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.

BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

“…, 2003; Markus et al. , 2009, 2011) and in some aspects resemble the LIS1-independent and p150-dynactin–dependent pathway described in filamentous fungi such as Aspergillus nidulans and Ustilago maydis (Zhang et al. , 2003; Lenz et al.…”

Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein

“…Dynein reaches the cortex by localizing to MT plus ends, either via kinesin-dependent transport or recruitment from the cytosol (Carvalho et al, 2004; Caudron et al, 2008; Markus et al, 2009). Dynein’s plus-end-localization, kinesin-dependent transport, and later “off-loading” to the cell cortex all require Lis1 (Lee et al, 2003; Li et al, 2005; Markus and Lee, 2011; Markus et al, 2009; 2011; Sheeman et al, 2003). The requirement of Lis1 for plus-end-localization and off-loading to the cortex is compatible with a model where Lis1 promotes a tight MT-binding state in dynein.…”

Transport of fungal RAB11 secretory vesicles involves myosin-5, dynein/dynactin/p25, and kinesin-1 and is independent of kinesin-3

“…Our data, however, show markedly decreased yet detectable accumulation of dynein in the plus ends of MTs of kinA∆ tips, indicating that kinesin-independent mechanisms for dynein’s plus-end accumulation, such as direct recruitment from the cytoplasm, may be operative (Zhang et al. , 2003; Markus et al. , 2011; Cianfrocco et al.…”