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The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release.

DeWitt MA, Cypranowska CA, Cleary FB, Belyy V, Yildiz A - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude more slowly than AAA1, and the two sites do not coordinate.Nucleotide hydrolysis at AAA3 lifts this 'MT gate' to allow fast motility.These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT-anchoring tasks in cells.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, Berkeley, Berkeley, California, USA.

ABSTRACT
Cytoplasmic dynein is an AAA+ motor responsible for intracellular cargo transport and force generation along microtubules (MTs). Unlike kinesin and myosin, dynein contains multiple ATPase subunits, with AAA1 serving as the primary catalytic site. ATPase activity at AAA3 is also essential for robust motility, but its role in dynein's mechanochemical cycle remains unclear. Here, we introduced transient pauses in Saccharomyces cerevisiae dynein motility by using a slowly hydrolyzing ATP analog. Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude more slowly than AAA1, and the two sites do not coordinate. ATPase mutations to AAA3 abolish the ability of dynein to modulate MT release. Nucleotide hydrolysis at AAA3 lifts this 'MT gate' to allow fast motility. These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT-anchoring tasks in cells.

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ATP hydrolysis cycle at AAA1 drives the stepping motility of dynein. (Left) When AAA3 is in the apo state, communication between AAA1 and the MTBD is blocked, resulting in slow, force-dependent MT release, and consequent slow progression through the AAA1 hydrolysis cycle (τcycle = 1.2 s)15,18. (Right) When ATP binds to AAA3 and is hydrolyzed, the allosteric circuit connecting AAA1 and the MTBD is completed. In this state, ATP binding at AAA1 leads to fast release from the MT, and subsequent progression through the AAA1 hydrolysis cycle (τcycle = 0.13 s). Cycle times are calculated from the bulk ATPase rates per head. This AAA3 controlled switch may play an essential role in repurposing of dynein for intracellular transport (i.e. fast MT release) and anchoring (i.e. slow MT release) functions.
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Figure 7: ATP hydrolysis cycle at AAA1 drives the stepping motility of dynein. (Left) When AAA3 is in the apo state, communication between AAA1 and the MTBD is blocked, resulting in slow, force-dependent MT release, and consequent slow progression through the AAA1 hydrolysis cycle (τcycle = 1.2 s)15,18. (Right) When ATP binds to AAA3 and is hydrolyzed, the allosteric circuit connecting AAA1 and the MTBD is completed. In this state, ATP binding at AAA1 leads to fast release from the MT, and subsequent progression through the AAA1 hydrolysis cycle (τcycle = 0.13 s). Cycle times are calculated from the bulk ATPase rates per head. This AAA3 controlled switch may play an essential role in repurposing of dynein for intracellular transport (i.e. fast MT release) and anchoring (i.e. slow MT release) functions.

Mentions: Combining these results with previous work, we propose that AAA3 acts as a switch that regulates AAA1-directed MT release (Fig. 7). In the apo or ATP-bound state (mimicked by AAA3 ATP-binding and hydrolysis mutants, respectively), AAA1 cannot allosterically direct MT release through the ring (Fig. 7, Left)15,19,26. In this switch off state, dynein remains processive, albeit at a substantially lower speed26, presumably via tension generated on the MTBD through the powerstroke of the linker17,28 (Fig. 1e,f). Slow MT detachment in turn slows the hydrolysis cycle at AAA1, resulting in reduced speed and strong MT attachment. After ATP hydrolysis at AAA3, the switch is on, and the allosteric circuit connects AAA1 to MTBD, allowing rapid, ATP-stimulated release from the MT (Fig. 7, right). This “switch on” post-hydrolysis state (either ADP-Pi or ADP) persists for an average of ~20 steps of the motor, allowing the motors to move at full speed. Conformational changes required for release from the MT is driven by ATP hydrolysis in AAA118,23. Eventual release of products at AAA3 turns the switch off until the next nucleotide cycle.


The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release.

