Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains

Enilza M. Espreafico, Richard E. Cheney, Michela Matteoli, Alexandra A C Nascimento, Pietro V. De Camilli, Roy E. Larson, Mark S. Mooseker

Research output: Contribution to journalArticlepeer-review

Abstract

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain pi 90 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the pi 90-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (he), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V he (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an ∼23-aa ∼IQ-motif.∼ All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil a helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells. Myosin-V staining was particularly intense in the cell bodies and dendrites of Purkinje cells. Double labeling with wheat germ agglutinin revealed colocalization of myosin-V with cytoplasmic, presumably Golgi-derived, membranes. In primary cultures of neurons and glia, myosin-V immunoreactivity had a punctate distribution more abundant in the region of the Golgi complex and at the tips of long processes such as growth cones. These results, together with the phenotypes of mutations described for the dilute and myo2 genes, suggest that the myosin-V family of unconventional myosins may be in part associated with cytoplasmic organelles.

Original languageEnglish
Pages (from-to)1541-1557
Number of pages17
JournalJournal of Cell Biology
Volume119
Issue number6
Publication statusPublished - Dec 1992

ASJC Scopus subject areas

  • Cell Biology

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