The Leber Congenital Amaurosis Protein AIPL1 Modulates the Nuclear Translocation of NUB1 and Suppresses Inclusion Formation by NUB1 Fragments

Jacqueline Van Der Spuy,Michael E Cheetham

doi:10.1074/jbc.m407871200

Abstract

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) cause the blinding disease Leber congenital amaurosis (LCA). The similarity of AIPL1 to AIP has led to suggestions that AIPL1 could function in a similar manner to AIP in facilitating protein translocation and as a component of chaperone complexes. AIPL1 interacts with the cell cycle regulator NEDD8 ultimate buster protein 1 (NUB1). As AIPL1 is predominantly cytoplasmic and NUB1 is predominantly nuclear, we tested the hypothesis that AIPL1 could modulate the nuclear translocation of NUB1. Co-transfection of AIPL1 with GFP-NUB1 resulted in a shift of GFP-NUB1 subcellular distribution toward the cytoplasm. Interestingly, AIPL1 was able to act in a chaperone-like fashion to efficiently suppress inclusion formation by NUB1 fragments. Co-transfection of AIPL1 with GFP-NUB1-N and GFP-NUB1-C resulted in an AIPL1-dependent suppression of GFP-NUB1-N perinuclear inclusions and GFP-NUB1-C intranuclear inclusions leading to the redistribution of these fragments in the cytoplasm. This chaperone-like function of AIPL1 was specific for NUB1, since AIPL1 was unable to suppress the inclusion formation by unrelated aggregation-prone proteins and AIP had no effect on NUB1 localization or inclusion formation. We examined the effect of a range of pathogenic and engineered mutations on the ability of AIPL1 to modulate NUB1 localization or inclusion formation. With the exception of W278X, which formed non-functional SDS-insoluble inclusions, all of the pathogenic mutations studied were soluble and could modulate NUB1 with varying efficiency compared with the wild-type protein. The effect of AIPL1 on NUB1 required the C-terminal region of AIPL1, as engineered C-terminal truncation mutations had no effect on NUB1. These data show that AIPL1 can modulate protein translocation and act in a chaperone-like manner and suggest that AIPL1 is an important modulator of NUB1 cellular function.

Highlights

aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) shares 49% identity and 69% similarity with human aryl hydrocarbon receptor-interacting protein (AIP), named XAP2 or ARA9 [1, 4]
Co-transfection of AIPL1 with GFPNUB1-N and GFP-NEDD8 ultimate buster protein 1 (NUB1)-C resulted in an AIPL1-dependent suppression of GFP-NUB1-N perinuclear inclusions and GFP-NUB1-C intranuclear inclusions leading to the redistribution of these fragments in the cytoplasm
Increasing amounts of the myc-AIPL1 plasmid led to a shift in the subcellular distribution of GFP-NUB1 toward the cytoplasm

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Mutagenesis—Full-length AIPL1 was amplified from human retinal cDNA using AIPL1-specific primers. ImageJ (rsb.info.nih.gov/ij/) was used to assess the OD of GFP-NUB1 fluorescence in the nucleus and whole cell of ϳ100 transfected cells. Cell Extracts and Western Blotting—24 h after transfection, the SK-N-SH cells were washed three times with Hank’s balanced salts without calcium and magnesium (HBSS) (Invitrogen Life Technologies) and trypsinized with trypsin-EDTA solution (1ϫ) (Sigma). The cells were lysed on ice for 3 min with gentle agitation in 10 volumes of phosphate-buffered saline containing 0.5% Nonidet P-40 and 10% mammalian protease inhibitor mixture (Sigma). Filter Trap Assay—Cell extracts were prepared as described above, but were lysed in 10 volumes of SDS-lysis buffer (10 mM Tris.HCl, pH 8.0; 150 mM NaCl; 2% SDS; 10% mammalian protease inhibitor mixture (Sigma)) and sonicated for 10 s. The membrane was removed and Western blotted as described above

RESULTS

DISCUSSION

Control