Abstract
Nonmuscle myosin IIs (NM IIs) are a group of molecular motors involved in a wide variety of cellular processes including cytokinesis, migration, and control of cell morphology. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene. These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. Most previous studies on NM II used tissue-purified protein or expressed fragments of the molecule, which presents potential drawbacks for characterizing individual paralogs of the intact protein in vitro. To circumvent current limitations and approach their native properties, we have successfully expressed and purified the three full-length human NM II proteins with their light chains, using the baculovirus/Sf9 system. The enzymatic and structural properties of the three paralogs were characterized. Although each NM II is capable of forming bipolar filaments, those formed by IIC tend to contain fewer constituent molecules than those of IIA and IIB. All paralogs adopt the compact conformation in the presence of ATP. Phosphorylation of the regulatory light chain leads to assembly into filaments, which bind to actin in the presence of ATP. The nature of interactions with actin filaments is shown with different paralogs exhibiting different actin binding behaviors under equivalent conditions. The data show that although NM IIA and IIB form filaments with similar properties, NM IIC forms filaments that are less well suited to roles such as tension maintenance within the cell.
Highlights
Three genes encode human nonmuscle myosin II (NM II) heavy chains, and the proteins have different intracellular roles and localizations
There are multiple paralogs of NM II present in most mammalian cells and tissues [15]. This hampers the determination of the properties of individual NM II paralogs when using tissue-purified protein
Purification of NM II from brain tissue should result in myosin, which is highly enriched in NM IIB, but no cell types are known in which exclusively NM IIB or NM IIC are expressed
Summary
Three genes encode human nonmuscle myosin II (NM II) heavy chains, and the proteins have different intracellular roles and localizations. There are three paralogs of the NM II heavy chain in humans (IIA, IIB, and IIC), each encoded by a separate gene These paralogs are expressed at different levels according to cell type and have different roles and intracellular distributions in vivo. The heavy chain begins with an N-terminal motor domain that is responsible for ATP hydrolysis and is the source of force generation Following this is a lever arm region that binds the light chains. Much of the information to date regarding the macromolecular assembly of NM II has been obtained by studying tail fragments of the molecule, usually expressed as recombinant proteins in Escherichia coli (29 –34) These tail fragments do not form uniform discrete bipolar assemblies such as those seen with full-length myosin and are not ideal as a system for comparing the properties of the native filaments. We report on the physical and biochemical properties of filaments formed from the full-length baculovirus expressed NM II paralogs and compare and contrast them to previous reports for the tissue-purified proteins
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