(Received for publication, June 28, 1995; and in revised form, August 31, 1995)
From the
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.
Binding through the Src homology domain, SH3, is a
recognized means of linking signal transduction
proteins(1, 2, 3) , but a function has not
been ascribed to the SH3 domain of the cytoskeletal protein myosin-I.
The isoforms of myosin-I that contain an SH3 domain include myosin-Is
from Acanthamoeba(4) , Dictyostelium(5) , Saccharomyces(6) ,
rat(7) , and human(8) . In the known myosin-I
sequences, the SH3 domain invariably resides in tandem with one or two
proline-rich domains(5, 9) ; a proline-rich sequence
of 3BP1 has been identified as a motif that binds to the SH3 domain of
Abl (10) . An interaction has not been demonstrated between SH3
and proline-rich domains of myosin-I, but proline-rich domains of
myosin-I have been shown to interact with
actin(11, 12, 13) . These results suggested
to us that the SH3 domain of myosin-I might be available for
interaction with another protein.
The proposal of proteins that interact with myosin-I is rooted in efforts to reconcile reconstitution results with cellular localization studies. In vitro binding (11, 14, 15) and motility (16) assays are consistent with myosin-I acting mechanically on the surface of any membrane containing the ubiquitous phospholipid 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, and
p47
(26, 27, 28) , and
dynamin(29, 30, 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.
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 added. Proteins were stable as
determined by SDS-PAGE during the time of the experiments.
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.).
To isolate SH3-binding proteins, affinity beads was prepared
with the ligand being the SH3 domain of Acanthamoeba myosin-IC
(SH3) expressed as a fusion protein of GST. The fusion
proteins GST, GST-SH3
, or GST-SH3
(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
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
(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
(data not shown). Thus, four proteins, Acan125, (
)Acan62, Acan55, and Acan47 (Fig. 1), are reversibly
associated with SH3
.
Figure 1:
Specific association
of Acan125 with the SH3 domain of Acanthamoeba myosin-IC. Each
lane represents a separate binding experiment and shows the proteins
that eluted from glutathione beads containing the following fusion
proteins: lane 1, none; lane 2, GST; lane 3,
GST-SH3; lane 4, GST-SH3
. a-d, proteins, Acan125, Acan62, Acan55, and Acan47, that
specifically bind to SH3
.
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 400 µg 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 SH3-containing 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-Acan125-reactive protein is consistent with the assertion that Acan125 is not a myosin-I.
Figure 2: Western blot of Acanthamoeba lysate showing the specificity of antibodies used. Lane 1, Coomassie Blue-stained gel showing proteins before transfer; lane 2, M1.8-reactive proteins; lane 3, M1.7-reactive proteins; lane 4, Anti-Acan125-reactive protein. The blotting surface between lanes 2 and 4 is continuous, thereby demonstrating that the anti-Acan125-reactive protein does not comigrate with the anti-myosin-I-reactive proteins.
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 two proteins is likely.
Figure 3: Immunoprecipitations showing that Acan125 and myosin-I form a complex. Western blot A, separate immunoprecipitations were performed using M1.8 (lane 2), M1.7 (lane 3), M2.42 (lane 4), or no antibody (lane 5). A standard of Acan125 protein was included on the same blot (lane 1). The entire blot was probed with anti-Acan125 antibodies. Western blot B, separate immunoprecipitations were performed using no antibody (lane a), preimmune serum (lane b), anti-Acan125 antibodies (lane c), and M1.7 (lane d). The entire blot was probed with M1.7.
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 fluorescein-labeled anti-Acan125 antibodies in double-labeled cells (Fig. 4B).
Figure 4: Double-stained Acanthamoeba show the colocalization of myosin-I- and Acan125-reactive proteins. The same field shows M1.7 staining in the rhodamine channel (panel A) and anti-Acan125 staining in the fluorescein channel (panel B). Marked are amoebastomes (arrow), defined by M1.7 staining, and one example of nucleoplasmic staining (n).
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. 4are 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 ()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.