Jcb_201401016 1..16

Spectrin and ankyrin are associated with the cytoplasmic surface of the plasma membrane where they cooperate in micrometer-scale organization of membrane-spanning proteins within specialized membrane domains in many vertebrate tissues (Bennett and Healy, 2009; Bennett and Lorenzo, 2013). A common organizational principle shared by spectrin/ankykrin-based domains, as presented in reviews and cartoons, is straightforward: membrane-spanning proteins, including cell adhesion proteins capable of responding to extracellular cues as well as membrane transporters, are “anchored” within the fluid bilayer by association with ankyrin, which in turn is coupled to an extended spectrin–actin network that is tightly associated with the plasma membrane (Bennett and Healy, 2009; Bennett and Lorenzo, 2013). However, these proteinbased models, although descriptive of steady-state protein composition, do not provide an explanation for how membrane domains are actually assembled and precisely localized in cells. Membrane lipids and lipid modifications play important roles in determining plasma membrane identity. For example, phosphoinositide lipids are increasingly recognized as critical determinants of plasma membrane organization in addition to their roles in intracellular organelles (Martin-Belmonte et al., 2007; Shewan et al., 2011; Hammond et al., 2012; Johnson et al., 2012; Nakatsu et al., 2012). In addition, the aspartatehistidine-histidine-cysteine (DHHC) family of 23 protein palmitoyltransferases, first discovered in yeast, now are known to function in vertebrates in targeting and trafficking of membrane proteins (Bartels et al., 1999; Roth et al., 2002; Fukata et al., 2004; Fukata and Fukata, 2010; Greaves and Chamberlain, 2011). -Spectrins contain a pleckstrin homology (PH) domain with preference for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2; Travé et al., 1995; Das et al., 2008). Moreover, ankyrin-G is S-palmitoylated at a conserved cysteine (C70; He et al., 2012). This palmitoylated Ankyrin-G and II-spectrin colocalize at sites of cell–cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and II-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that IIspectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and II-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G–II-spectrin interaction or II-spectrin– phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with II-spectrin, and II-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells. Ankyrin-G palmitoylation and II-spectrin binding to phosphoinositide lipids drive lateral membrane assembly