Other components involved with basolateral transport are the actin-regulatory GTPase cdc42, the exocyst complex, and the GTPase Rab8. adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1BCdependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells. ROR agonist-1 Introduction In polarized epithelial cells, the plasma membrane is divided into functionally and morphologically distinct apical and basolateral domains that have different protein and lipid compositions and are separated by tight junctions. The polarized distribution of proteins is achieved by the sorting of newly synthesized proteins at the TGN and/or the recycling endosome into separate carriers destined for the apical or basolateral domain (Mostov et al., 2000). In addition, recycling proteins are sorted in the endosomal compartment after endocytosis from the plasma membrane. Sorting of basolateral transmembrane proteins is guided by peptide motifs present in their cytoplasmic tails, such as dileucine motifs or tyrosine-containing sequences, including Yxx and other less well-characterized signals (Muth and Caplan, 2003; Rodriguez-Boulan et al., 2005). Some, if not all, of these motifs bind to the clathrin adaptor protein complexes (AP)-1 and/or -4, which mediate incorporation of the cargo protein into basolateral carriers. Sorting to the apical domain is less well defined and may involve several pathways; depending on the specific membrane protein, it may require glycosylation of its extracellular domain, lipid raft association, or the presence of cytoplasmic peptide sequences (Muth and Caplan, 2003; Rodriguez-Boulan et al., 2005). Polarized epithelial cells express a specific variant of AP-1, called AP-1B, which is important for basolateral targeting of several transmembrane proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) and the low-density lipoprotein receptor (LDLR; Folsch et al., 1999; ROR agonist-1 Ohno et al., 1999; Gan et al., 2002; Sugimoto et al., 2002). Both of these proteins contain tyrosine-dependent sorting motifs (Matter et al., 1992, 1994; Thomas et al., 1993). In contrast, sorting of the Fc receptor isoform BII (FcRIIB), which contains a dileucine motif, appears to be AP-1B independent (Hunziker and Fumey, 1994; Matter et al., 1994; Roush et al., 1998; Folsch et al., 1999). Other components involved in basolateral transport are the actin-regulatory GTPase cdc42, the exocyst complex, and the GTPase Rab8. Functional defects caused by overexpression of cdc42 mutants highlight the importance of the actin cytoskeleton for transport of proteins from the TGN to the basolateral domain (Kroschewski et al., 1999; Musch et al., 2001). The exocyst is a protein complex of eight subunits that mediates the tethering of secretory vesicles to docking ROR agonist-1 sites on the plasma membrane. In MDCK cells, this exocyst complex is required for delivery of membrane proteins to the basolateral domain, but not the apical domain (Grindstaff et al., 1998). In yeast, the function of the exocyst complex is regulated by the Rab GTPase Sec4 (Guo et ROR agonist-1 al., 1999). The mammalian homologue of Sec4 is Rab8, which is a key regulator of exocytic membrane traffic from the Golgi complex to the plasma membrane (Huber et al., 1993; Moritz et al., 2001). In polarized MDCK cells, Rab8 specifically regulates AP-1BCdependent transport to the basolateral domain (Ang et al., 2003). In these cells, Rab8 and AP-1B can be found in recycling endosomes, which is an important sorting station for certain basolateral proteins that are en route to the plasma membrane Keratin 18 (phospho-Ser33) antibody (Ang et al., 2004). In nonpolarized cells, Rab8 has been shown to bind to optineurin, a conserved 67-kD protein containing multiple.