Domain structure of a mammalian myosin 113 - Europe PMC

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actin-binding sites, followed by an 8.5-kDa domain with three ...... Collins, K., Sellers, J. R. & Matsudaira, P. (1990) J. Cell Biol. 110, 1137-1147. 16. Ruppert, C.
Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 6349-6353, July 1994

Biochemistry

Domain structure of a mammalian myosin 113 (caduln bldng/phspholpld binding)

OFER REIZES*, BARBARA BARYLKO*, CAI Lit, THOMAS C. SODHOFti, AND JOSEPH P. ALBANESI*§ Departments of *Pharmacology and tMolecular Genetics and tHoward Hughes Medical Institute, University of Texas-Southwestern Medical Center at Dallas, Dallas, TX 75235

Communicated by A. J. Hudspeth, November 30, 1993 (received for review September 20, 1993)

(contrast refs. 12 and 15). Recently, the cDNA sequence and the deduced amino acid sequence of another mammalian myosin I heavy chain were reported. This protein, which was called MMIa in the mouse (13) and myrl in the rat (16), also shows a widespread tissue distribution and binds to calmodulin. A protein which probably corresponds to MMIa/myrl has been purified to apparent homogeneity from rat liver (17). Despite the similarities among these three forms of vertebrate myosin I, BBMI, MMIa/myrl, and MMI(8 display a distinct pattern of expression (13) and limited immunological crossreactivity (unpublished observations). Further, the sequence of a cDNA coding for a portion of MMI( from rat demonstrated that it is a unique member of the myosin I family. To characterize mammalian myosin I( further, we determined its primary structure, expressed constructs corresponding to its putative functional domains, and analyzed these domains for their ability to bind calmodulin, phospholipids, and specific monoclonal antibodies. The complete sequence consists of an open reading frame of 3084 bp encoding a protein of 1028 aa.1 In addition to the highly conserved amino-terminal catalytic and actin-binding domain (residues 1-685), MMI.8 has three calmodulin-binding motifs (residues 698-767) and a basic carboxyl-terminal tail (residues 768-1028). Analysis of the tail sequence confirms that MMIP is a distant relative of BBMI and MMIa/myrl.

ABSTRACT We have determined the primary structure of a myosin I (called m myosin I, MMI3) from bovine brain and Identified Its functional domain. The protein was previously purified from brain and adrenal gand. Several constructs were generated and expressed in Escherichia cofl as glutathione S-traferase fusion proteins and the recombinant proteins were recognized by monoclona antibodies that recof native myosin I. A ognize either "head" or "tail" dom gel overlay method was used to confirm that dulin binds to the consensus calmodulin-binding sequence in MMIIJ. Binding assays were used to detect interaction with anionic phosconsists of an pholipid vesicles. We conclude that MMI_3 amino-terminal 80.5-kDa domain that contains the ATP- and actin-binding sites, followed by an 8.5-kDa domain with three calmodulin-bindng sequences and a basic 30-kDa carboxylterminal tail segment that binds to anionic phospholipids and membranes.

Myosins are a diverse family of force-generating enzymes that catalyze the actin-dependent hydrolysis of ATP (1, 2). All myosins are composed of conserved amino-terminal domains containing ATP- and actin-binding sites fused to a wide variety of carboxyl-terminal domains suited for distinct tasks (1, 3, 4). Myosin I enzymes consist of single heavy chains ranging from 110 to 150 kDa and, in vertebrates, from three to six calmodulin light chains. Unlike conventional myosins (myosin II), myosin I molecules do not interact to form bipolar filaments. Most of the biochemical and genetic information on myosin I was generated from the study of Acanthamoeba castelanii, Dictyostelium discoideum, and the avian intestinal brush border (reviewed in refs. 1-3, 5, and 6). Although these investigations have not unambiguously revealed the function of any form of myosin I, considerable evidence suggests that these enzymes are motors for motile events at plasma and organellar membranes (7-11). We purified and characterized a myosin I from bovine adrenal gland and brain (12). This protein, which we now call mammalian myosin Id (MMI() [using the nomenclature of Sherr et al. (13) who isolated a partial clone of the rat ortholog], is enriched at the plasma membrane and has a broad tissue and cell distribution (14). MMI( shares some features with the well-characterized avian intestinal brushborder myosin I (BBMI) found almost exclusively in intestinal tissue. Like the avian enzyme, MMI(3 has a 118-kDa heavy chain and multiple calmodulin light chains. Calmodulin binds to the BBMI and MMI(3 heavy chains with a higher affinity in the absence than in the presence of Ca2+, in marked contrast to most calmodulin-target protein interactions. The actin-activated ATPase activities of both BBMI and MMI(3 are regulated by Ca2+/calmodulin, although the nature ofthis regulation appears to be different for the two enzymes

MATERIALS AND METHODS General Techniques. Standard molecular biology methods were used (18). cDNA clones were subcloned into M13 vectors and sequenced by dideoxy chain termination (19) on an Applied Biosystems model 373A DNA sequencer. RNA was isolated by the guanidinium thiocyanate/CsCl procedure (20). Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography (18). Sequence analysis was performed with Genetics Computer Group software developed for the VAX computer (21). Immunoblot analysis was carried out by the method of Towbin et al. (22) as described previously (14). Protein was assayed by the method of Bradford (23). Cloning Strategy. PCR (24) with degenerate oligonucleotides was used to amplify a cDNA fragment encoding a myosin I from cDNA from bovine adrenal medulla (18). The 5' PCR primer (PI) was based on a tryptic fragment (HYAGEVTYN) of adrenal myosin I (12) thought to be localized within the catalytic domain. The 3' PCR primers (P2 and P3) corresponded to a highly conserved peptide [P(H/ N)YIRCIK] believed to be the high-affinity actin-binding site. The sequences of these oligodeoxynucleotide primers were as follows: P1, 5'-ATCATGCATTACGCCGGRGARGTNACNTAMAA-3'; P2, 5'-CTTAAGCTTGATGAbbreviations: MMIV8, mammalian myosin IP; BBMI, brush-border myosin I; GST, glutathione S-transferase. tTo whom reprint requests should be addressed. IThe sequence reported in this paper has been deposited in the GenBank data base (accession no. Z22852).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Biochemistry: Reizes et al.

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Proc. Natl. Acad. Sci. USA 91 (1994)

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FIG. 1. Amino acid sequence comparison of bovine MMII3 with rat MMIf3, BBMI, mammalian myosin Ia (MMIa), and chicken skeletal myosin II aligned with the PILEUP program of the Genetics Computer Group software package. (a) N-terminal two-thirds of the proteins. The 145-448 (B); BBMI, aa 1-694 (C); murine MMIa, aa 1-702 (D); chicken skeletal sequences used were bovine MMIV, aa 1-6% (A); rat MMI(, myosin II, aa 1-780 (E); and the consensus (F). (b) Carboxyl-terminal third of the proteins. Sequences compared are MMI1,, aa 685-1028 (A); BBMI, aa 683-1043 (B); MMIa, aa 690-1079 (C); and the consensus (D). an

Biochemistry: Reizes et al.

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Proc. Natl. Acad. Sci. USA 91 (1994)

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agene) were screened with a uniformly 32P-labeled MMIP cDNA probe. A single genomic clone of 7 kb was isolated. Proteins and Binding Studies. Plasmids encoding five different domains of MMIVB were constructed as fusion proteins with glutathione S-transferase (GST) in pGEX (Pharmacia), using PCR to amplify the desired fragments. GST fusion proteins were expressed in Escherichia coli and purified on aglutathione-Sepharose 4B column (Pharmacia) (25). Myosin I was purified according to our published procedure (12). Calmodulin was purified from bovine brain according to Watterson et al. (26). Calmodulin (2 mg) was labeled with 125I to a specific activity of 2 x 108 cpm/mmol by use of Iodo-Beads (Pierce). The gel overlay procedure was based on a method described by Glenney and Weber (27). Phospholipid binding studies were performed according to Hayden et al. (28) except that large unilamellar vesicals prepared by the extrusion technique (LUVETs) were used instead of small unilamellar vesicles (SUVs). Egg yolk phosphatidylcholine and phosphatidylglycerol and bovine brain phosphatidylsernme were purchased from Avanti Polar Lipids. Phospholipids were dried under nitrogen, resuspended in 10 mM imidazole buffer at pH 7.0, and subjected to 10 freeze-thaw cycles to form multilamellar vesicles (MLVs). The liposomes were then extruded 10 times through a liposome extruder (Lipex Biomembranes, Vancouver, B.C.) to form LUVETs. Phospholipid concentrations were determined by phosphate analysis (29). Binding was performed in 10 mM imidazole, pH 7.0/2.5 mM MgCk2/75 mM KCl/1 mM EGTA/0.4 mM dithiothreitol/0.02% bovine serum albumin. Native myosin I or recombinant myosin I fragments were incubated on ice with 1 mM phospholipid vesicles for 20 min and then were ~~~~~centrifuged at 100,000 x g for 1 hr to separate free from bound

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FIG. 2. Immunoblotting and calmodulin overlay of recombinant MMII3 fiagments. The diagam at the top represents the five fragments expressed from MMI,3. LB medium was inoculated with bacteria containing head (H) or tail (T) constructs pH1 (aa 42-697),

pT1 (aa 703-768), pT2 (aa 820-1025), pT3 (aa 862-1025), or pT4 (aa 675-1025). When the cultures reached an OD600 of 0.6, fusion protein expression was induced with 0.4 mM isopropyl 3-D-thiogalactopyranoside. After an additional 3 hr, the proteins expressed were analyzed by SDS/PAGE in a 5-20%6 gradient polyacrylamide gel. Samples were then either stained with Coomassie blue, fixed and overlaid with calmodulin, or transferred to nitrocellulose and immunoblotted with antibodies mH5 and mT2, respectively. Native adrenal MMIB was used as a control (MI). Since these constructs are fused to GST the migration in the gels is slower than the expected mobility of the expressed fragments alone.

CAVCGRATRTARTKNGG-3'; P3, 5'-CTTAAGCTTGATGCAYCTRATRTARTKNGG-3'. An amplified PCR product of the correct size (,225 bp) (encoding aa 512-592 in the full-length protein) was sequenced and a 32P-labeled oligonucleotide of 22 bases corresponding to aa 573-580 (probe I) was used to screen a randomly primed bovine brain cDNA library in AZAP. From 10 cloning events, four overlapping clones were isolated and sequenced, and an open reading frame of 3039 nt was identified. The deduced amino acid sequence was found to lack an initiator methionine. Therefore, 106 plaques from a bovine genomic DNA library (Strat-

RESULTS Cloning and Sequence of MMI. The sequence of bovine MMI(3 was obtained by using PCR and cDNA and genomic cloning. Purified myosin I was digested with trypsin and several of the tryptic peptides were sequenced. One peptide (HYAGEVTYN) was similar to a sequence found in all myosins I and is z70 aa from the amino terminus of the conserved actin-binding site (RCIKPN) (30, 31). A directed PCR strategy using primers based on these peptides yielded a PCR fragment encoding a novel myosin I. A bovine brain cDNA library was screened with an oligonucleotide from the PCR product. The initiator methionine, lacking from the cDNA clones, was determined from a genomic clone. Although the 5' untranslated exons were not identified, the first two translated exons were sequenced. Exon A contains the initiator methionine (aa 1) through aa 42, while exon B codes for aa 43-81. The initiator methionine was identified by three criteria: (i) it meets Kozak's consensus (32), (ii) an in-frame stop codon was found 54 bp upstream, and (iii) the full-length sequence codes for a protein of the same size as purified myosin I (118-kDa heavy chain). The sequences of two tryptic peptides from purified myosin I (LTVIDFTEDEVE, aa 256-267; HYAGEVTYN, aa 511-519), matched the deduced sequence of the clone exactly. The MMIB open reading frame predicts a protein of 1028 aa (118, 124 Da) containing highly conserved ATP-binding (GESGAGKT) and actin-binding (RCIKPN) sequences in the amino-terminal two-thirds of the molecule. Comparison of the amino acid sequences shows that MMIB is more like BBMI and MMIa/myrl than it is like the protozoan forms of myosin I, while BBMI and MMIa/myrl are more closely related to each other than either is to MMII3 (Fig. la). The myosins I are least similar in sequence in the regions carboxyl-terminal to the catalytic and actin-binding domains (Fig. lb). The carboxyl-terminal domains of vertebrate my-

6352

Biochemistry: Reizes et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

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Liposome

FIG. 3. Interaction of myosin I and pT2 with phospholipid vesicles. Binding to vesicles composed of different phospholipid head groups. Adrenal myosin I (black bars) or pT2 (gray bars) was incubated on ice for 20 min either with buffer or with liposomes composed of phosphatidylserine (PS), phosphatidylglycerol (PG), or phosphatidylcholine (PC). Samples were then centrifuged at 100,000 x g for 60 min and supernatants and pellets were analyzed by immunoblotting with anti-myosin I mT2. Blots were then incubated with rabbit anti-mouse antibodies followed with 125I-labeled goat anti-rabbit antibodies (Fab fiagments), and radioactivity was detected on a Molecular Dynamics Phosphorlmager. Quantification was performed with the IMAGEQUANT software (version 3.3) purchased from Molecular Dynamics. Various concentrations of pure myosin I were used to verify the linearity of the assay. GST alone does not bind to liposomes (data not shown).

osins I can be further subdivided into a junction or "neck" region containing the putative calmodulin-binding sequences and a basic "tail" region which has been implicated in membrane and phospholipid binding. Unlike most interactions between calmodulin and target proteins, the affinity of calmodulin for myosin I heavy chains is greater in the absence than in the presence of Ca2+, a characteristic shared with several other calmodulin-binding proteins, including neuromodulin/GAP43 (33, 34), ninaC (35), and myosin V/dilute (36, 37). These proteins all contain one or more copies of a sequence, now called the IQ motif (5), consisting of a basic 23-aa unit including a common sequence of IQX3RGX3R (where X is any amino acid) shown to bind calmodulin (34, 37, 38). The bovine form of MMIP has three similar IQ motifs located in the "neck" region of the protein (aa 698-767). The carboxyl-terminal tail domain of MMI,8 (residues 768-1028, =29 kDa) is highly charged, having 26 acidic residues, 37 basic residues, and an isoelectric point of 10.5. Of the three myosin I domains, the tail segment of MMIP3 is least similar in sequence to corresponding domains of other vertebrate myosins I (36% identical and 52% similar to bovine BBMI; 29%6 identical and 51% similar to MMIa). Nevertheless, the overall basic nature of vertebrate myosin I tail domains is conserved, with bovine MMI(8, bovine BBMI, and MMIa/myr I tails having net positive charges of 11, 18, and 14, respectively. The basic character of this portion of the molecule could account for the anomalous migration of the expressed constructs pT2 and pT3 (see Fig. 2). Binding of Expressed MMIJ Fragments to Calmoduin and Phospholds. Digestion of MMI(3 with chymotrypsin in the presence of Ca2+ generates a 78-kDa amino-terminal fragment and a 40-kDa calmodulin-binding fragment (12). Consistent with this result, three putative calmodulin-binding sequences (IQ motifs) were identified within the carboxylterminal 37.5-kDa domain of MMI,3. To examine the association of calmodulin with this domain directly, five MMI(3 fragments were constructed as GST fusion proteins and were assayed by a calmodulin overlay (Fig. 2). Only the IQcontaining constructs pTI and pT4 bound calmodulin; pH1, pT2, and pT3 did not. The weak signal observed with pH1 and pT2 was probably due to background staining ofthe proteins, although weak binding of calmodulin to these constructs cannot be excluded. This result confirms that the 8.5-kDa

fragment immediately following the conserved aminoterminal domain is able to bind to calmodulin. Previously, we had shown that the putative "tail"-specific antibodies mT1, mT2, and mT3 recognized an epitope within the 40-kDa CaM-binding chymotryptic fragment, while "head"-specific antibodies mH4, mH5, mH6, mH7, and mH8 recognize an epitope in the 78-kDa catalytic domain (14). [The previously described monoclonal antibodies (14) have been renamed to reflect their epitope location and clone number. Antibodies previously named Ml, M2, M3, M4, M5, M6, M7, and M8 are now named mTl, mT2, mT3, mH4, mH5, mH6, mH7, and mH8, respectively.] The fusion constructs were used to determine whether the epitope for the "tail" antibodies indeed lay in the carboxyl-terminal domain of MMIfi. This analysis (Fig. 2 and data not shown) showed that antibodies mTl, mT2, and mT3 recognized an epitope within the carboxyl-terminal 18 kDa of MMIp. Furthermore, all "head"' antibodies recognized construct pH1. Fig. 3 shows that adrenal myosin 1(3 binds to anionic phospholipid vesicles. The myosin I preparation used in these experiments was contaminated by two proteins of 100 and 38 kDa which did not cosediment with phospholipids. To determine whether the basic carboxyl-terminal domain of MMI(3 interacts with phospholipid vesicles, binding of pT2 to liposomes was studied. Binding of both intact myosin I and pT2 to anionic phospholipid vesicles was inhibited by high ionic strength.

DISCUSSION A form of myosin I corresponding to the bovine adrenal enzyme previously purified in our laboratory (12, 14) was cloned and sequenced from a bovine brain cDNA library. A portion of the sequence, aa 145-448, is nearly identical to a partial sequence of rat MMIp, which was derived from the neuronal pheochromocytoma cell line PC12 (13). Hence, we refer to this enzyme as bovine MMIj,. Northern analysis using MMI(3 probe revealed hybridization to a single mRNA transcript of 4 kb in many tissues, with particularly high expression in the adrenal gland, kidney, and lung (data not shown) (see also ref. 13). Of the 11 forms of protozoan and vertebrate myosin I that have been completely sequenced to date, the closest relative to MMII3 is the bovine brush-border enzyme, which is 50% identical within the 80.5-kDa head

Biochemistry: Reizes et al. domain and 37% identical in the tail. The relatively weak similarity between the tail domain of MMI(3 and the brushborder enzyme may account for the failure to observe antigenic crossreactivity with monoclonal antibodies raised against the two proteins (12). Like other characterized vertebrate myosins I, MMI/3 has a calmodulin-binding domain which lies between the catalytic domain and the membrane-binding domain. The function of the calmodulin light chains in myosin I is not understood, although they have been implicated in regulating ATPase activity (39), motility (15), and membrane binding (40). Purified adrenal MMI(3 and the recombinant tail construct pT2 bind to anionic phospholipid vesicles. In similar experiments Doberstein and Pollard (41) localized the phospholipidbinding domain of Acanthamoeba myosin IC to a basic region in the carboxyl third of the molecule. Although all forms of myosin I appear to associate with membranes (1, 14), the nature of the interaction is still unclear. The highly basic character of the carboxyl-terminal domains of all sequenced myosin I molecules can explain their interaction with anionic phospholipids but fails to account for the apparent specificity of targeting to organellar membranes (9). Therefore, a membrane-associated receptor may be hypothesized to link these myosins to specific cellular targets (7). We thank Richard Dixon for the use of the AZAP bovine brain cDNA library. We thank Yuri Ushkaryov for help with the northern blots and Andrea Hopkins for assistance in sequencing. We thank Henry Zot for use of the liposome extruder and Clive Slaughter for the protein sequence. We are grateful to Dr. Melanie H. Cobb for helpful suggestions and critical reading of the manuscript. This work was supported by National Institutes of Health Grant GM38567 (to J.P.A.). J.P.A. is an Established Investigator of the American Heart Association. O.R. was supported by a National Institutes of Health Predoctoral Training Grant GM07062. This work was completed in partial fulfillment of the dissertation requirements of O.R. at Southwestern Graduate School of Biomedical Sciences. 1. Pollard, T. D., Doberstein, S. K. & Zot, H. G. (1991) Annu. Rev. Physiol. 53, 653-681. 2. Kiehart, D. P. (1990) Cell 60, 347-350. 3. Korn, E. D. & Hammer, J. A., III (1990) Curr. Opin. Cell Biol. 2, 57-61. 4. Bement, W. M. & Mooseker, M. S. (1993) Nature (London) 365, 785-786. 5. Cheney, R. E. & Mooseker, M. S. (1992) Curr. Opin. Cell Biol. 4, 27-35. 6. Titus, M. A. (1993) Curr. Opin. Cell. Biol. 5, 77-81. 7. Zot, H. G., Doberstein, S. K. & Pollard, T. D. (1992) J. Cell Biol. 116, 367-376. 8. Baines, I. C. & Korn, E. D. (1990) J. Cell Biol. 3, 1895-1904. 9. Baines, I. C., Brzeska, H. Y. & Korn, E. D. (1992) J. CellBiol. 119, 1193-1203. 10. Gillespie, P. G., Wagner, M. D. & Hudspeth, A. J. (1993) Neuron 11, 581-594.

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