2544 Research Article Introduction Multiple sorting signals that reside at the N-terminus of the cytoplasmic domain of the chicken kidney AE1-4 anion exchanger direct the basolateral sorting and Golgi recycling of this membrane transporter in polarized MDCK cells (Adair- Kirk et al., 1999; Adair-Kirk et al., 2003). Recent studies have shown that one of these signals, which is located between amino acids 38 and 63 of AE1-4, is sufficient to direct basolateral sorting when fused to a cytoplasmic tailless murine IgG F c RII B2 receptor. However, this chimeric receptor, F c 38- 63, primarily accumulates in the trans-Golgi-network (TGN) at steady state. This localization profile is dependent upon recycling of F c 38-63 from the basolateral membrane to the TGN. Mutagenesis studies have shown that the TGN recycling of F c 38-63 is dependent upon a tyrosine residue located in the tetrapeptide Y 47 VEL, which matches the sequence of a YXX motif, where X is any amino acid and  is a hydrophobic residue. This motif directs the clathrin-dependent endocytosis of membrane-spanning proteins (Collawn et al., 1990) through its association with the AP2 clathrin adaptor complex (Aguilar et al., 2001; Boehm and Bonifacino, 2001). Other TGN resident proteins, such as furin (Schafer et al., 1995) and TGN38 (Bos et al., 1993; Wong and Hong, 1993), also undergo recycling from the cell surface to the TGN and their internalization from the cell surface requires the tyrosine- based endocytic signals YKGL and YQRL, respectively. The endocytic signals of TGN38 and furin each bind the mu- subunit of the clathrin AP2 adaptor complex (Owen and Evans, 1998; Teuchert et al., 1999). Furthermore, the endocytosis of TGN38 and furin is dependent upon the tyrosine and the hydrophobic residue in their YXX motifs (Humphrey et al., 1993; Owen and Evans, 1998). Some lipids (Sharma et al., 2003; Singh et al., 2003) and proteins (Le et al., 2002) that recycle from the plasma membrane to early compartments of the secretory pathway are internalized by non-clathrin-dependent endocytic pathways. Cargo internalized by clathrin-independent pathways often traverse caveolin 1-positive endosomes prior to their delivery to early secretory pathway compartments (Pelkmans et al., 2001; Nichols, 2002; Sharma et al., 2004). However, the endocytosis of these clathrin-independent cargos from the plasma membrane can occur through vesicular carriers coated with caveolin 1 or through uncoated vesicular carriers (Kirkham et al., 2005; Damm et al., 2005; Cheng et al., 2006). In this report we have further characterized the recycling pathway of F c 38-63 in MDCK cells. Studies using small interfering RNA (siRNA) and dominant-negative mutants show that even though the endocytosis of F c 38-63 is dependent upon a YXX motif, it is internalized from the plasma membrane through a caveolin-dependent pathway. Moreover, co-precipitation studies indicate that caveolin 1 is associated with F c 38-63 and the AE1-4 anion exchanger in this kidney epithelial cell type. Mutations in the cytoplasmic Y 47 VEL tetrapeptide of these proteins disrupt their interaction with caveolin 1. The fact that these mutations also inhibit F c 38-63 and AE1-4 trafficking suggests a novel role for YXX motifs in targeting membrane-spanning proteins to caveolin- dependent sorting pathways. Previous studies showed that the sequence between amino acids 38 and 63 of the chicken AE1-4 anion exchanger is sufficient to direct basolateral sorting and recycling to the Golgi when fused to a cytoplasmic tailless F c RII B2 receptor. Further characterization of the recycling pathway has indicated that the chimera F c 38-63 colocalizes with caveolin 1 in the basolateral membrane of MDCK cells, and in early endosomes following its internalization from the cell surface. Studies using small interfering RNA (siRNA) and dominant-negative mutants revealed that F c 38-63 endocytosis is primarily caveolin-dependent and clathrin- independent. The endocytosis of the chimera is also dependent upon cholesterol and dynamin. Co-precipitation studies indicated that caveolin 1 associates with F c 38-63. Mutation of the tyrosine or leucine residues in the cytoplasmic sequence Y 47 VEL of F c 38-63 disrupts this interaction and inhibits the endocytosis of the chimera. Additional analyses revealed that AE1-4 also associates with caveolin 1. Mutation of the leucine in the Y 47 VEL sequence of AE1-4 disrupts this interaction, and blocks the recycling of this transporter from the basolateral membrane to the Golgi. The Y 47 VEL tetrapeptide matches the sequence of a YXX motif, and our results indicate a novel role for this motif in directing caveolin-dependent sorting. Key words: Caveolin, Sorting signal, Endosomes, AE1 anion exchanger Summary A novel role for a YXX motif in directing the caveolin- dependent sorting of membrane-spanning proteins Frank C. Dorsey*, Thangavel Muthusamy, Michael A. Whitt and John V. Cox ‡ Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA *Present address: Department of Biochemistry, St Jude Children’s Research Hospital, Memphis, TN, USA ‡ Author for correspondence (e-mail: jcox@utmem.edu) Accepted 21 May 2007 Journal of Cell Science 120, 2544-2554 Published by The Company of Biologists 2007 doi:10.1242/jcs.002493 Journal of Cell Science