Abstract
The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine‐tune motor activity in time and space. These motor–adaptor–cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling‐based proteomics strategy to map the interactome of the unique minus end‐directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6‐adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG‐Induced F‐actin for Tethering) complex that controls endosome positioning and motility through RHO‐driven actin polymerisation; and the DISP (DOCK7‐Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.
Highlights
In eukaryotic cells, myosin motor proteins regulate the distribution of a wide variety of cytoplasmic cargo by mediating short-range transport or tethering of organelles, vesicles, mRNA and protein complexes
BirA*MYO6 cargo-binding domain (CBD) replicates were analysed by mass spectrometry (MS) and compared against a bank of 10 BirA* only negative control experiments using the online tool at CRAPome.org [25]
This confirms our previous observations that binding of DAB2 and other adaptors is not isoform specific [8,10,14], but targeting of the large insert” isoform (LI) isoform to clathrin-coated structures is directed by the large insert [27]
Summary
Myosin motor proteins regulate the distribution of a wide variety of cytoplasmic cargo by mediating short-range transport or tethering of organelles, vesicles, mRNA and protein complexes. The functional and phenotypic diversity associated with MYO6 arises from interactions with multiple cargo adaptors including disabled-2 (DAB2), GAIP-interacting protein C-terminus (GIPC1), target of Myb 1 (TOM1), lemur tyrosine kinase 2 (LMTK2), optineurin (OPTN), TAX1 binding protein 1 (TAX1BP1) and nuclear dot protein 52 (NDP52) [8,9,10,11,12,13] These interactions occur at two major protein binding motifs, the RRL and WWY (named after their amino acid composition), which are located within two distinct subdomains of a unique C-terminal cargo-binding tail [10,11]. Two distinct ubiquitin-binding sites—a motif interacting with ubiquitin (MIU) and a MYO6 ubiquitin-binding domain (MyUb)—in the tail region may bind ubiquitinated cargo or regulate other interactions [15,16] These adaptor interactions mediate targeting of the motor to its appropriate cellular location, making them a critical determinant of motor function
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