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.
Highlights
MethodsYeast cultures and protein production of the single-headed dynein, GFP-Dyn1331 kDa, and its mutants was done as described previously[24] with the modifications described below
Blocking AAA1-ATP binding has a dominant effect on stalk registries and induces the α registry under backward load and the γ registry under forward load irrespective of the nucleotide state of AAA4
The AAA4 E/Q mutant in the presence of 1 mM ATP behaved to Dyn1331kDa-β CL, the Dyn1331kDa mutant with the cross-linked β registry (Fig. 3c and Tables 1, 2). These results demonstrate that ATP binding to AAA4 is required for transition of the stalk helices into the β-registry, supporting the hypothesis that ATP binding to AAA4 is required for linker dissociation from AAA5 and subsequent transition of the stalk helices into the β-registry
Summary
Yeast cultures and protein production of the single-headed dynein, GFP-Dyn1331 kDa, and its mutants was done as described previously[24] with the modifications described below. The parent strain used in this work was VY137, which expresses a tail-truncated minimal S. cerevisiae MD containing the linker, AAA+ ring, stalk, buttress, and MTBD. This construct retains its motor activities[42,52,56] and is equivalent to the Dictyostelium discoideum MD used in key biochemical studies[19,22,37,39,70,71,72]. For single-headed dynein, expression was induced by growing the cells in 2× YPG medium (4% [w/v] galactose) to a final OD600 between
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