NRFL-1, the C. elegans NHERF orthologue, interacts with amino acid transporter 6 (AAT-6) for age-dependent maintenance of AAT-6 on the membrane.
The NHERF (Na(+)/H(+) exchanger regulatory factor) family has been proposed to play a key role in regulating transmembrane protein localization and retention at the plasma membrane. Due to the high homology between the family members, potential functional compensations have been a concern in sorting out the function of individual NHERF numbers. Here, we studied C. elegans NRFL-1 (C01F6.6) (nherf-like protein 1), the sole C. elegans orthologue of the NHERF family, which makes worm a model with low genetic redundancy of NHERF homologues. Integrating bioinformatic knowledge of C. elegans proteins into yeast two-hybrid scheme, we identified NRFL-1 as an interactor of AAT-6, a member of the C. elegans AAT (amino acid transporter) family. A combination of GST pull-down assay, localization study, and co-immunoprecipitation confirmed the binding and characterized the PDZ interaction. AAT-6 localizes to the luminal membrane even in the absence of NRFL-1 when the worm is up to four-day old. A fluorescence recovery after photobleaching (FRAP) analysis suggested that NRFL-1 immobilizes AAT-6 at the luminal membrane. When the nrfl-1 deficient worm is six-day or older, in contrast, the membranous localization of AAT-6 is not observed, whereas AAT-6 tightly localizes to the membrane in worms with NRFL-1. Sorting out the in vivo functions of the C. elegans NHERF protein, we found that NRFL-1, a PDZ-interactor of AAT-6, is responsible for the immobilization and the age-dependent maintenance of AAT-6 on the intestinal luminal membrane.
Division of Bio-system Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan.
Kohei
K
Hagiwara
Shushi
S
Nagamori
Yasuhiro M
YM
Umemura
Ryuichi
R
Ohgaki
Hidekazu
H
Tanaka
Daisuke
D
Murata
Saya
S
Nakagomi
Kazuko H
KH
Nomura
Eriko
E
Kage-Nakadai
Shohei
S
Mitani
Kazuya
K
Nomura
Yoshikatsu
Y
Kanai
D026905:Amino Acid Transport Systems / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D026905:Amino Acid Transport Systems; D029742:Caenorhabditis elegans Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor