The motor function of vertebrate unconventional myosins is not well understood. In this study, we initiated the baculovirus expression system to characterize a novel myosin I from bovine adrenal gland that we had previously cloned [Zhu, T., & Ikebe, M. (1994) FEBS Lett. 339, 31-36], which is classified as myosin I beta. The expressed myosin I beta was well extracted when calmodulin was coexpressed in Sf9 cells. The recombinant myosin I beta cosedimented with actin in an ATP dependent manner. The purified myosin I beta was composed of one heavy chain and three calmodulins. The electron microscopic image of myosin I beta confirmed its single-headed structure with a short tail, which is similar to that of brush border myosin I (BBMI). Myosin I beta showed high K+,EDTA--ATPase activity (approximately 0.14 mumol/min/mg) and Ca(2+)-ATPase activity (approximately 0.32 mumol/min/mg), and the KCl/pH dependence of these activities was different from that of conventional myosin. Mg(2+)-ATPase activity of myosin I beta alone was increased above pCa 6, while the actin dependent activity was not affected by Ca2+. Actin sliding velocity of myosin I beta in the absence of Ca2+ was 0.3-0.5 microns/s at 25 degrees C, which is much greater than that of BBMI (< 0.05 microns/s). The actin sliding activity was abolished above pCa 6, and the sliding activity was restored when exogenous calmodulin was added in the absence of Ca2+. Within similar Ca2+ concentrations, one of the three calmodulins was dissociated from myosin I beta. The results suggest that Ca2+ dependent association of calmodulin may function as a regulatory mechanism of myosin I beta motor activity and that the motor activity of mammalian myosin I is largely different among distinct myosin I isoforms.

We coexpressed myosin I␤ heavy chain with three different calmodulin mutants in which the two Ca 2؉ -binding sites of the two N-terminal domain (E12Q), C-terminal domain (E34Q), or all four sites (E1234Q) are mutated in order to define the importance of these Ca 2؉ binding sites to the regulation of myosin I␤. The calmodulin mutated at the two Ca 2؉ binding sites in N-terminal domain and C-terminal domain lost its lower affinity Ca 2؉ binding site and higher affinity Ca 2؉ binding site, respectively. We found that, based upon the change in the actin-activated ATPase activities and actin translocating activities, myosin I␤ with E12Q calmodulin has the regulatory characteristics similar to myosin I␤ containing wild-type calmodulin, while myosin I␤ with E34Q or E1234Q calmodulin lose all Ca 2؉ regulation. While the increase in myosin I␤ ATPase activity paralleled the dissociation of 1 mol of calmodulin from myosin I␤ heavy chain for both wild type (above pCa 5) and E12Q calmodulin (above pCa 6), the Ca 2؉ level required for the inhibition of actin-translocating activity of myosin I␤ was lower than that required for dissociation of calmodulin, suggesting that the conformational change induced by the binding of Ca 2؉ at the high affinity site but not the dissociation of calmodulin is critical for the inhibition of the motor activity. Our results suggest that the regulation of unconventional myosins by Ca 2؉ is directly mediated by the Ca 2؉ binding to calmodulin, and that the C-terminal pair of Ca 2؉ -binding sites are critical for this regulation.

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A 3.5 kb cDNA clone was isolated from bovine adrenal gland cDNA library. The clone contained a full-length 3.1 kb open reading frame, encoding a novel myosin I. The deduced amino acid sequence was highly homologous to other known myosin Is in the N-terminal 2 kb region which corresponds to the myosin head domain, while no strong homology was detected in the tail region. The head-tail junction contained the Ca"-independent calmodulin binding consensus sequence, suggesting that the novel myosin I binds calmodulin. This was confirmed by calmodulin overlay which showed the binding of '2SI-calmodulin to the recombinant myosin I expressed in E. coli. Northern blots with probes from head and tail regions of this myosin I revealed that this novel myosin I is widely distributed among various tissues.

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