Intracellular trafficking and ubiquitin-dependent catabolism of AF1q by the proteasome. (A) Comparison of the amino acid sequence of AF1q orthologs. The AF1q AHD1 and AHD2, NES, and CBR are indicated. ClustalW alignments were performed using the Accelrys Gene v2.5 software. Conserved amino acids are shown in red; consensus residues are in green. Synonymous amino-acid substitutions are indicated in lower-case letters; conserved basic residues of the CBR are underlined. (B) The centrosomal localization of AF1q follows its nuclear export. SupT1 cells were treated with 2 ng/mL of LMB for 2 hours before labeling with AF1q (red) and γ-tubulin (green) antibodies. (C-D) Subcellular localization of wild-type AF1q and of its Δ-NES L30/32A (A2M) or L27/29/30/32A (A4M) mutant derivatives. HeLa cells were transfected with vectors encoding HA-tagged AF1q (C) or the mutant proteins (D). Cells were treated or not with 2 ng/mL of LMB for the last 2 hours and labeled with AF1q (red) and LAP2 (green) antibodies. LAP-2 is a marker of the inner nuclear envelope. Confocal microcopy analysis was performed 48 hours after transfection. (E-F) Constitutive nuclear relocation of AF1q slows down its catabolism. HEK-AF1q (right panel) and HEK-A2M cell lines (left panel) were treated for the indicated periods with CHX and analyzed by immunoblot (E) followed by protein quantification by densitometry (F). Results are normalized relative to protein levels detected in control cells cultured under the same conditions but without CHX. (G) AF1q degradation depends on its prior ubiquitylation. HEK-AF1q cells were transfected or not with a His-tagged ubiquitin (6xHis-Ubiquitin) vector, and treated or not with 10μM Lactacystin (Lacta) for 12 hours before immunoblotting with AF1q antibodies. Left panel shows total cell extracts; right panel, His-purified proteins.