Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Nucleotide binding sites

The photoaffinity analogs 2-azidoadenosine 5'-tri(di)-phosphate (2-N3AT(D)P) and 8-azidoadenosine 5'-triphosphate (8-N3ATP) have been used to probe the substructural organization of the nucleotide binding pockets within the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Both 2-N3ATP and 8-N3ATP are competitive inhibitors of dynein ATP hydrolysis, and both analogs are themselves hydrolyzed by the alpha-beta dimer. Following vanadate-dependent photolysis at the V1 site (by UV irradiation in the presence of Mg2+, ATP, and vanadate), both probes exclusively labeled the larger fragment from the alpha chain. In contrast, within the beta chain the predominant insertion sites for the two analogs were located on opposite sides of the V1 site. Therefore, the hydrolytic pockets of these two molecules have different substructures. Vanadate-dependent photolysis of the alpha and beta chains at the V2 sites (by UV irradiation in the presence of vanadate and Mn2+) profoundly affected the predominant modification sites; for example, following photolysis at the V2a site neither fragment of the alpha chain was photolabeled by 2-N3ATP or 8-N3ATP. Based on the photolabeling patterns obtained, the single V2 site within the beta chain is predicted to be analogous to the V2b site within the alpha chain. The results support the hypothesis that the V2 sites occur within the ATP binding pockets, and indicate that these functional domains are composed of portions of the heavy chains which are linearly separated by up to at least 100,000 daltons. Thus, the central region of each dynein heavy chain must be extensively folded so as to bring the widely separated photocleavage and photolabeling sites together within a single catalytic unit.     

Structure of the alpha and beta 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-dependent cleavage of the alpha and beta 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 alpha chain yields fragments of Mr 290,000 and 190,000. Fragments of Mr 255,000 and 185,000 are obtained from the beta chain. Ultraviolet irradiation of the alpha and beta 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 V1 sites. The single V2 site within the beta chain is located 75,000 daltons from the site of V1 cleavage within the Mr 255,000 V1 fragment. The alpha chain contains three distinct sites of V2 cleavage; all are located within the Mr 290,000 V1 fragment, 60,000, 90,000, and 100,000 daltons from the site of V1 cleavage. From these studies, we estimate the masses of the alpha and beta heavy chains to be 480,000 and 440,000 daltons, respectively.     

Structure of the gamma heavy chain of the outer arm dynein from Chlamydomonas flagella

We describe here the vanadate-dependent photocleavage of the gamma heavy chain from the Chlamydomonas outer arm dynein and the pathways by which this molecule is degraded by endoproteases. UV irradiation in the presence of ATP, Mg2+, and vanadate cleaves the gamma chain at a single site (termed V1) to yield fragments of Mr 235,000 and 180,000. Irradiation in the presence of vanadate and Mn2+ results in cleavage of the gamma chain at two other sites (termed V2a and V2b) to yield fragment pairs of Mr 215,000/200,000 and 250,000/165,000. The mass of the intact chain is therefore estimated to be 415,000 D. We have located the major tryptic and staphylococcal protease cleavage sites in the gamma chain, determined the origins of the resulting fragments, and identified the regions which contain the epitopes recognized by two different monoclonal antibodies. Both antibodies react with the smaller V1 fragment; the epitope recognized by antibody 25-8 is within 9,000-52,000 D of the original gamma-chain terminus contained in that fragment, whereas that recognized by antibody 12 gamma B is within 16,000 D of the V1 site. The data permit the construction of a linear map showing the structural organization of the polypeptide. The substructure of the gamma chain is similar to that of the alpha and beta chains of the outer arm dynein with regard to polarity as defined by the sites of vanadate-dependent photocleavage, and to that of the beta chain with regard to a highly sensitive protease site located approximately 10,000 D from the original terminus contained in the smaller V1 fragment.     

Distinct roles of 1alpha and 1beta heavy chains of the inner arm dynein I1 of Chlamydomonas flagella

The Chlamydomonas I1 dynein is a two-headed inner dynein arm important for the regulation of flagellar bending. Here we took advantage of mutant strains lacking either the 1α or 1β motor domain to distinguish the functional role of each motor domain. Single- particle electronic microscopic analysis confirmed that both the I1α and I1β complexes are single headed with similar ringlike, motor domain structures. Despite similarity in structure, however, the I1β complex has severalfold higher ATPase activity and microtubule gliding motility compared to the I1α complex. Moreover, in vivo measurement of microtubule sliding in axonemes revealed that the loss of the 1β motor results in a more severe impairment in motility and failure in regulation of microtubule sliding by the I1 dynein phosphoregulatory mechanism. The data indicate that each I1 motor domain is distinct in function: The I1β motor domain is an effective motor required for wild-type microtubule sliding, whereas the I1α motor domain may be responsible for local restraint of microtubule sliding.     

A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain

A novel Chlamydomonas flagellar mutant (oda-11) missing the alpha heavy chain of outer arm dynein but retaining the beta and gamma heavy chains was isolated. Restriction fragment length polymorphism analysis with an alpha heavy chain locus genomic probe indicated that the oda-11 mutation was genetically linked with the structural gene of the alpha heavy chain. In cross-section electron micrographs, the oda-11 axoneme lacked the outermost appendage of the outer arm, indicating that the alpha heavy chain should be located in this region in the wild-type outer arm. This mutant swam at 119 microns/s at 25 degrees C, i.e., at an intermediate speed between those of wild type (194 microns/s) and of oda-1 (62 microns/s), a mutant missing the entire outer dynein arm. The flagellar beat frequency (approximately 50 Hz) was also between those of wild type (approximately 60 Hz) and oda-1 (approximately 26 Hz). These results indicate that the outer dynein arm of Chlamydomonas can be assembled without the alpha heavy chain, and that the outer arm missing the alpha heavy chain retains partial function.     

A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.     

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.     

Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function

Mutants with outer dynein arm defects or deficiencies all show a major reduction in beat frequency to about half the normal value; some of these mutants show an additional decrease in sliding velocity associated with reduced shear amplitude and an additional reduction in beat frequency, as well as other more minor modifications of the normal forward mode bending pattern. New mutants (ida98, pf30), which appear to be deficient in a subset of inner dynein arms show a reduction in sliding velocity that is primarily associated with a reduction in shear amplitude, with only a small reduction in beat frequency. These differences in motility phenotype between inner and outer dynein arm mutants suggest that inner and outer dynein arms may have distinct functions. The relatively large decrease in sliding velocity associated with partial loss of inner arms is consistent with earlier observations on pf23, a nonmotile mutant lacking inner arms, suggesting that inner arms may have an essential function in motility. The ability to generate reverse mode bending patterns is retained in some inner or outer dynein arm mutants, but appears to be decreased in those mutants which show reduced shear amplitude for the forward mode bending pattern.     

Conformational changes of dynein: mapping and sequence analysis of ATP/Vanadate-dependent trypsin-sensitive sites on the outer arm dynein b heavy chain from sea urchin sperm flagella

Conformational changes of dynein during ATP hydrolysis are demonstrated by the difference in the tryptic fragments of the dynein heavy chain between in the absence and presence of ATP and vanadate. Here tryptic sites in the presence of ATP and vanadate (Tav sites) have been mapped on the betaheavy chain of outer arm dynein from sea urchin sperm flagella. Tav sites are located not only near the central catalytic domain which includes four P-loops, but also near the carboxyl-terminal coiled-coil region. The Tav2 site is located in the most carboxyl-terminal region, which is nearly 850 amino acid residues apart from the the fourth P-loop (P4 site). The region from the most amino-terminal Tav site (Tav1 site) to the Tav2 site covers approximately 2,100 amino acid residues, which is almost half the whole betaheavy chain. Comparison of the sequences around the tryptic sites of the sea urchin b chain and those of the dynein heavy chains from other organisms reveals that the sequence around the Tav1 site is highly conserved in both cytoplasmic and axonemal dyneins but that around Tav2 sites is only conserved in axonemal dyneins, suggesting functional differences in the Tav2 region between the two subfamilies of dynein.     

Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.     

Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes.

A procedure was developed for isolating Chlamydomonas outer-arm dynein that can functionally combine with the axoneme of an outer-arm-missing mutant, oda1. Previous studies showed that the outer-arm dynein of this organism, containing three heavy chains (alpha, beta, gamma), dissociates upon extraction with a high-salt-concentration buffer solution into an 18-S particle containing the alpha and beta heavy chains and a 12-S particle containing the gamma heavy chain. It was found, however, that the three heavy chains did not dissociate if the high-salt extract was centrifuged in the presence of Mg2+; the three chains constituted a single species (23-S dynein) sedimenting at about 23 S and displayed a three-headed bouquet configuration in electron micrographs. Furthermore, the 23-S dynein had the activity to bind to the axonemes of oda1 and increase the reactivated motility of detergent-extracted cell models; its addition increased the beat frequency from 28 Hz to 53 Hz, a frequency comparable to that of wild-type axoneme. The 18-S and 12-S dyneins, on the other hand, were unable to increase the motility of oda1 axonemes even when added together. The new protocol thus enables purification of outer-arm dynein that retains its functional activity. It will provide a useful experimental system with which to study the mechanism of outer-arm function.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

Domains in the 1alpha dynein heavy chain required for inner arm assembly and flagellar motility in Chlamydomonas.

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1alpha Dhc of the I1 inner dynein arm. The 1alpha Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH(2)- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1alpha Dhcs were recovered. The truncated 1alpha Dhc fragments lacked the dynein motor domain, but still assembled with the 1beta Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1alpha motor domain in the I1 structure and further revealed the role of the 1alpha Dhc in flagellar motility and phototactic behavior.     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.     

Structure of the alpha-, beta-, and gamma-heavy chains of 22 S outer arm dynein obtained from Tetrahymena cilia

Here we document the UV-induced, vanadate-dependent cleavage of the alpha-, beta-, and gamma-heavy chains of 22 S outer arm dynein obtained from Tetrahymena cilia. All three polypeptides have a single site of photocleavage in the presence of Mg2+, ATP, and vanadate (termed V1 cleavage). The alpha-chain yields complementary fragments with masses of 232 and 185 kDa, the beta-chain has complementary fragments with masses of 225 and 195 kDa, and the gamma-chain has complementary fragments with masses of 242 and 161 kDa. In the absence of ATP, only the beta-chain undergoes V1 cleavage. All three polypeptides have one single site of V2 cleavage, which are unaffected by the presence of nucleotide and only require the presence of Mn2+ and vanadate. V2 cleavage always occurs on the larger V1 fragments and is separated from the V1 site by 52, 48, and 57 kDa for the alpha-, beta-, and gamma-heavy chains, respectively. We have also found a third type of UV-induced vanadate-dependent cleavage which we have termed VMT cleavage. VMT cleavage occurs when dynein is bound to microtubules in an ATP-sensitive manner under V1 solution conditions that should only support cleavage of the beta-chain (i.e. vanadate, Mg2+, and absence of ATP). Under these conditions V1 cleavage of the beta-chain and V2 cleavage of all three chains occur. This is the first documented evidence of V2 cleavage occurring under V1 solution conditions and implies a change in dynein structure when it binds to a microtubule. Using a combination of polyclonal and monoclonal antibodies, we have been able to construct linear polypeptide maps of all three heavy chains. Their relationship to the polypeptide maps previously obtained for heavy chains obtained from the dynein of Chlamydomonas and sea urchin axonemes is discussed.     

Light chain 1 from the Chlamydomonas outer dynein arm is a leucine-rich repeat protein associated with the motor domain of the gamma heavy chain.

The LC1 light chain from Chlamydomonas outer arm dynein is tightly bound to the gamma heavy chain. Molecular cloning revealed that LC1 is a member of the SDS22+ subclass of the leucine-rich repeat protein family and as such is likely involved in mediating interactions between dynein and the components of a signal transduction pathway. Through the combination of covalent cross-linking and vanadate-mediated photolysis, LC1 was found to associate with that portion of the gamma HC that is C-terminal to the P1 loop. This region comprises most of the globular head domain of the heavy chain and includes the stalk-like structure that is involved in microtubule binding. Attachment of LC1 to this region represents the only known example of an accessory polypeptide directly associated with a dynein motor domain. Additional cross-linking experiments revealed that LC1 also interacts directly in situ with an approximately 45 kDa axonemal component; this interaction is disrupted by the standard high salt treatment used to remove the outer arm from the axoneme. These data suggest that LC1 acts to mediate the association between this 45 kDa axonemal polypeptide and the motor unit of the gamma HC.     

Substructure of the outer dynein arm

The substructure of the outer dynein arm has been analyzed in quick-frozen deep-etch replicas of Tetrahymena and Chlamydomonas axonemes. Each arm is found to be composed of five morphologically discrete components: an elliptical head; two spherical feet; a slender stalk; and an interdynein linker. The feet make contact with the A microtubule of each doublet; the stalk contacts the B microtubule; the head lies between the feet and stalk; and the linker associates each arm with its neighbor. The spatial relationships between these five components are found to be distinctly different in rigor (ATP-depleted) versus relaxed (ATP- or vanadate plus ATP-treated) axonemes, and the stalk appears to alter its affinity for the B microtubule in the relaxed state. Images of living cilia attached to Tetrahymena cells show that the relaxed configuration is adopted in vivo. We relate our observations to morphological and experimental studies reported by others and propose several models that suggest how this newly described dynein morphology may relate to dynein function.     

Mapping of ATP-dependent trypsin-sensitive sites on the beta chain of outer-arm dynein from sea urchin sperm flagella.

The beta chain of sea urchin outer-arm dynein showed a peculiar tryptic digestion pattern in the presence of ATP (or ADP) plus Vi. Examination of the molecular mass of the products formed by photocleavage of tryptic fragments indicated that the trypsin-sensitive sites on the 165-kDa ATP-binding polypeptide in the presence of ATP and Vi are located 15 kDa apart from its amino-terminus, 2 kDa apart from its carboxy-terminus, and near the middle portion between the adenine- and gamma-Pi-binding sites. On the other hand, the carboxy-terminal region of the beta chain, the 135-kDa polypeptide, was cleaved into a 96-kDa polypeptide by tryptic digestion in the presence of ATP and Vi. Peptide mapping of 135-kDa, 96-kDa, and carboxy-terminally truncated polypeptides of the 135-kDa polypeptide revealed that the 96-kDa region is located at the amino-terminal portion of the 135-kDa region. These results indicate that the changes of trypsin susceptibility of dynien beta chain caused by binding ADP and Vi occur not in local region but over an extensive region on the beta chain.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)