Rat myr 4 defines a novel subclass of myosin I: identification, distribution, localization, and mapping of calmodulin-binding sites with differential calcium sensitivity.

Abstract

Abstract. We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin Fs. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences.The Ca2+-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca 2÷ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca 2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca 2+. A further Ca 2+-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca 2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin.Myr 4 was demonstrated to be expressed in many .cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F-actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.

Production of Reagents and Optimization of Methods for Studying Calmodulin-Binding Proteins

“…It seems likely that the short myosin is MyoIE, which is the only myosin I in D. discoideum that possesses more than one clearly identifiable IQ (CaM-binding) motif. Alternatively, the different behavior of short-and long-tailed myosins I could be explained by the fact that IQ motifs differ in their Ca 2ϩ requirements for CaM binding (39). Surprisingly, myosin II was found to bind to the matrix, which likely reflects the ability of its two IQ motifs to bind CaM in vitro.…”

Two‐dimensional electrophoresis with cationic detergents: A powerful tool for the proteomic analysis of myelin proteins. Part 2: Analytical aspects

“…Myo1D (rat: myr4) is an unconventional myosin protein (Bahler et al, 1994;Kohler et al, 2005) and is linked to membrane trafficking along the recycling pathway (Huber et al, 2000). In developmental analysis of myelin fraction, Myo1D was detected from 3 weeks of age and increased with age in the same way as major myelin proteins (PLP, DM-20, and CNP; Fig.…”

“…The expression of Myo1D (myr4) was reported to start at about postnatal day 14 and increase until the adult stage in rat forebrain (Bahler et al, 1994). In addition, the expression of Myo1D was recently detected in maturing oligodendrocytes in culture (Nielsen et al, 2006).…”

The axonal transport of mitochondria

“…Although it has not been reported to be associated with mitochondria, the monomeric motor myosin I has been implicated in organelle transport (Barylko et al, 2000). Myosin I is present in neurons, in which it has a punctate and cortical distribution (Wagner et al, 1992;Bahler et al, 1994;Lewis and Bridgman, 1996). In axons much of it is distributed in the cortex, where it may drive vesicle-plasma-membrane fusion or plasma-membrane-actin interactions (Lewis and Bridgman, 1996), perhaps during slow axonal transport (Lund et al, 2005).…”