Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains.

Carboni, Joan M.; Conzelman, Karen A.; Adams, Rick A.; Kaiser, Dale; Pollard, Thomas D.; Mooseker, Mark S.

doi:10.1083/jcb.107.5.1749

Cited by 65 publications

(56 citation statements)

References 41 publications

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“…Antibody directed against the head domain of chicken BBMI (CX-1; ref. 33) allowed us to detect three polypeptides in the BWTG3 hepatoma cell line, whose size is in agreement with the size of members of the myosin-I family (E.C., unpublished data). However, further analysis is necessary to demonstrate that these immunologically related proteins are indeed endogenous myosins-I associated with the endocytic pathway.…”

Section: Discussionsupporting

confidence: 55%

“…BBMI tail (730-1040 aa) lacks the entire aminoterminal motor domain as well as two of its four putative calmodulin binding sites, whereas BBMIA446 (446-1040 aa) lacks the ATP binding site. The production of chicken BBMI and truncated variants in these cell lines was determined by immunoblotting using a monoclonal antibody directed against the carboxy-terminal domain of the chicken BBMI (CX-7 antibody) (33). In control cells that have been transfected with the expression vector alone, the CX-7 antibody did not detect any polypeptide corresponding to the electrophoretic migration of a myosin-I (Fig.…”

Section: Truncated Bbmi Proteins Were Recovered In Fractionsmentioning

confidence: 99%

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Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Dürrbach

Collins

Matsudaira

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins I, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing a2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of a2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

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Section: Discussionsupporting

confidence: 55%

Section: Truncated Bbmi Proteins Were Recovered In Fractionsmentioning

confidence: 99%

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Dürrbach

Collins

Matsudaira

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…The tail domain of CBB-MI is thought to contain the CAM binding sites [8,9]. To determine whether the 29-residue insert might contain a CAM binding site, clones A and B were expressed in E. coli as fusions to a 37 kDa fragment of the E. coli protein trpE.…”

Section: Resultsmentioning

confidence: 99%

“…1). Peptide mapping studies indicate that the CAM binding sites reside somewhere within the unconventional, -37 kDa tail domain [8,9].…”

Section: Introductionmentioning

confidence: 99%

A second isoform of chicken brush border myosin I contains a 29‐residue inserted sequence that binds calmodulin

Halsall

Hammer

1990

FEBS Letters

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Chtcken brush border myosm I (CBB-MI) IS a stngle-headed, nonfilamentous. myosm-hke mechanoenzyme which. as isolated, has 3 mol of calmodulm (CAM) 'hght chams' bound per mole of 119 kDa heavy cham. We have isolated a parttal cDNA clone for CBB-MI that encodes the C-termmal -35 kDa of the heavy cham The sequence of thts clone IS identtcal to that of an authentic. near-full-length CBB-MI cDNA clone reported recently, except for an 87-bp29-residue msertion occurrmg -32 kDa from the C-termmus Thts Insert, which is probably generated by an alternate sphcmg event. IS expressed m brush horder as part of a message of the stze predicted for the CBB-MI heavy cham. although the steady state level of this transcript is -X-fold lower than for transcripts lackmg the Insert iZSI-CAM overlays of this cDNA clone (expressed as a trpE fusion protem m E toll) Indicate that it hmds one more calmodulm than does a second cDNA clone that lacks the 29-residue Insert. A synthettc pepttde correspondmg to the Insert sequence hinds ttghtly to CAM-Sepharose.demonstrates a shift and enhancement of fluorescence m the presence of CAM. and btnds CAM m solution with a Ko of 190 nM (m 100 mM KCl) We conclude that a second, low-abundance tsoform of CBB-MI contams an addtttonal (and possibly fourth) CAM htndmg site as a result of a 29-restdue peptide that is Inserted mto the tail domain by an apparent alternate sphcmg event.Chicken brush horder myosm I. Calmodulm bmdmg; Alternate sphcmg

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“…8) and a monoclonal antibody, MaB CX-1, made to BB myosin-I that reacts with the N-terminal third of the BB myosin-I head as well as with BM-V ("head antibody"; Ref. 16) were used for immunoblot analysis.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Characterization and Functional Domain Mapping of Brain Myosin-V

Nascimento

Cheney

Tauhata

et al. 1996

Journal of Biological Chemistry

120

131

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Although the conventional myosins (myosins-II) responsible for processes such as muscle contraction and cytokinesis have been intensively studied, relatively little is known about the properties of the unconventional myosins. One widely distributed group of unconventional myosins, the class V myosins, have been suggested to play a role in organelle transport or membrane targeting (reviewed in Refs. 1 and 2). Brain myosin-V (BM-V 1 ), which was originally identified as a calmodulin (CaM)-binding protein in vertebrate brain, is the only member of this class of unconventional myosins to be purified and characterized biochemically (3-7). The class V myosins share a common domain structure consisting of an N-terminal head domain containing the actin-binding and ATP hydrolysis sites, an extended neck domain containing six IQ motifs (which form binding sites for CaM and/or related light chains), and a tail domain consisting of a region predicted to form coiled coils attached to a globular region of unknown function (7,8). The hypothetical functions of the class V myosins and their novel domain structure, particularly the extended neck domain, raise the obvious question of how the basic biochemical properties of the class V myosins compare with those of other types of myosins.Among the critically important properties of a molecular motor are its steady-state ATPase activity and the factors which regulate this activity. Known myosins are regulated either indirectly via actin-associated proteins such as troponin/ tropomyosin (as in vertebrate skeletal muscle myosin) or directly via the subunits of the myosin molecule itself. In the latter type of direct "myosin-linked" regulation, the light chains associated with the neck domain often function as the regulating subunits. The myosin light chains are all members of the CaM superfamily of proteins, although not all of them have retained the ability to bind to Ca 2ϩ . Vertebrate non-muscle and smooth muscle myosins-II are "turned on" by phosphorylation of their regulatory light chains by Ca 2ϩ /CaM-dependent myosin light chain kinase (9), whereas molluscan myosin-II and vertebrate brush border (BB) myosin-I are regulated by the direct binding of Ca 2ϩ to their light chains (10, 11). Like BB myosin-I, BM-V has multiple CaM light chains that remain bound in the absence of Ca 2ϩ (3,5,7). The neck domain of BM-V has been shown to be the precise site of CaM binding (8), suggesting that this myosin might also be directly regulated by Ca 2ϩ binding. The initial reports of BM-V ATPase activity also indicated that this protein is activated by Ca 2ϩ (5, 7), although the K ATPase for Ca 2ϩ and F-actin were not determined. The tail domains of myosins are unique to each class in the myosin superfamily and probably reflect the specific functions

show abstract

Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains.

Cited by 65 publications

References 41 publications

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

A second isoform of chicken brush border myosin I contains a 29‐residue inserted sequence that binds calmodulin

Enzymatic Characterization and Functional Domain Mapping of Brain Myosin-V

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