GRIF1 binds Hrs and is a new regulator of endosomal trafficking

Abstract

depletion of GRIF1 inhibit the degradation of internalized epidermal growth factor receptors and block the trafficking of the receptors from early endosomes to the lysosomal pathway. Our results indicate, for the first time, a functional role for GRIF1 in the regulation of endosomal trafficking. Interestingly, overexpression of full-length GRIF1, but not the Hrs-or kinesin-interacting GRIF1 deletion mutants, causes a perinuclear clustering of early endosomes. Our findings suggest that GRIF1 may also participate in microtubule-based transport of early endosomes by acting as an adaptor linking Hrs-containing endosomes to kinesin.

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

“…DNA fragments encoding torsinA WT, WT⌬40, ⌬E, ⌬323-328, K108A, and E171Q were subcloned into mammalian vectors expressing C-terminal HA, Myc, or FLAG tags. A rabbit polyclonal anti-printor antibody was raised against a synthetic peptide encoding amino acids 1-18 of human printor and affinity purified using the immunogen peptide coupled to a Pierce column as we described previously (35)(36)(37). Other anti-bodies used in this study are as follows: anti-torsinA (16); anti-EEA1 and anti-TIM23 (BD transduction); anti-LAMP2 (H4B4; DSHB, University of Iowa); anti-FLAG (M2; Sigma); anti-calnexin and anti-KDEL (Stressgen); anti-␤-actin (Sigma); mouse monoclonal anti-HA (12CA5); and anti-Myc (9E10) (35).…”

“…Yeast Two-hybrid Screen-The bait plasmids were constructed by subcloning the full-length human torsinA (WT) or N-terminal-truncated torsinA (WT⌬40) into the vector pPC97 (35,37,38). For yeast two-hybrid screens, the yeast strain CG-1945 (Clontech) was sequentially transformed with the WT or WT⌬40 bait plasmid and a rat hippocampal/cortical two-hybrid cDNA library (35,36,39).…”

“…For yeast two-hybrid screens, the yeast strain CG-1945 (Clontech) was sequentially transformed with the WT or WT⌬40 bait plasmid and a rat hippocampal/cortical two-hybrid cDNA library (35,36,39). Positive clones were selected on 3-aminotriazole-containing medium lacking leucine, tryptophan, and histidine, and confirmed by filter assay for ␤-galactosidase activity.…”

“…Cell lysates were prepared from transfected cells and immunoprecipitations were carried out as described previously (16,35,(41)(42)(43) using the indicated antibodies, followed by the recovery of immunocomplexes with protein G-Sepharose beads (Upstate). For co-immunoprecipitations of endogenous proteins, the Seize Primary Mammalian Immunoprecipitation Kit (Pierce) was used according to the manufacturer's instructions.…”

“…DNA fragments encoding torsinA WT, WT⌬40, ⌬E, ⌬323-328, K108A, and E171Q were subcloned into mammalian vectors expressing C-terminal HA, Myc, or FLAG tags. A rabbit polyclonal anti-printor antibody was raised against a synthetic peptide encoding amino acids 1-18 of human printor and affinity purified using the immunogen peptide coupled to a Pierce column as we described previously (35)(36)(37) …”