JCB_201702022 3061..3072

As the number and diversity of newly discovered interorganelle contacts increase, it is clear that these contacts impact many aspects of cell biology (Helle et al., 2013; Prinz, 2014; EisenbergBord et al., 2016; Gatta and Levine, 2017; Kraft and Lackner, 2017). Many proteins and protein complexes that function to directly tether organelles have been identified (Prinz, 2014; Eisenberg-Bord et al., 2016; Gatta and Levine, 2017). Some serve solely to physically bridge organelles, whereas others actively participate in interorganelle communication and function. Importantly, these proteins establish, maintain, and alter contacts in response to different physiological contexts. Thus, gaining an understanding of the tethering mechanism, activity, and regulation of these proteins provides insight into how interorganelle contacts and their critical functions are regulated. In budding yeast, the mitochondria–ER–cortex anchor (MECA) functions to tether mitochondria to the cortical ER and plasma membrane, bringing the three cellular membranes in close proximity (Cerveny et al., 2007; Hammermeister et al., 2010; Klecker et al., 2013; Lackner et al., 2013; Ping et al., 2016). MECA interacts directly with mitochondria and the plasma membrane via two distinct lipid-binding domains within its core protein component, Num1. A membrane-binding region within the N-terminal coiled-coil (CC) region of Num1 interacts directly with the mitochondrial outer membrane (MOM; Tang et al., 2012; Lackner et al., 2013; Ping et al., 2016), and a C-terminal pleckstrin homology domain interacts with PI4,5P2 in the plasma membrane (Yu et al., 2004; Tang et al., 2009). Num1CC also mediates Num1 self-interactions and is required for Num1 to assemble into clusters at the cell cortex (Tang et al., 2012; Lackner et al., 2013). Mutations that interfere with the ability of Num1 to assemble into clusters decrease the tethering capacity of MECA, suggesting that assembly of Num1 increases the avidity of Num1 for its target membranes (Lackner et al., 2013; Ping et al., 2016). Therefore, the regulation of Num1 assembly likely serves as a mechanism to regulate the tethering function of MECA. In addition to functioning as a cortical anchor for mitochondria, Num1 plays a well-characterized role as a cortical anchor for dynein (Heil-Chapdelaine et al., 2000; Farkasovsky and Küntzel, 2001; Markus and Lee, 2011). At the cell cortex, dynein captures and walks along astral microtubules, generating a pulling force to position the mitotic spindle across the mother–bud neck during mitosis (Eshel et al., 1993; Li et al., 1993; Adames and Cooper, 2000). Mutants that interfere with Num1 assembly exhibit compromised dynein activity (Tang et al., 2012). Together, these observations suggest that Num1 assembly strengthens its interaction with all binding partners, including dynein and phospholipid membranes. Whereas Num1 assembly facilitates its function as an anchor for mitochondria and dynein, factors influencing Num1 assembly are poorly understood. Here, we report that the assembly of Num1 requires an interaction with mitochondria. Additionally, we find that mitochondria-assembled Num1 clusters function as cortical attachment sites for dynein and that disrupting Interorganelle contacts facilitate communication between organelles and impact fundamental cellular functions. In this study, we examine the assembly of the MECA (mitochondria–endoplasmic reticulum [ER]–cortex anchor), which tethers mitochondria to the ER and plasma membrane. We find that the assembly of Num1, the core component of MECA, requires mitochondria. Once assembled, Num1 clusters persistently anchor mitochondria to the cell cortex. Num1 clusters also function to anchor dynein to the plasma membrane, where dynein captures and walks along astral microtubules to help orient the mitotic spindle. We find that dynein is anchored by Num1 clusters that have been assembled by mitochondria. When mitochondrial inheritance is inhibited, Num1 clusters are not assembled in the bud, and defects in dynein-mediated spindle positioning are observed. The mitochondria-dependent assembly of a dual-function cortical anchor provides a mechanism to integrate the positioning and inheritance of the two essential organelles and expands the function of organelle contact sites. Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein