I).

Trypsin Resistance of Mutant Dimers Transiently Expressed in COS-7 Cells-- Immunoprecipitated dimers derived from lysates of [³⁵S]methionine-labeled COS-7 cells were digested with trypsin and analyzed on SDS-PAGE. The two bands corresponding to the carboxyl- and amino-terminal fragments are indicated in Fig. 6A. Note that the amino-terminal fragment is bigger in mutant H₁ or wild-type H₁ than in endogenous  because of the hexahistidine tag. All the mutants, except H₁[D3] and H₁[D7], gave an approximately normal amount of fragments. The intensity of the carboxyl-terminal fragment band was measured and normalized to the amount of H₁ found in the undigested sample (see Fig. 6B and Table I). Consistent with the results from in vitro translated protein, H₁[D3] formed dimers that were not resistant to tryptic cleavage and were presumably not quite properly folded. Nevertheless, H₁[D3] is able to bind  reasonably well, confirming that the overall structure is close to native. Dimers containing H₁[D7] were only partly resistant to trypsin (45%), suggesting that the structure of the -propeller must be subtly different from that of the wild type. The only double mutants that made a substantial amount of dimers included mutations in blades 3 and 7. Both produced little, if any, stable tryptic product.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Trypsin digestion of H1 Asp mutants transiently expressed in COS-7 cells. A, COS-7 cells were transiently transfected with pcDNA3 vector alone (Vect), HA-2 alone (HA-), or HA-2 together with wild-type H1 or the H1 single Asp mutants. The dimers were immunoprecipitated from 35S-labeled cell lysates with an anti-HA antibody, and then half of the sample was treated with trypsin (+trypsin). The fragments were resolved on SDS-PAGE and visualized by autoradiography. The repeat in which the Asp was mutated is indicated at the top of each pair of lanes. The 1st lane of each pair shows the undigested sample. The two tryptic fragments (carboxyl- and amino-terminal) are shown by arrows. The bands corresponding to endogenous G or to transfected H1 are also indicated. B, the carboxyl-terminal tryptic fragment produced by each mutant was quantitated by densitometry, normalized to the amount of H1 found in the corresponding undigested sample (trypsin), and expressed as percent with respect to the amount produced by the wild-type subunit. The bar graph summarizes the mean (± S.E.) of three independent experiments. Where no error bars are shown, the error was too small to plot.

Cross-linking of Mutant Dimers Transiently Expressed in COS-7 Cells-- The  subunit contains two structurally distinct regions as follows: the seven WD repeats that give rise to the seven bladed -propeller, and an amino-terminal segment of approximately 20-30 amino acids that forms a coiled-coil with the amino terminus of the  subunit (3, 5). The trypsin digestion assay provides important information about the integrity of the propeller itself. However, it does not tell us if the amino-terminal -helix, and hence the coiled-coil, are properly formed. These aspects are better studied by analyzing the ability of G to be cross-linked to the ₃ subunit using BMH. We have previously shown that this reagent specifically cross-links a cysteine residue in the -helix of ₁ (cysteine 25) to cysteine 30 in 3 (15). Because the cross-linker has a very limited flexibility, this assay is very sensitive to changes in the structure that might alter the distance between these two residues. Wild-type and mutated H₁ subunits labeled with [³⁵S]methionine were co-immunoprecipitated through HA-tagged ₃ (Fig. 7). BMH cross-linked wild-type and mutant H₁ to ₃ to give a major cross-linked product of ~50 kDa corresponding to H₁-HA₃ (3). The second, fainter band observed right below corresponds to endogenous  subunits cross-linked to HA-₃. This band becomes more evident when H₁ is not included in the transfection (see 1st two lanes, labeled HA-3). These results indicate that the aspartic acid mutations do not induce changes in the structure of the amino-terminal part of G or in the relative orientation of the G subunit in that region. The fact that even the mutants that showed an abnormal behavior on trypsin digestion (such as H₁[D3] or H₁[D7]) are cross-linked to ₃ confirms that the -propeller and the amino-terminal extension are independent regions and that alterations in one do not necessarily alter the conformation of the other.

View larger version (43K): [in this window] [in a new window]   Fig. 7.   Cross-linking of H1 Asp mutants to HA-3. COS-7 cells were transiently transfected with HA-3 alone (HA-) or HA-3 together with wild-type H1 or the H1 single Asp mutants. The dimers formed were immunoprecipitated through the HA epitope in 3, and half of the sample was treated with the cross-linking reagent (BMH). Reactions were stopped by addition of Laemmli sample buffer containing 15% -mercaptoethanol and subjected to 11% SDS-PAGE followed by autoradiography. Control, uncross-linked samples are shown in the 1st lane of each pair, and the numbers on top indicate the WD repeat in which the Asp was mutated. The cross-linked product of approximately 50 kDa obtained in BMH-treated samples from cells transfected with HA-3 alone corresponds to endogenous  cross-linked to transfected HA-3 and can be detected in all other samples. H1 cross-linked to HA-3 gives a slightly bigger and more intense product, present in all other treated samples. Shown is an autoradiogram of a representative experiment that was repeated three times with similar results. Note that in this particular experiment the amount of H1[D6] and H1[D7] that was immunoprecipitated through HA-3 was somewhat lower than wt H1. However, it is within the range of variability consistent with the results shown in Fig. 5.

