title: Arfaptin-1 negatively regulates Arl1-mediated retrograde transport.
abstract: The small GTPase Arf-like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN). In this study, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1b (arfaptin-1b) as an Arl1-interacting protein and characterized a novel function for arfaptin-1 (including the arfaptin-1a and 1b isoforms) in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A), consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.
authors: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors: Lien-Hung
authors: LH
authors: Huang
authors: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors: Wei-Chung
authors: WC
authors: Lee
authors: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors: Shu-Ting
authors: ST
authors: You
authors: Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
authors: Chia-Chen
authors: CC
authors: Cheng
authors: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan.
authors: Chia-Jung
authors: CJ
authors: Yu
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000704:Analysis of Variance; D001324:Autoantigens / Q000378:metabolism; D002846:Chromatography, Affinity; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D034622:RNA Interference; D022641:Shiga Toxin 2; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: C105088:ARFIP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D001324:Autoantigens; C098772:GOLGA4 protein, human; D000077614:Golgi Matrix Proteins; C450721:Golgi complex autoantigen, 97-kDa; D008565:Membrane Proteins; D022641:Shiga Toxin 2; D033921:Vesicular Transport Proteins; C450841:shiga toxin 2 B subunit; C097313:ADP-ribosylation factor related proteins; D020727:ADP-Ribosylation Factors