Quantitative analysis of Pac1/LIS1-mediated dynein targeting: Implications for regulation of dynein activity in budding yeast.

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.

A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment.

During mitosis in budding yeast, cortically anchored dynein exerts pulling forces on cytoplasmic microtubules, moving the mitotic spindle into the mother-bud neck. Anchoring of dynein requires the cortical patch protein Num1, which is hypothesized to interact with PI(4,5)P(2) via its C-terminal pleckstrin homology (PH) domain. Here we show that the PH domain and PI(4,5)P(2) are required for the cortical localization of Num1, but are not sufficient to mediate the cortical assembly of Num1 patches. A GFP fusion to the PH domain localizes to the cortex in foci containing approximately 2 molecules, whereas patches of full-length Num1-GFP contain approximately 14 molecules. A membrane targeting sequence containing the CAAX motif from the yeast Ras2 protein can compensate for the PH domain to target Num1 to the plasma membrane as discrete patches. The CAAX-targeted Num1 exhibits overlapping but largely distinct localization from wild-type Num1. However, it is fully functional in the dynein pathway. More importantly, cortical PI(4,5)P(2) is dispensable for the localization and function of the CAAX-targeted Num1. Together, these results demonstrate that cortical assembly of Num1 into functional dynein-anchoring patches is independent of its interaction with PI(4,5)P(2).

Motor- and tail-dependent targeting of dynein to microtubule plus ends and the cell cortex.

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.