Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1.
In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid-base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS(812)LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI(623)LL and QVVA(635)LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI(623)LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI(623)LL with a highly conserved pocket in their γ1-σ1A hemicomplex.
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065 boulan@med.cornell.edu dig2003@med.cornell.edu edelafuente@ucn.cl.
Erwin
E
de la Fuente-Ortega
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065 boulan@med.cornell.edu dig2003@med.cornell.edu edelafuente@ucn.cl.
Diego
D
Gravotta
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065.
Andres
A
Perez Bay
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065.
Ignacio
I
Benedicto
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065.
Jose Maria
JM
Carvajal-Gonzalez
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065.
Guillermo L
GL
Lehmann
Department of Endocrinology, School of Medicine, Pontificia Universidad Católica de Chile, Santiago Centro 8330074, Santiago, Chile Facultad de Ciencia, Universidad San Sebastián, Providencia 7510157, Santiago, Chile.
Carlos F
CF
Lagos
Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, New York, NY 10065 boulan@med.cornell.edu dig2003@med.cornell.edu edelafuente@ucn.cl.
Enrique
E
Rodríguez-Boulan
D033961:Adaptor Protein Complex 1 / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D004285:Dogs; D061985:Madin Darby Canine Kidney Cells; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport
D033961:Adaptor Protein Complex 1; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; C000715428:Clcn2 protein, mouse