Structure of the a! and @ Heavy Chains of the Outer Arm Dynein from Chlamydomonas Flagella MASSES OF CHAINS AND SITES OF ULTRAVIOLET-INDUCED VANADATE-DEPENDENT CLEAVAGE*

We report here on the UV-induced vanadate-depen- dent cleavage of the a and B heavy chains of the outer arm dynein from Chlamydomonas flagella. Both polypeptides are cleaved at a single site (termed the V1 site) by UV irradiation in the presence of Mg2+, ATP, and vanadate. The a chain yields fragments of M, 290,000 and 190,000. Fragments of M, 255,000 and 185,000 are obtained from the B chain. Ultraviolet irradiation of the a and chains in the presence of vanadate and Mn2+ (but no nucleotide) induces cleavage of both molecules at sites (termed the V2 sites) distinct from the VI sites. The single V2 site within the 0 chain is located 75,000 daltons from the site of V1 cleavage within the M, 255,000 V1 fragment. The a chain contains three distinct sites of V2 cleavage; all are located within the M, 290,000 VI fragment, 60,000, 90,000, and 100,000 daltons from the site of V1 cleavage. From these studies, we estimate the masses of the a and B heavy chains to be 480,000 and 440,000 daltons, respectively.

In the biflagellate green alga Chlamydomonas, the outer dynein arm is composed of three discrete subunits. During purification the (Y and p subunits remain associated and are isolated together as an a-/3 dimer,' whereas the y subunit is ~~ ~ *This work was supported by Grants GM30626 and CA12708 awarded by the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed.
In accordance with recently agreed upon nomenclature for dyneins and their component subunits (I. R. Gibbons, manuscript in isolated as a separate particle (Pfister and Witman, 1984;Pfister et al., 1982;Piperno and Luck, 1979;Watanabe and Flavin, 1976). Each subunit contains one very high molecular weight polypeptide, the a, /3, and y heavy chains (M, > 400,000), each of which has a site of ATP hydrolysis (Pfister et al., 1984(Pfister et al., , 1985. In addition, the a subunit contains a single light chain of M, 16,000; the y subunit contains two light chains (M, 18,000 and 22,000); and the p subunit contains two intermediate chains (Mr 78,000 and 69,000) and seven distinct light chains (M, 8,000-20,000)? The heavy chains from the outer dynein arm are immunologically distinct from each other and also from the intermediate chains of the / 3 subunit Mitchell and Rosenbaum, 1986).
Our goal has been to identify and locate sites of structural and functional interest within the heavy chains of the Chlamydomonas outer arm dynein and to examine the relationships between the various components of these very large protein complexes Witman, 1985,1986). Recently, Lee-Eiford et al. (1986) described a procedure that resulted in a single site-specific cleavage of each heavy chain from sea urchin sperm flagella dynein, yielding fragments of M , 228,000 and M, 200,000. To achieve this, the dynein preparation was first incubated with ATP and vanadate to form an ADP.
vanadate complex, which acts as a dead-end kinetic block within the catalytic sites of these enzymes (Shimizu and Johnson, 1983). Prolonged ultraviolet irradiation of the dynein. ADP. vanadate complex then resulted in cleavage of the heavy chains through a reaction mechanism that is not yet understood. This reaction showed an absolute requirement for the presence of both the vanadate anion and the nucleotide, suggesting that the sites of cleavage (designated the V1 sites) occurred within the ATP-binding domains of these molecules Lee-Eiford et al., 1986).
A second site of vanadate-dependent cleavage has also been found within each of the sea urchin dynein heavy chains . Cleavage at these sites (designated V2), located approximately 100,000 daltons from the V1 sites, was obtained by UV irradiation of dynein in the presence of vanadate and Mn2+ and was completely inhibited by the addition of ATP or dithiothreitol. It is at present preparation), the complex composed of the a and 0 subunits from the Chlamydomonos outer arm, which was previously termed "18 S dynein," is now referred to as the a-/3 dimer. Similarly, the third subunit of the outer arm (previously 12 S dynein) is referred to as the y subunit.
