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.

Eriko Kage-Nakadai,Kazuko H Nomura,Anne C Hart,Saya Nakagomi,Yasuhiro M Umemura,Shushi Nagamori,Kohei Hagiwara,Kazuya Nomura,Yoshikatsu Kanai,Hidekazu Tanaka,Shohei Mitani,Daisuke Murata,Ryuichi Ohgaki

doi:10.1371/journal.pone.0043050

Abstract

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.

Highlights

Proper localization and maintenance of transmembrane proteins in the plasma membrane are essential for appropriate cellular function
Identification of NRFL-1 as an Acid Transporter 6 (AAT-6) Interactor The C-terminus of AAT-6 protein ends in threonine, arginine, and methionine (-T-R-M), which makes up a class I PDZ binding motif (-S/T-X-F-COOH, where X denotes any residue and F denotes a hydrophobic residue) [23]
In search for proteins that associate with AAT-6 via PDZ interactions, C. elegans proteins were first screened for PDZ domain by a molecular architecture research database, SMART [24]

Summary

Introduction

Proper localization and maintenance of transmembrane proteins in the plasma membrane are essential for appropriate cellular function. The highly homologous primary structures of their PDZ domains allow them to share some of the target proteins such as CFTR (cystic fibrosis transmembrane conductance regulator) [5,6,7], NHE3 (sodium-hydrogen exchanger 3) [8,9,10] and organic solute transporters [11,12,13]. This redundancy in expression profile and interaction, yielding potential functional compensations between the family members, has made it difficult to separate the in vivo functions of individual NHERF family proteins

Methods

Results

Conclusion