Effect of Aspartic Acid Mutations in Another WD Repeat Protein, Sec13-- To determine if the results obtained with the G protein ₁ subunit were generalizable to other members of the WD repeat family, we undertook the same analysis on another WD repeat protein, Sec13, a yeast protein involved in vesicular traffic. This protein differs from G in three important respects: it has six repeats with no amino- or carboxyl-terminal extensions; it has no -like partner protein; and it folds even in E. coli, showing no requirement for mammalian over bacterial chaperonins (9). We applied the same strategy as with ₁, mutated the conserved aspartic acid to glycine in each repeat and analyzed the ability of the mutants to fold into a native structure. To facilitate our subsequent analysis, mutations were introduced in a Sec13 construct that had been previously tagged with an HA epitope at the amino terminus (HA-Sec13). We know from our previous studies that Sec13 translated in vitro forms a globular, symmetric protein with a Stokes radius of 26 Å. The compact structure is resistant to tryptic cleavage, despite the presence of multiple (28) potential cleavage sites throughout the sequence (12). We synthesized the mutant Sec13 proteins in a rabbit reticulocyte lysate and measured their Stokes radius and resistance to proteolysis by trypsin. All of the mutants were equally well translated, and all eluted from a calibrated AcA 34 column with a Stokes radius of 26-27 Å (data not shown, see Ref. 12 for an example of the elution pattern). The width of each peak at half-height was the same for mutant and wild-type protein, indicating that there was no detectable increased size heterogeneity in the mutant. Therefore, none of the aspartic acid mutations prevented folding in vitro. However, mutation of Asp in repeats 2, 3, 4, or 5 eliminated the resistance to tryptic cleavage (Fig. 8A). Most likely, these mutants form a less rigid structure that allows some tryptic sites to become exposed. Mutations in blades 1 and 6 were normal in both assays. The double mutant, HA-Sec[D1-6], was also resistant to tryptic cleavage (data not shown). The same results were obtained when the HA-Sec13 mutants were transfected in COS-7 cells, immunoprecipitated through the HA epitope, and digested with trypsin (see Fig. 8B and Table I). These experiments confirmed our previous observation that not all of the conserved aspartic acids are equivalent, and that each contributes to a different degree to the stabilization of the  propeller. They further support the idea that not all aspartic acids are essential for a WD protein to fold properly. In addition, our results indicate that the effect of mutating one of the conserved aspartic acids cannot be predicted based on the position of the repeat in which that residue is found, because the location of the essential or non-essential aspartic acids is not conserved in different WD repeat proteins.

View larger version (48K): [in this window] [in a new window]   Fig. 8.   Trypsin digestion of HA-Sec13 Asp mutants translated in vitro or transiently expressed in COS-7 cells. A, wild-type or mutated HA-Sec13 were translated in vitro in the presence of [35S]Met and [35S]Cys and then incubated without () or with (+) trypsin. The reaction products were then resolved by SDS-PAGE (11%) and visualized by autoradiography. B, wild-type or mutated HA-Sec13 were transiently expressed in COS-7 cells and immunoprecipitated from 35S-labeled cell lysates with an anti-HA antibody. The immunoprecipitates were then treated without () or with (+) trypsin, and the products were analyzed on 11% SDS-PAGE followed by autoradiography. For control, a sample from cells transfected with vector (Vect) alone was also included. A and B, the position of undigested HA-SEC13 is indicated by an arrow. The numbers on top of each pair of lanes correspond to the WD repeat in which the Asp was mutated.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

These studies show that no single mutation of a conserved aspartic acid residue in G to a glycine prevents the formation of a compact and substantially folded structure. Under optimal conditions (e.g. in a mammalian cell) and given enough time, each of the single mutant G subunits can form a dimer with G, bind G, allow the ADP-ribosylation of  by pertussis toxin, and be cross-linked by BMH. However, the propeller may be locally abnormal because mutations in repeats 2 and 3 diminish, but do not eliminate, the affinity for . Blades 2 and 3 are the site of several residues that contact , so decreased affinity because of local distortion in these blades could be expected. In addition, mutation in repeat 3 exposes additional sites to digestion by trypsin. Nevertheless, the end state for the mutants is close to normal.

Synthesis of mutant in COS-7 cells allows us to evaluate the final state but does not allow us to analyze the effect of the mutation on the folding process. Such analysis can be done with subunits synthesized separately in vitro, then mixed, and assembled. Folding and assembly are intimately linked for G and G. We have shown previously that without G, G synthesized in vitro does not fold into a native structure (9, 11). At best, the reticulocyte lysate is inefficient at folding and/or assembling , and only 20-30% of the wild-type  synthesized forms a dimer. In vitro dimerization of G stops after about 30 min, perhaps because an essential protein chaperone or cofactor is degraded or depleted. Adding fresh lysate increases the dimerization of both wild-type and mutant protein, but we were not able to drive the reaction to completion.