* The molecular weights of the individual polypeptides of the a-0 dimer are 480, 000, 440,000, 78,000, 69,000, 20,000, 19,000, 16,000, 14,000, 14,000, 11,000,8,000, and 8,000. Quantitative densitometry of Coomassie Blue-stained gels indicates that all polypeptides are present in 1:1 molar ratios except for the two M , 8,000 light chains, both of which appear to be present at a stoichiometry of five copiesla-0 dimer (K. K. Pfister and G. B. Witman, unpublished results). uncertain whether the Mn2+. vanadate-dependent sites of cleavage also occur within the ATP-binding site or whether they define a separate vanadate-binding domain within each heavy chain.
In this report we describe the vanadate-dependent cleavage products of the a and /3 heavy chains from the Chlamydomonas outer arm dynein. Both polypeptides are cleaved at a single site (VI) in the presence of vanadate and ATP. The /3 chain also contains a single V2 site, whereas there are three discrete sites within the a chain at which V2 cleavage may occur. By using monoclonal antibodies specific for each polypeptide, we have been able to unambiguously locate the sites at which cleavage occurs within each molecule. Finally, examination of the complementary fragments has allowed us to accurately determine the M, of these very large proteins. These results will be useful for locating other regions of structural and functional significance, such as sites of ATP binding (Pfister et al., 1985) and phosphorylation Piperno and Luck, 1981), within these polypeptides.

MATERIALS AND METHODS
Purification of Outer Arm Dynein-Flagella were isolated from Chlamydomonas reinhurdtii strain 1132D (mating type (-)) by the dibucaine procedure , demembranated with Nonidet P-40, and the resulting axonemes extracted with 0.6 M KCl. The a+ dimer was then separated from the y subunit by sucrose density gradient centrifugation. For some experiments the dimer was further purified by hydroxylapatite column chromatography Pfister et al., 1982).
Monoclonal Antibodies-The generation and characterization of monoclonal antibodies which specifically recognize the high molecular weight components of the Chlamydomonas outer dynein arm have been described previously . Two antibodies which recognize different epitopes within the a chain are designated 18aA and 18aB; two that react exclusively with different regions of the 8 chain are designated 18pB and 188C. Supernatants from hybridoma cultures were used directly as the source of primary antibody in the immunostaining procedure described below.
UV-induced Vanadate-dependent Cleavage at the VI Sites-The purified a-8 dimer was dialyzed against 1500 volumes of 30 mM Tris. HCl, pH 7.5,0.5 mM EDTA, 5 mM MgSO,. 7Hz0, 1 mM phenylmethylsulfonyl fluoride (TEM buffer) for 6-18 h. Twenty-microliter aliquots containing -5 pg of protein were placed in 500-pl micro test tubes (Bio-Rad) to each of which was then added 10 p1 of a 400 p~ stock solution of NaV03.HZ0 (no. 7260, Fluka Chemical Corp., Ronkonkoma, NY) in TEM and 10 pl of either 40 or 400 p~ 8azidoadenosine 5'-triphosphate (A 2392, Sigma), or adenosine 5'triphosphate (A 5394,Sigma). Samples were mixed by vortexing and incubated at room temperature for 15 min. To examine the effects of vanadate or ATP alone, the appropriate volume of TEM buffer was added in place of the anion or nucleotide. After incubation, 5 pl of 10 mM dithiothreitol (D 0632, Sigma) was added as a free radical scavenger, and the tubes were then placed on ice and irradiated for 20-90 min with UV light at 254 nm (UVG-11 lamp, Ultraviolet Products Inc., San Gabriel, CA; power output, 580 microwatts cm-' at a distance of 15 cm) or 365 nm (Spectroline EN-280L lamp, Spectronics Corp., Westbury, NY; power output, 1300 microwatts cm-' at 15 cm), with either lamp placed directly on top of the tubes. After irradiation, 10 pl of 5 X concentrated electrophoresis sample buffer and 5 pl of 0-mercaptoethanol were added, and the samples were boiled for 5 min prior to gel electrophoresis.
UV-induced Vanadate-dependent Cleavage at the V2 Sites-The a-8 dimer, purified by sucrose density gradient centrifugation, was dialyzed overnight against 500 volumes of 10 mM HEPES; pH 7.5. One hundred microliter aliquots were placed in micro test tubes (Bio-Rad) and 1 r n~ NaV03. HzO and 10 mM MnClz. 4Hz0 were added to final concentrations of 150 p~ and 1 mM, respectively. The samples were mixed by vortexing, incubated at room temperature for 15 min, and then placed on ice and irradiated for 0-6 h at 365 nm. Samples were prepared for gel electrophoresis as described above.
