Summary Background Cytoplasmic dynein mediates spindle positioning in budding yeast by powering sliding of microtubules along the cell cortex. Although previous studies have demonstrated cortical and plus-end targeting of dynein heavy chain (Dyn1/HC), the regulation of its recruitment to these sites remains elusive. Results Here we show that separate domains of Dyn1/HC confer differential localization to the dynein complex. The N-terminal tail domain targets Dyn1/HC to cortical Num1 receptor sites, whereas the C-terminal motor domain targets Dyn1/HC to microtubule plus-ends in a Bik1/CLIP-170 and Pac1/LIS1 dependent manner. Surprisingly, the isolated motor domain blocks plus-end targeting of Dyn1/HC, leading to a dominant negative effect on dynein function. Overexpression of Pac1/LIS1, but not Bik1/CLIP-170, rescues the dominant negativity by restoring Dyn1/HC to plus-ends. In contrast, the isolated tail domain has no inhibitory effect on Dyn1/HC targeting and function. However, cortical targeting of the tail construct is more robust than full-length Dyn1/HC, and occurs independently of Bik1/CLIP-170 or Pac1/LIS1. Conclusions Our results suggest that the cortical association domain is normally masked in the full-length dynein molecule. We propose that targeting of dynein to plus-ends unmasks the tail, priming the motor for off-loading to cortical Num1 sites.

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SUMMARY Cytoplasmic dynein mediates spindle orientation from the cell cortex through interactions with astral microtubules, but neither the mechanism governing its cortical targeting, nor the regulation thereof, are well understood. Here we show that yeast dynein offloads from microtubule plus ends to the daughter cell cortex. Mutants with an engineered peptide inserted between the tail domain and the motor head retain wild-type motor activity but exhibit enhanced offloading and cortical targeting. Conversely, shortening the ‘neck’ sequence between the tail and motor domains precludes offloading from the microtubule plus ends. Furthermore, chimeric mutants with mammalian dynein ‘neck’ sequences rescue targeting and function. These findings provide direct support for an active microtubule-mediated delivery process that appears to be regulated by a conserved masking/unmasking mechanism.

LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik1 recruits approximately equal amounts of Pac1 and Dyn1. Overexpression of Pac1 enhanced plus end targeting of Dyn1 and vice versa, while affinity-purification of Dyn1 revealed that it exists in a complex with Pac1 in the absence of Bik1, leading us to conclude that the Pac1-Dyn1 complex preassembles in the cytoplasm prior to loading onto Bik1-decorated plus ends. Strikingly, we found that Pac1-overexpression augments cortical dynein activity through a mechanism distinct from loss of She1, a negative regulator of dynein-dynactin association. While Pac1-overexpression enhances the frequency of cortical targeting for dynein and dynactin, the stoichiometry of these complexes remains relatively unchanged at the plus ends compared to that in wild-type cells (~3 dynein to 1 dynactin). Loss of She1, however, enhances dynein-dynactin association at the plus ends and the cell cortex, resulting in an apparent 1:1 stoichiometry. Our results reveal differential regulation of cortical dynein activity by She1 and Pac1, and provide a potentially new regulatory step in the off-loading model for dynein function.

Upon offloading to Num1 cortical receptor sites in budding yeast, cytoplasmic dynein motility is switched “on” by a mechanism that likely involves Num1-mediated dissociation of the Pac1 inhibitor, a homologue of human LIS1.