Identification of Acan125 as a myosin-I-binding protein present with myosin-I on cellular organelles of Acanthamoeba.

We have discovered the first protein to bind to a nonfilamentous myosin, aside from actin. This protein, Acan125, is a 125-kDa protein from Acanthamoeba that associates with the SH3 domain of Acanthamoeba myosin-IC and not the SH3 domain of human fodrin. Antibodies raised against Acan125 recognize a single protein of 125 kDa from a whole cell lysate of Acanthamoeba; antibodies to myosin-I (M1.7 and M1.8) do not recognize Acan125 on the same blot. Double labeling of Acanthamoeba show Acan125 and myosin-I to be present on the same intracellular organelle, most likely amoebastomes. Immunoprecipitation with either anti-myosin-I or anti-Acan125 antibodies coprecipitates both Acan125 and myosin-I from a lysate of Acanthamoeba, demonstrating that Acan125 interacts with native myosin-I.

lipid phosphatidylserine. But immunostaining shows myosin-I to be excluded from most cell membranes and to be concentrated at the leading edges of migrating cells (17,18) and on selected organelles (19). The contractile vacuole of Acanthamoeba has been demonstrated to selectively bind the myosin-IC isoform (20). Myosin-IA and myosin-IB were found, using immunogold, to be associated along one side of fractionated membranes (21), as though bound to proteins.
Myosin-I could associate with other proteins on membrane surfaces via interactions with SH3. SH3 domains have been shown to mediate specific associations between SH3-containing proteins and various binding partners, including phosphatidylinositol 3-kinase (22)(23)(24)(25), p22 phox , and p47 phox (26 -28), and dynamin (29 -31). In each of these studies, bacterially expressed fusion proteins of SH3 domains were used as affinity ligands to selectively extract the binding partner from a cell lysate. Selectivity may be dictated by the structures of both the SH3 domain and its binding partner (32,33). Thus, binding partners for myosin-I might be identified by their association with the SH3 domain of myosin-I.
We report here one protein from Acanthamoeba, Acan125, which binds to the SH3 domain of myosin-IC and colocalizes with myosin-I on cellular organelles.

EXPERIMENTAL PROCEDURES
Preparation of GST Fusion Proteins-GST (glutathione S-transferase) fusion protein constructs were prepared from polymerase chain reaction products of SH3 domains of Acanthamoeba myosin-IC and human nonerythroid spectrin (fodrin). The DNA corresponding to amino acids 981-1031 of Acanthamoeba myosin-IC was amplified from the plasmid p4.5L (gift from T. D. Pollard, Johns Hopkins University). The polymerase chain reaction primers were 5Ј-CCGGATCCGCGCGT-GCGCTGTA (sense) and 5Ј-GATGAATTCGACGTAGGA (antisense), which included BamHI and EcoRI sites in the sense and antisense primers, respectively. We subcloned the product into Bluescript (Stratagene, La Jolla, CA) and verified the nucleotide sequence. The bona fide DNA was subcloned directly into the BamHI and EcoRI cloning sites of the bacterial expression vector pGEX-2TK (Pharmacia, Piscatawa, NJ). We received the sequence verified DNA for the SH3 domain of human nonerythroid spectrin (fodrin) corresponding to residues 974-1030 in the pGEX vector (gift from J. P. Albanesi, University of Texas Southwestern Medical Center). Both constructs and the empty vector were transformed into Escherichia coli DH5␣ cells for expression.
GST fusion proteins were expressed in bacteria and purified by affinity chromatography on glutathione-agarose (Pharmacia Biotech Inc.). The protein was eluted with glutathione, dialyzed, and stored at 4°C with NaN 3 added. Proteins were stable as determined by SDS-PAGE during the time of the experiments.
Affinity Chromatography of Acanthamoeba Lysate-Acanthamoeba (ϳ2 g) were harvested, resuspended in 2 ml of 0.5 ϫ TBS (1 ϫ TBS: 25 mM Tris-HCl, pH 7.4, 3.7 mM KCl, 138 mM NaCl) supplemented with 1 mM phenylmethylsulfonyl fluoride, 100 g/ml leupeptin, 10 g/ml pepstatin A, 100 units/ml aprotinin, 1 mg/ml diisopropyl fluorophosphate, and 1 mM dithiothreitol, and lysed with 10 strokes in a Dounce homogenizer. The lysate was cleared by centrifugation at 400,000 ϫ g for 10 min and the supernatant passed through a 0.45-m filter. A 0.5-ml volume of the filtrate was mixed with 0.2 ml of glutathione beads coupled with 250 g of fusion protein for 15 min at 4°C. The beads were washed five times with 1.5 ml of 0.5 ϫ TBS and then eluted with 0.2 ml of 5 ϫ TBS. The proteins were separated on 10% SDS-PAGE and stained with Coomassie Blue.
Control serum was collected before immunizing the rabbit.
Antibodies to myosin were obtained from other laboratories. From T. D. Pollard (Johns Hopkins University), we received mouse monoclonal antibodies, M1.7 and M1.8, to Acanthamoeba myosin-I, and M2.42, to Acanthamoeba myosin-II. From I. C. Baines (National Institutes of Health), we received rabbit polyclonal antibodies to Acanthamoeba myosin-IC. These antibodies have been characterized elsewhere (20,34).
Specificity of the anti-Acan125 antibodies was determined by Western blot. A whole cell lysate of Acanthamoeba was separated on SDS-PAGE and a strip of the gel was cut and stained with Coomassie Blue. The remainder of the gel was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and used for a Surf Blot (Idea Scientific, Minneapolis, MN) in which sealed wells of solution containing the antibodies overlay the filter. This creates lanes for antibody reactivity on a continuous blotting surface and eliminates the problems associated with aligning strips cut from a blot. Peroxidase coupled secondary antibodies to mouse or rabbit IgG (American Qualex, La Mirada, CA) were added to the wells, and the peroxidase reaction was developed on the whole blotting surface with chemiluminescent reagent (ECL, Amersham Corp.).
Immunoprecipitations of Acan125 and Myosin-I-Lysate (0.7 ml) of Acanthamoeba (described above) was mixed with 0.7 ml of 0.5 ϫ TBS and with 20 l of protein A beads alone (Pharmacia Biotech Inc. and Sigma) or an equal volume of protein A beads coupled with ϳ7 g of IgG. The suspensions were incubated 60 min at 4°C with constant gentle mixing. The beads were recovered by gentle centrifugation (82 ϫ g for 1 min) and washed five times with 1 ml of 0.5 ϫ TBS. Proteins were eluted from the beads in 100 l of 5 ϫ TBS, separated on SDS-PAGE, and transferred to a PVDF membrane. The blots were incubated with the primary antibodies indicated and developed using peroxidasecoupled secondary antibodies and chemiluminescent reagent.
Immunostaining of Acanthamoeba-M1.7 and anti-Acan125 immune serum in phosphate-buffered saline were directly labeled with fluorescent dyes (35). M1.7 was conjugated with tetramethylrhodamine 6-isothiocyanate (Molecular Probes, Eugene, OR) and purified on G-25 (A 548 / A 280 ϭ 0.40), and anti-Acan125 immune serum was conjugated with fluorescein isothiocyanate (Molecular Probes) and purified on protein A (A 495 /A 280 ϭ 0.12). Acanthamoeba castellanii, Neff strain (American Type Culture Collection, Rockville, MD), were grown in normal liquid culture on polystyrene and split 12-24 h prior to staining. Cells (2-5 ϫ 10 5 ) were seeded on polylysine-coated coverslips and allowed to attach 60 min in culture medium. One-step fixation was used (19); coverslips were immersed in 1% paraformaldehyde and methanol at Ϫ20°C and incubated 5 min. Nonspecific binding was blocked with 1% bovine serum albumin in TBS. For staining, coverslips were incubated 60 min in 110 l containing 1.4 g of rhodamine-labeled M1.7, 78 g of fluorescein labeled anti-Acan125 IgG, and 1% bovine serum albumin in TBS. Fluorescence was observed with a Zeiss Axiovert microscope outfitted with 100X oil immersion lens and standard filter sets for fluorescein and rhodamine. Raw images were captured with a CCD camera, digitized, and stored on disk. Digital images were processed with Adobe Photoshop only to match backgrounds in the two channels and then printed directly by dye sublimation.

RESULTS AND DISCUSSION
To isolate SH3-binding proteins, affinity beads was prepared with the ligand being the SH3 domain of Acanthamoeba myosin-IC (SH3 AmyoIC ) expressed as a fusion protein of GST. The fusion proteins GST, GST-SH3 AmyoIC , or GST-SH3 HuFod (human fodrin SH3-negative control) were immobilized on glutathione beads and then mixed with a lysate of Acanthamoeba; best results were obtained using the lysate clarified by centrifugation at 400,000 ϫ g. High salt was used to elute amoeba proteins without disrupting the association between fusion proteins and the beads. The same proteins that eluted from all beads containing fusion proteins were shown to be nonspecifically bound to the beads alone (Fig. 1). Specifically bound proteins were detected exclusively in the high salt wash from the beads containing GST-SH3 AmyoIC (Fig. 1, a-d). Subsequent release of all proteins from the beads with SDS revealed that the same amount of fusion protein was bound to all beads and that no other proteins were specifically associated with SH3 AmyoIC (data not shown). Thus, four proteins, Acan125, 2 Acan62, Acan55, and Acan47 (Fig. 1), are reversibly associated with SH3 AmyoIC .
Acan125 was selected for further study because it was the least likely to be a proteolytic fragment, the most abundant, and the most well separated from other proteins on the gel. A large preparation yielded ϳ400 g of the Acan125, which was sufficient for the production of antibodies and the attainment of microsequence data.
Given that the SH3 of Src binds a proline-rich motif (10) and that a proline-rich region is present in the sequence of SH3containing isoforms of myosin-I (5), the SH3 domain of myosin-IC could interact with the proline-rich region of another, possibly uncharacterized, myosin-I. To determine if Acan125 is a myosin-I, we transferred Acan125 to a PVDF membrane for microsequence determination. Attempts to obtain sequence directly failed, indicating that the N terminus is blocked. The bound Acan125 was digested with endoproteinase lysC and the peptides separated by high performance liquid chromatography. Several peptide sequences were obtained (data not shown) and searches of protein data bases (PIR 42 and Swiss-Prot 30) did not reveal a match to a known myosin-I.
Polyclonal antibodies were raised in rabbits immunized with Acan125 that was excised from SDS-PAGE. A single protein of 125 kDa was recognized on a Western blot of Acanthamoeba whole cell lysate using anti-Acan125 antibodies (Fig. 2); immune serum, protein A purified immune IgG, and blot purified antibodies gave identical results. On the same blot, mouse monoclonal antibodies to myosin-I (M1.7 and M1.8) recognized multiple bands of proteins (Fig. 2). Previously, M1.7 was shown to react with myosin-IA, myosin-IC, and myosin-II; M1.8 was shown to react with the same proteins plus myosin-IB (34). The M1.7-and M1.8-reactive proteins on the blot of Acanthamoeba lysate (Fig. 2) correlate with the sizes of myosins, 130 -190 kDa. The fact that the broad spectrum myosin antibodies M1.7 and M1.8 recognize proteins larger than the anti-Acan125reactive protein is consistent with the assertion that Acan125 is not a myosin-I.
To demonstrate that Acan125 interacts with myosin-I, we immunoprecipitated the complex from a lysate of Acanthamoeba using M1.7 and M1.8. The myosin-II antibody M2.42 was used as a control. Immunoprecipitations with M1.7 and M1.8, but not with M2.42, showed a single band of reactivity to anti-Acan125 antibodies on a Western blot (Fig. 3A). Thus, Acan125 is precipitated specifically by myosin-I antibodies, indicating that a direct association between the  a-d, proteins, Acan125, Acan62, Acan55, and Acan47, that specifically bind to SH3 AmyoIC . two proteins is likely.
To verify Acan125 association with myosin-I, myosin-I was coprecipitated with Acan125 antibodies from a lysate of Acanthamoeba. Anti-Acan125 and preimmune sera were used to form immunoprecipitates, but only the anti-Acan125 antibodies coprecipitated proteins that reacted with M1.7. The blot shows at least two bands of M1.7 reactivity in the immunoprecipitation (Fig. 3B), indicating that Acan125 interacts with more than one isoform of myosin-I. We identified one isoform, myosin-IC, in the lower band in Fig. 3B using antibodies specific for myosin-IC (data not shown); we have not yet identified a specific isoform of myosin-I in the upper band. The ability of anti-Acan125 antibodies to precipitate myosin-I isoforms from a soluble lysate suggests that complexes of Acan125 and myosin-I exist in Acanthamoeba.
To assess potential interactions in vivo, we stained Acanthamoeba cells for both myosin-I and Acan125. Myosin-I was detected using M1.7 labeled with rhodamine, and Acan125 was detected using protein A-purified anti-Acan125 antibodies labeled with fluorescein. Rhodamine-labeled M1.7 staining of Acanthamoeba was characterized by diffuse fluorescence throughout the cytoplasm excluding the interior of vacuoles, by intense fluorescence in the nuclear region, and by occasional intense fluorescence circumscribing a single round structure (Fig. 4A). The same round structure was stained by fluoresceinlabeled anti-Acan125 antibodies in double-labeled cells (Fig. 4B).
Staining in the nuclear region by rhodamine-labeled M1.7 was observed in all cells (one cell is marked and two cells are unmarked in Fig. 4A). This fluorescence arises from intense staining of the nucleoplasm that surrounds a single large unstained nucleolus (visible in the unmarked cells in Fig. 4A). The nucleoplasmic staining is absent from identically prepared cells stained with unlabeled M1.7 and a labeled secondary antibody. Although the nucleoplasmic staining appears to be an artifact of the rhodamine-labeled M1.7, this provides a convenient internal control for the present experiment. The nucleoplasm is not stained with anti-Acan125 antibodies (Fig.  4B); however, the round structures in Fig. 4 are double-stained, revealing the colocalization of myosin-I and Acan125.
These round structures appear to be similar to structures described earlier as vacuoles in Acanthamoeba stained with M1.7 (19). But a more recent exhaustive study 3 indicates that M1.7 stains myosin-I on tubular structures known as amoebastomes, which appear similar to vacuoles in cross-section. All amoebastomes that stained with M1.7 also stained with anti-Acan125 antibodies in our experiments. No structures were observed to be stained exclusively by anti-Acan125 antibodies, and we found no more than a single organelle per cell to be stained by both antibodies. Colocalization of Acan125-and myosin-I-reactive proteins on M1.7-stained organelles, which are probably amoebastomes, further indicates the formation of a complex of myosin-I and Acan125 in the cell.
Acan125 is presently the only protein aside from actin known to interact with myosin-I. However, we think it is likely that others will be identified including Acan62, Acan55, and Acan47, which bind myosin-I SH3 and do not react with anti-Acan125 antibodies on a blot. The SH3 domain of myosin-I may regulate or target myosin-I's interactions with these proteins in response to cellular signals. The presence of Acan125 and myosin-I in the high speed supernatant suggest that they are both present in the cytoplasm, and the immunoprecipitation results demonstrate that Acan125 and myosin-I are competent to form a complex. The complex may be recruited to organelle surfaces where attachment could be mediated by either Acan125 or myosin-I. A role for the complex will become clearer when a function can be ascribed to Acan125.