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

Abstract

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.