The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

Abstract

Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain

“…In this control strain, the gpdA-∆C-hookA-S allele was present to ensure that dynein mainly moves towards the microtubule minus end. We found that wild-type dynein moved robustly in the motility assay, and the full-length wB-AAA3 dynein also moves processively along the microtubule (when 3mM ATP instead of 1mM ATP was used), albeit with a very low speed (Figure S4A, S4B and S4C), consistent with previous in vitro data obtained from the yeast dynein motor domain (Cho et al, 2008;Bhabha et al, 2014;DeWitt et al, 2015;Nicholas et al, 2015;DeSantis et al, 2017;X. Liu et al, 2020).…”

“…Despite the differences in the mechanisms of plus-end dynein localization, the dynein motor mechanism is most likely conserved in different organisms. Consistent with previous results on the yeast wB-AAA3 dynein motor domain (Cho et al, 2008;Bhabha et al, 2014;DeWitt et al, 2015;Nicholas et al, 2015;DeSantis et al, 2017;X. Liu et al, 2020), the wB-AAA3 mutation also significantly inhibits the in vitro motility of full-length dynein in A. nidulans extract (Figure S4).…”

“…The minus-end-directed movement of dynein requires multiple elements of the dynein heavy chain, including the AAA1-6 domains, the linker, the buttress, the stalk and its distal microtubule-binding domain (Bhabha et al, 2016;Can et al, 2019;Niekamp et al, 2019;Rao et al, 2019;X. Liu et al, 2020).…”

“…The copyright holder for this preprint (which this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439451 doi: bioRxiv preprint Rao et al, 2019). ATP hydrolysis at AAA3 and ATP binding at AAA4 regulate the AAA1-dependent release of dynein from microtubule (Silvanovich et al, 2003;Kon et al, 2004;Cho et al, 2008;Bhabha et al, 2014;DeWitt et al, 2015;Nicholas et al, 2015;X. Liu et al, 2020).…”

Structure and Mechanics of Dynein Motors

“…Therefore, the ATP hydrolysis activity of this site may be greatly reduced. Recent work has shown that blocking ATP binding to AAA4 fully inhibits dynein, whereas blocking ATP hydrolysis has little effect on motor velocity (100). These results indicate that AAA4 remains mostly in the ATP-bound state and that the nucleotide plays a structural role for the AAA1 site to drive the priming stroke of the linker and sliding of the stalk coiled-coils (100).…”