The motor function of Drosophila melanogaster myosin-5 is activated by calcium and cargo-binding protein dRab11

Abstract

In the Drosophila melanogaster compound eye, myosin-5 (DmM5) plays two distinct roles in response to light stimulation: transport of pigment granules to the rhabdomere base to decrease light exposure and transport of rhodopsin-bearing vesicles to the rhabdomere base to compensate for the rhodopsin loss during light exposure. However, little is known of how the motor function of DmM5 is regulated at the molecular level. In the present study, we overexpressed DmM5 in Sf9 insect cells and investigated its regulation using purified proteins. We found that the actin-activated ATPase activity of DmM5 is significantly lower than that of the truncated DmM5 having the C-terminal globular tail domain (GTD) deleted, indicating that the GTD is the inhibitory domain. The actin-activated ATPase activity of DmM5 is significantly activated by micromolar levels of calcium. DmM5 associates with pigment granules and rhodopsin-bearing vesicles through cargo-binding proteins Lightoid (Ltd) and dRab11 respectively. We found that GTP-bound dRab11, but not Ltd, significantly activates DmM5 actin-activated ATPase activity. Moreover, we identified Gln(1689) in the GTD as the critical residue for the interaction with dRab11 and activation of DmM5 motor function by dRab11. Based on those results, we propose that DmM5-dependent transport of pigment granules is directly activated by light-induced calcium influx and the DmM5-dependent transport of rhodopsin-bearing vesicle is activated by active GTP-bound dRab11, whose formation is stimulated by light-induced calcium influx.

Rab GTPases and their interacting protein partners: Structural insights into Rab functional diversity

“…9C ) or the presence of μM level of Ca 2+ activates the motor in vitro by destabilizing the auto-inhibited state of MyosinV. 103 However, in the cell, endogenous Rab11 is not sufficient to recruit the auto-inhibited motor to the membranes. 104 Simultaneous binding of the actin regulator Spir and Rab11 to 2 distinct sites of the GTD is necessary to trigger the recruitment of MyosinV to vesicles ( Fig.…”

Unconventional Myosins: How Regulation Meets Function

“…Structural studies have revealed that myosin Vb binds both the GDP-and GTPforms of Rab11, with around 30-fold higher affinity for the GTP-form [134]. Although the binding to Rab11 has been proposed to activate the Drosophila myosin V (DmM5) [144], the mechanism of this activation and the effect on the motor properties remains to be addressed. Similarly, given the broad range of binding partners and the diverse mechanisms underlying their interaction with the myosin V isoforms, it remains unclear whether the melanophilin-induced activation and the changes in myosin Va's biophysical properties is a generally applied mechanism or not.…”

Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease

“…Double-headed class 5 myosins are evolutionary ancient, prototypic actin-based molecular motors implicated in the spatiotemporal segregation and the transport of organelles across phylogeny (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). A prerequisite for efficient transport is that a single motor can either take multiple discrete steps on an actin filament without detaching, a property also referred to as processivity, or that the motor works in an ensemble with other myosins-5 to efficiently move a cargo.…”