Mutation of the Asp in repeat 1 decreases the recovery of to 25% of wild type. Kinetic analysis shows that over the first 30 min of folding and assembly, the rate of formation by this mutant is 28% of wild type. Thus, the low recovery of H₁[D1] dimers reflects the slowed rate of formation. The recovery of other mutants varies from nearly undetectable for repeat 7 to nearly normal for repeat 2. The dimerization rate of the mutants that were further analyzed (H₁[D3], H₁[D4], H₁[D5]) was also found to be low, and varied from one to another, consistent with their specific yield of dimers. The slower rate of folding and assembly of the mutants may be due to the flexibility introduced by glycine that increases the number of potential conformations and so increases the time needed to find the correct one. Alternatively, mutation may lower the affinity of the protein for an essential chaperone. Whatever the mechanism, these results show that mutations in each repeat do not have the same consequences for folding and assembly, and they are not equivalent.

The comparison of in vivo and in vitro results indicates that the Asp mutants may be folding pathway mutants; they are able to achieve a normal or near normal structure, but the folding process is slowed. Therefore, they can be considered as conditional mutants whose phenotype is only evident under non-permissive conditions, such as a reticulocyte lysate, where elements important for folding are limiting. These mutants may provide an assay to identify factors and/or conditions that suppress or diminish their phenotype in a reticulocyte lysate. Identifying such factors might help understand the folding process of this WD repeat protein and perhaps of other members of the family.

Each of the Asp to Gly mutants in Sec13 were able to fold into a compact globular structure (presumably a propeller) with the same Stokes radius as the native protein. Unlike G, they are capable of doing so even in vitro, consistent with our previous finding (8, 11) that Sec13 has a less stringent requirement for chaperonins, or can use chaperonins from very different sources (E. coli to mammalian cells).

Although all the mutant Sec13 proteins had a normal Stokes radius, not all of them were resistant to trypsin. Because the 28 potential tryptic cleavage sites in Sec13 are randomly distributed, the cleavage assay is extremely sensitive and can detect even subtle conformational changes. We propose that the Asp to Gly mutation provides a certain freedom for the propeller to "breathe" and thus allows access of the enzyme to otherwise hidden sequences.

Comparison of the effect of Asp mutations in  with the equivalent mutations in Sec13 shows that the repeat positions that have the least effect on the final structure are different for the two proteins (repeat 2 in G and repeats 1 and 6 in Sec13). All known propeller proteins have a mechanism for closing the ring (reviewed in Ref. 6). In G, and presumably in all other WD proteins, the outermost  strand of the last blade is provided by the amino-terminal variable region of the first repeat, thereby bringing together the two ends of the molecule to create the circular structure. Thus, one would expect that the first and last repeats would be more sensitive than others to any mutation. Our results prove that this assumption is wrong because, in Sec13, the first and last repeats are precisely the ones where Asp mutations have no detectable phenotype.

Our finding, both in G and in Sec13 that no mutation of the conserved Asp entirely prevents folding, suggests that there is no obligatory folding order of the repeats. Harrison and Durbin (7) proposed that evolution favors multiple paths leading to the same final folded state. One can easily imagine that formation of the D-containing tight turn between strands b and c is one of the early folding steps acting as a seed for each blade. So long as at least a majority of blades can initiate folding under optimal conditions, nearly any folded subset of blades will initiate the overall propeller. Nevertheless, analysis of in vitro folding and assembly of G suggests that not all pathways are equally favorable because mutation of a critical residue in one blade has a different effect from mutation in another. We propose that different WD repeats initiate different folding pathways with different rates or probabilities, which may differ in different WD proteins.

Wall et al. (5) and Lambright et al. (3) pointed out that the conserved Asp forms intra- and interblade hydrogen bonds in a triad with His in the GH motif, and a Ser/Thr in the second  strand. Such a full triad occurs in only four (1, 3, 4, and 7) out of seven repeats in G, so triad hydrogen bonding is not absolutely essential for proper folding or stability. However, our results suggest that the presence or absence of the His that is hydrogen-bonded to Asp can affect the final state of the molecule when Asp is mutated. In G, repeat 2 is the only one that lacks the His. It is also the only one where mutation of the Asp does not slow folding in vitro. Analysis of the double mutants is consistent with the conclusion that mutation in this repeat is better tolerated because H₁[D2-3] and H₁[D2-7] fold with similar efficiency as the single mutants, H₁[D3] and H₁[D7]. In contrast, all other double mutants tested were poorly folded. In Sec13, the only repeat with no His is repeat 6, and indeed, the double mutant, H₁[D1-6], shows the same phenotype as the single mutants, H₁[D1] and H₁[D6]. We could not test any more double mutants because all other single