Tryptic Digestion-Proteolytic digestion was initiated by adding 60 pl of 0-50 pg/ml trypsin (T 8003,Sigma) in TEM to 300-pl aliquots of the purified dynein (-40 pg of protein). After vortexing, samples were incubated at room temperature for 5 min. The proteolytic digestion was stopped by the addition of a 10-fold excess (by mass) of soybean trypsin inhibitor (T 9003, Sigma). Following addition of vanadate to 100 p~ and a 15-min incubation, dithiothreitol was added to 1 mM and the samples irradiated for 45 min at 254 nm prior to preparation for electrophoresis.
Sodium Dodecyl Sulfate-Polyacrylumide Gel Electrophoresis and Protein Blotting-Dynein polypeptides and the fragments generated by vanadate-dependent cleavage and trypsin digestion were separated in either 0-2.4 M glycerol, 5-15% acrylamide, or 2-8 M urea, 3-5% acrylamide gradient gels as described previously Pfister et al., 1982). The latter formulation was required to obtain adequate resolution of polypeptides of M, > 200,000, whereas the former was used to examine smaller molecules and fragments. After electrophoresis, polyacrylamide gels were silver-stained by the procedure of Merril et al. (1981). Alternatively, the separated polypeptides were electrophoretically transferred to nitrocellulose sheets (BA-83; Schleicher and Schuell). The buffer and power conditions necessary for the efficient elution of very high molecular weight species from polyacrylamide gels and for their subsequent retention on the nitrocellulose sheets have been described previously in considerable detail Otter et al., 1987). Nitrocellulose sheets were stained to reveal monoclonal antibody reactivity using a peroxidase-conjugated secondary antibody and color development with 4-chloro-1-naphthol, as described previously . The portion of the nitrocellulose sheet to which the molecular weight markers were transferred was stained with Amido Black.

RESULTS
UV-induced Cleavage in the Presence of Vanadate, ATP, and Mg2+ (the VI Sites)-When purified a-/3 dimer in TEM was incubated with 10 or 100 p~ ATP or 8-N3ATP (a hydrolyzable photoaffinity analog of ATP) and 100 p~ vanadate and then subjected to a prolonged (>20 min) UV irradiation at 254 nm, the amounts of a and / 3 heavy chains detectable in silver-stained gels decreased. Concomitantly, four discrete fragments (Mr 290,000, 255,000, 190,000, and 185,000) were formed ( Fig. 1, lanes 6-13). Estimation of the masses of these fragments is described below. When samples in TEM were incubated and then irradiated in the presence of 100 p~ vanadate without nucleotide, only the M, 290,000 and 190,000 fragments were obtained (Fig. 1, lane 3). Incubation of the dynein preparation with either 0,10, or 100 p~ 8-N3ATP (or ATP), but no vanadate, followed by irradiation for 60 min resulted in no detectable cleavage products from either polypeptide ( Fig. 1, lanes 2, 4, and 5, respectively). However, the amounts of a and /3 heavy chains remaining in these samples were greatly reduced, probably as a result of random UVinduced breakage of the polypeptides. This conclusion is supported by the observation that increasing concentrations of 8-N3ATP had an increasingly protective effect on the proteins (Fig. 1, lane 5), presumably due to screening of the proteins from UV irradiation. Nonspecific degradation was also greatly reduced by irradiation at 365 nm, which induced cleavage in the presence of vanadate and ATP (data not shown). Cleavage of both polypeptides was dependent upon the nature of the divalent cation present in the reaction mix. Cleavage in the presence of nucleotide occurred when the divalent cation in the buffer was 5 mM M$+, Ca2+, or Zn2+; however, when Mn2+ was substituted for M$+ no specific fragments were obtained from either heavy chain (data not shown). Both the M , 78,000 and 69,000 intermediate chains are labeled by photoaffinity analogs of ATP, suggesting that these polypeptides also contain sites of ATP binding (Pfister et al., 1985). However, no detectable cleavage products were obtained from either intermediate chain following UV irradiation of the a-p dimer in the presence of M$+, ATP, and vanadate (results not shown).
Origin of the VI Fragments-In order to unambiguously identify the polypeptide from which each V1 fragment was derived, the immunoreactivity of the fragments was determined ( Fig. 2). This analysis made use of four monoclonal antibodies, the specificities of which have been described previously . Two antibodies (18aA and 18aB), which react exclusively with the a heavy chain, recognized the fragments generated by UV cleavage in the presence of vanadate alone. Antibody l8aA reacted with the M, 190,000 fragment, whereas 18aB bound specifically to the larger M, 290,000 fragment. Neither antibody recognized both fragments nor were any other detectable fragments generated directly from the CY chain. Thus, the M , 190,000 and M , 290,000 fragments are complementary portions of the CY heavy chain. Antibody 18aB also detected a minor fragment (M, 200,000) in the irradiated but not the control sample; this fragment is derived from "band 11" by cleavage at the V1 site (see below and Fig. 4).
Of the two additional fragments obtained by the cleavage of dynein in the presence of both vanadate and S-NdTP, the M , 185,000 fragment was recognized by two monoclonal antibodies (BOB and 188C) which react specifically with the p chain. Therefore, this fragment is derived from the p chain. The fourth fragment (MI 255,000) was not recognized by any of the monoclonal antibodies used. However, it was only observed in samples which contained the M , 185,000 fragment and was always present in amounts approximately equimolar to the M, 185,000 fragment as judged by the intensity of the bands in silver-stained gels. This strongly suggests that the M , 255,000 fragment represents that portion of the / 3 chain complementary to the M , 185,000 immunoreactive fragment.
Tryptic Digestion and Mass of the Dynein Heavy Chains-Two of the fragments obtained by V1 cleavage of the a and p chains migrate, in acrylamide gradient gels, within the linear range defined by well characterized molecular weight standards, including myosin ( M , 205,000). The relative molecular masses of these fragments, therefore, can be estimated directly from the molecular weight standards to be M , 190,000, for that derived from the a chain, and M, 185,000, for that derived from the fi chain.
The complementary fragments migrate more slowly than myosin. In order to obtain an accurate estimate of the molecular mass of the larger of these fragments, i.e. that derived from the a chain and recognized by antibody 18aB, the a-p dimer was digested with increasing concentrations of trypsin (Figs. 3 and 4) and then subjected to the V1 cleavage procedure (Fig. 4). At the trypsin concentrations used, very little or no digestion of the j 3 chain was observed in either silver-stained gels (Fig. 3) or on nitrocellulose blots probed with antibodies against this polypeptide (data not shown). However, trypsin treatment did result in a single cleavage of the a chain to yield the very large fragment previously designated band 11 (Pfister and Witman, 1984;Pfister et al., 1982) and a complementary smaller peptide of M, 90,000 (Fig. 3). The band 11 fragment, which was recognized by both 18aA and 18aB ( 4, upper p a n e l s ) , was then subjected to V1 cleavage, and the resulting fragments identified by immunoblot analysis (Fig.   4, lower panels). Antibody l8aA recognized a fragment of M , 190,000; this fragment is identical, in mass and immunoreactivity, to that obtained by cleavage of the intact a chain.

UV-induced Cleavage in the Presence of Vanadate and Mn2+
(the V2 Sites)-When the a-/3 dimer was incubated with 1 mM Mn2+ and 150 p~ vanadate and then subjected to long wavelength UV irradiation, cleavage of both heavy chains was observed (Figs. 6 and 7). The amount of a chain present in the samples decreased rapidly, and this polypeptide was undetectable in samples irradiated for longer than 3.5 h. Concomitant with the decrease in the amount of a chain, six discrete fragments (Mr 290,000, 280,000, 250,000, 230,000, 200,000, and 190,000) appeared. The M, 290,000,280,000, and 250,000 fragments were recognized by both 18aB (Fig. 7) and 18aA (data not shown), whereas the remaining fragments were not immunoreactive. Because the mass of the a chain is 480,000 daltons, the complementary pairs of fragments must be 250,000 and 230,000,280,000 and 200,000, and 290,000 and 19O,ooO, the sites of cleavage which generated these pairs are referred to as V2a, V2b, and V~C , respectively (see Table I and Fig. 8). Inasmuch as the larger fragment from each pair was recognized by both 18aA and 18aB, the sites of cleavage 280,000,230,000, and 200,000 fragments was observed, with a corresponding increase in the intensity of the bands which migrated at M , 250,000 and 190,000. This indicates that, within a given molecule, V2 cleavage occurred at more than one site with the result that the smallest fragments containing the original termini of the a chain accumulated with time. This predicts that additional fragments of M , 40,000, 30,000, and 10,000 should also exist. An M , 40,000 fragment apparently derived from cleavage of the a chain at both the V2a and V2c sites has been observed in silver-stained gels, but because this fragment is not immunoreactive it has not been possible to unambiguously determine its origin.
The /3 chain was more resistant to V2 cleavage; detectable amounts of this polypeptide were observed in samples irradiated for as long as 5 h. However, after extensive irradiation, V2 cleavage of the /3 chain did occur at a single site yielding fragments of M , 260,000 and 180,000 (Fig. 6). The former fragment contained the epitopes recognized by l8DC (Fig. 7) and 18/3B (data not shown). Thus, the single site of V2 cleavage within the /3 chain must occur within the M , 255,000 V1 fragment, 180,000 daltons from the terminus of the original molecule (Fig. 8).
FIG. 6. UV-induced vanadate-dependent cleavage of the a and fl chains at the V2 sites. This figure shows the high molecular weight region of a silver-stained 3-5% acrylamide gradient gel. Lanes were loaded, from left to right, with 4.1 pg of hydroxylapatite-purified a-p dimer irradiated at 365 nm for 0, 2, 5, 10, 20, 30, 45, 60, 90, 120, 150, 180,210,240,300, and 360 min, respectively, in the presence of 150 pM vanadate and 1 mM MnZ+. The positions of the a and B heavy chains and the eight major fragments (M, 290,000, 280,000, 260,000, 250,000, 230,000, 200,000, 190,000, and 180,000) are indicated. 7. Immunoreactivity of the V2 cleavage fragments. Nitrocellulose replica of a 3-5% acrylamide gradient gel that was loaded with the same samples of hydroxylapatite-purified a-8 dimer (irradiated for various lengths of time in the presence of Mn2+ and vanadate) as shown in Fig. 6. The blot was first probed with antibody 18pC and then with 18aB; the combined result of the two probes is shown.

DISCUSSION
Ultraviolet irradiation of the a-/3 dimer from the Chlamydomonas outer dynein arm, after incubation with M F , ATP, and vanadate, resulted in the specific cleavage of both the a and /3 heavy chains. The a chain yielded two fragments. The larger fragment ( M , 290,000) was recognized by antibody 18aB, whereas the smaller fragment (MI 190,000) was recognized by antibody 18aA. The ( 3 chain was cleaved to fragments of M , 255,000 and 185,000. The latter fragment contained the epitopes recognized by both 18@B and 18/3C. Irradiation of the CY-@ dimer after incubation with Mg2+ and vanadate but no nucleotide resulted in the cleavage of the a chain alone. Again fragments of M , 290,000 and 190,000, recognized by 18aB and 18aA, respectively, were obtained. Thus, by the criteria of mass and immunoreactivity of the fragments, V1 cleavage of the a chain in the presence of either vanadate and ATP or vanadate alone occurred at the same site. Substitution of Ca2+ or Zn2+ for M e also supported V1 cleavage of the a and /3 chains. However, when Mg2+ was replaced with Mn2+, cleavage did not occur in the presence of nucleotide, even though MnATP2-is a suitable substrate for dynein-mediated ATP hydrolysis (Ogawa and Mohri, 1972;King and Witman, 1987).
When the a-/3 dimer was irradiated at 365 nm in the presence of vanadate and Mn2+ but no nucleotide, both heavy chains were cleaved at sites distinct from the V1 sites. The /3 chain was cleaved to yield fragments of M , 260,000 and 180,000. The M , 260,000 fragment was recognized by antibodies 18@B and 18/3C; therefore, the site of V2 cleavage occurred with the M , 255,000 V1 fragment at a point 75,000 daltons from the V1 site. V2 cleavage of the a chain occurred at three discrete sites (designated V2a, V2b, and V2c). Cleavage at the V2a site resulted in fragments of M , 250,000 and 230,000. The larger fragment was recognized by both anti-a chain antibodies; therefore, this site of cleavage occurred within the M , 290,000 V1 fragment, 60,000 daltons from the V1 site. Cleavage of the a chain at the V2b and V2c sites yielded fragments of M , 280,000 and 200,000, and 290,000 and 190,000, respectively. Again, the larger fragments were immunoreactive, indicating that these sites of cleavage also occurred within the M , 290,000 V1 fragment (90,000 and 100,000 daltons from the

TABLE I Fragments of the a and @ chins obtained by UV-induced vanadatedependent cleavage
Polypeptide "~~~" Fragments Immunoreactivity V 1 site, respectively). As the time of UV irradiation was increased, the intensity of the bands migrating at M , 290,000, 280,000, 230,000, and 200,000 decreased with a concomitant increase in the intensity of those migrating at M, 250,000 and 190,000. This indicates that cleavage of the a chain at one V2 site does not preclude cleavage of that same polypeptide at a second or a third site. The sites of V1 cleavage are presumed to occur within the ATP hydrolytic domains of the dynein heavy chains (Lee-Eiford et . This conclusion is based on the observation that vanadate-sensitized photocleavage of sea urchin sperm dynein at the V1 site occurs only in the presence of ATP, ADP, or other substrates, which probably help stabilize the vanadate anion at the y-phosphate binding site . Our finding that V1 cleavage of the a chain of Chlamydomonas outer arm dynein can occur in the absence of nucleotide indicates that, at least in Chlamydomonas, Mg2'vanadate alone is capable of binding to the hydrolytic site. The specific mechanism of vanadate- sensitized cleavage is not clear. However, the vanadate anion may rapidly esterify the hydroxyl group of tyrosine (Tracey and Gresser, 1986), and the oxovanadium (IV) cation (VO") may complex with the imidazole ring of histidine (Mulks et al., 1982). These observations have led to the proposal that such aminoacyl residues within the catalytic sites might be essential for vanadate-dependent cleavage . The locations of the V2 sites are less certain. Under the conditions of pH and vanadate concentration used here to obtain V2 cleavage, the vanadate anion probably exists in both monomeric (H2V0:) and polymeric forms (Cotton and Wilkinson, 1972). The latter include trimeric (V30$-) (Ingri and Brito, 1959;Howarth and Richards, 1965;Wells and Bagshaw, 1984) and tetrameric (V40:;) (Simon and Jahr, 1964;Tytko and Mehmke, 1983) species, both of which are thought to be cyclic structures (for review of the formation and structure of isopolyvanadates in solution, see Pope and Dale, 1968). It is possible that paramagnetic Mn2+ (but not diamagnetic M P ) coordinates with trimeric or tetrameric vanadate to form a complex which binds to part(s) of the hydrolytic domain (such as the sites normally occupied by the adenine and/or ribose sugar rings or by the a and p phosphates of ATP) not available in the presence of nucleotide. The interaction of trimeric or tetrameric vanadate with three separate aminoacyl residues within the a chain would then explain the observed presence of three sites of V2 cleavage in this polypeptide. In this case, the fact that only a single V2 site was observed within the / 3 chain would suggest that there are differences in the primary structure of the hydrolytic domains from these two molecules. Assuming that there is only one hydrolytic domainlheavy chain, each chain would have to be extensively folded to bring its vanadate-dependent cleavage sites, which are separated by 60,000-100,000 daltons, within the hydrolytic domain.
Alternatively, the V2 sites may define one or more vanadate-binding domains separate from the ATP hydrolytic sites.
Such sites could represent either catalytic or noncatalytic nucleotide or phosphate-binding domains. Experiments are currently underway to investigate these possibilities. By determining the masses of complementary fragments obtained by tryptic and V1 cleavage of the a chain we were able to estimate the mass of the intact chain. Low concentrations of trypsin cleaved the a chain at a specific site to yield two discrete fragments: one of M, 90,000 that was not immunoreactive and a large complementary fragment (previously referred to as band 11 Pfister and Witman, 1984)) that was recognized by both 18aA and 18aB. Cleavage of band 11 at the V1 site resulted in a fragment of M, 190,000, identical to that obtained by V1 cleavage of the a chain, and a second fragment of M , 200,000 that was recognized by 18aB. Thus, the larger V1 fragment from the a chain must contain the tryptic site that is cleaved to yield band 11; this site is located 90,000 daltons from one terminus of the molecule and 200,000 daltons from the site of V1 cleavage. These results indicate that the larger V1 fragment from the a chain has a mass of 290,000 daltons. From the sums of the masses of their fragments, the sizes of the intact a and /3 heavy chains are estimated at 480,000 and 440,000 daltons, respectively.
These values for the intact chains are considerably larger than previous estimates based on electrophoretic studies where it was necessary to extrapolate from markers of known mass Piperno and Luck, 1979). However, the new values for the masses of the a and /3 chains agree extremely well with the value obtained for the native mass of the a-/3 dimer by scanning transmission electron microscopy (Witman et al., 1983). From the STEM analysis the mass of the dimer was found to be 1,220,000 daltons with a standard deviation from the mean of 117,000 daltons. Using the new values for the a and /3 heavy chains, the sum of the masses of the individual polypeptide chains of the a-/3 dimer is 1,240,000 daltons. ' The STEM study also demonstrated that the a-/3 dimer is composed of two globular domains interconnected via a Yshaped stalk. Each globular unit had a mass of approximately 375,000 daltons. Therefore, each head is too small to accommodate an entire heavy chain; both the a and /3 chains must contribute mass (perhaps as much as 100,000 daltons in the case of the LY chain) to the stalk components of the dynein complex.
The a and /3 chains share a defined polarity; in both polypeptides the site(s) of V2 cleavage occurs within the larger V1 fragment (Fig. 8). Therefore, we predict that both molecules exhibit the same orientation with respect to their NH, and COOH termini. The maps shown in Figs. 5 and 8 have been drawn so as to reflect this prediction.
The studies reported here have highlighted a number of differences between the heavy chains of the a$ dimer. First, the a chain may be cleaved at the V1 site following incubation with M$+.vanadate alone, whereas the /3 chain also requires the presence of ATP (or ADP). This suggests that M$+. vanadate residues in the catalytic site of the a chain sufficiently long even in the absence of nucleotide to allow cleavage to occur, whereas a M$+ -ADP -vanadate complex is required to immobilize the anion within the active site of the /3 chain.
Second, three distinct sites of V2 cleavage have been identified within the a chain, but only one was observed within the /3 chain. Third, the mass of the a chain is approximately 40,000 daltons greater than that of the fi chain. Other differences between the Chlamydomonas dynein a and /3 heavy chains and their active sites have been noted previously. Pfister and Witman (1984) found the M$+-ATPase activity of the purified a subunit to be 0.6 pmol of phosphate released/min/mg, whereas the activity of the /3 subunit was approximately 10-fold higher (7.9 pmol of phosphate released/min/mg).  used monoclonal antibodies to demonstrate that the LY and /3 heavy chains are immunologically distinct from each other. Also, the a chain is phosphorylated in uiuo whereas the /3 chain is not Piperno and Luck, 1981). The large number of differences between these two chains suggests that they have distinctly different roles in the production and/or regulation of shearing forces during flagellar movement.
The vanadate-dependent cleavage procedures have also been applied to the outer arm dynein from sea urchin sperm flagella (Lee-Eiford et al., 1986;Gibbons et aE., 1987). Both heavy chains, although separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, give rise to V1 fragments which migrate at M , 228,000 and 200,000. V2 fragment pairs of M, 170,000 and 260,000 and 175,000 and 255,000 are obtained from the a and p chains, respectively; the V2 sites are located within the larger of the V1 fragments. The relative positions of the V1 and V2 sites in the dynein heavy chains are thus similar in Chlamydomonas and sea urchin sperm.
The V1 and V2 cleavage procedures will prove to be extremely useful in mapping the locations of other domains within the a and /3 heavy chains, because 1) they have allowed accurate estimates of the masses of these very large polypeptides and 2) they mark specific sites within these molecules, with respect to which other sites of interest may be mapped. We have utilized the sites of V1 and V2 cleavage, in conjunction with a number of endoproteases, to further define the regions which contain the epitopes recognized by our antibodies, to locate protease-sensitive sites, and to define the domains covalently modified by photoaffinity analogs of ATP Witman, 1986,1987).