Dynein's C-Terminal Domain Plays a Novel Role in Regulating Force Generation

Cytoplasmic dynein is a homodimeric, minus-end-directed microtubule motor protein involved in a wide range of both low and high force requiring functions in metazoans. In contrast, yeast cytoplasmic dynein is involved in a single, nonessential function, nuclear positioning. Interestingly, whereas mammalian isoforms exhibit a stall force of 1-2 piconewton (pN), S. cerevisiae dynein stalls at 5-7 pN. In addition, in the absence of load, mammalian dyneins move faster than yeast dynein (800-1,100 nm/s vs. 100 nm/sec respectively), and, under opposing force, maintain attachment to microtubules much less tenaciously (milliseconds to seconds vs. tens of seconds, respectively). The basis for these functional differences is unknown. However, the major structural difference between mammalian and yeast dyneins is an ∼30 kDa C-terminal extension (CT) present in higher eukaryotic dyneins, but missing in yeast. To test whether the CT accounts for the differences in function, we produced recombinant rat dynein motor domains (MD) with (WT-MD) and without (ΔCT-MD) the CT region, using a baculovirus expression system. Amino-terminal glutathione S-transferase (GST) tags induced formation of a dimeric, "two-headed" motor. We found that, like yeast dynein, the ΔCT-MD ATPase lacks the signature vanadate inhibition characteristic of higher eukaryotic dyneins, and exhibited a strikingly higher Km(ATP). To characterize motor function, we performed single-molecule optical trapping studies. Single WT-MD stalls at ∼1 pN and detaches from microtubules after brief stalls. In sharp contrast, but similar to yeast dynein, ΔCT-MD stalls at 6 ± 1 pN (mean ± SD), with stall durations up to tens of seconds. These results identify the CT as an important new regulatory element for controlling cytoplasmic dynein mechanochemistry, perhaps gating ATP access. The CT thus appears to represent the structural basis for differences in mechanochemical function between yeast and higher eukaryotic dyneins.