DeWitt MA, Cypranowska CA, Cleary FB, Belyy V, Yildiz A - Nat. Struct. Mol. Biol. (2014)

ATP hydrolysis cycle at AAA1 drives the stepping motility of dynein. (Left) When AAA3 is in the apo state, communication between AAA1 and the MTBD is blocked, resulting in slow, force-dependent MT release, and consequent slow progression through the AAA1 hydrolysis cycle (τcycle = 1.2 s)15,18. (Right) When ATP binds to AAA3 and is hydrolyzed, the allosteric circuit connecting AAA1 and the MTBD is completed. In this state, ATP binding at AAA1 leads to fast release from the MT, and subsequent progression through the AAA1 hydrolysis cycle (τcycle = 0.13 s). Cycle times are calculated from the bulk ATPase rates per head. This AAA3 controlled switch may play an essential role in repurposing of dynein for intracellular transport (i.e. fast MT release) and anchoring (i.e. slow MT release) functions.
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Related In: Results  -  Collection

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Figure 7: ATP hydrolysis cycle at AAA1 drives the stepping motility of dynein. (Left) When AAA3 is in the apo state, communication between AAA1 and the MTBD is blocked, resulting in slow, force-dependent MT release, and consequent slow progression through the AAA1 hydrolysis cycle (τcycle = 1.2 s)15,18. (Right) When ATP binds to AAA3 and is hydrolyzed, the allosteric circuit connecting AAA1 and the MTBD is completed. In this state, ATP binding at AAA1 leads to fast release from the MT, and subsequent progression through the AAA1 hydrolysis cycle (τcycle = 0.13 s). Cycle times are calculated from the bulk ATPase rates per head. This AAA3 controlled switch may play an essential role in repurposing of dynein for intracellular transport (i.e. fast MT release) and anchoring (i.e. slow MT release) functions.
Mentions: Combining these results with previous work, we propose that AAA3 acts as a switch that regulates AAA1-directed MT release (Fig. 7). In the apo or ATP-bound state (mimicked by AAA3 ATP-binding and hydrolysis mutants, respectively), AAA1 cannot allosterically direct MT release through the ring (Fig. 7, Left)15,19,26. In this switch off state, dynein remains processive, albeit at a substantially lower speed26, presumably via tension generated on the MTBD through the powerstroke of the linker17,28 (Fig. 1e,f). Slow MT detachment in turn slows the hydrolysis cycle at AAA1, resulting in reduced speed and strong MT attachment. After ATP hydrolysis at AAA3, the switch is on, and the allosteric circuit connects AAA1 to MTBD, allowing rapid, ATP-stimulated release from the MT (Fig. 7, right). This “switch on” post-hydrolysis state (either ADP-Pi or ADP) persists for an average of ~20 steps of the motor, allowing the motors to move at full speed. Conformational changes required for release from the MT is driven by ATP hydrolysis in AAA118,23. Eventual release of products at AAA3 turns the switch off until the next nucleotide cycle.

Bottom Line: Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude more slowly than AAA1, and the two sites do not coordinate.Nucleotide hydrolysis at AAA3 lifts this 'MT gate' to allow fast motility.These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT-anchoring tasks in cells.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, Berkeley, Berkeley, California, USA.

ABSTRACT
Cytoplasmic dynein is an AAA+ motor responsible for intracellular cargo transport and force generation along microtubules (MTs). Unlike kinesin and myosin, dynein contains multiple ATPase subunits, with AAA1 serving as the primary catalytic site. ATPase activity at AAA3 is also essential for robust motility, but its role in dynein's mechanochemical cycle remains unclear. Here, we introduced transient pauses in Saccharomyces cerevisiae dynein motility by using a slowly hydrolyzing ATP analog. Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude more slowly than AAA1, and the two sites do not coordinate. ATPase mutations to AAA3 abolish the ability of dynein to modulate MT release. Nucleotide hydrolysis at AAA3 lifts this 'MT gate' to allow fast motility. These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT-anchoring tasks in cells.

Show MeSH
Related in: MedlinePlus