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.
Highlights
The ADP-ribosylation factors (ARFs) are members of a small GTPase family of the Ras superfamily that are involved in membrane transport regulation, organelle integrity maintenance, membrane lipid modification, cytoskeletal dynamics, and signal transduction [1]
To gain a better understanding of the essential role that Arf-like protein 1 (Arl1) plays in vivo, we isolated Arl1 protein complexes from HeLa cells by differential affinity chromatography using immobilized GST-Arl1 (Arl1QL, the putative active form of Arl1; Arl1TN, the dominant-negative form of Arl1)
The protein components were identified by tandem mass spectrometry, as shown in S1 Fig. We identified 38 unique proteins that interacted with GTP-bound Arl1
Summary
The ADP-ribosylation factors (ARFs) are members of a small GTPase family of the Ras superfamily that are involved in membrane transport regulation, organelle integrity maintenance, membrane lipid modification, cytoskeletal dynamics, and signal transduction [1]. Arf-like proteins (Arls) share 40–60% sequence identity [2], and more than twenty Arls have been identified in humans [3]. Arl controls intracellular ion levels [4,5] and regulates endosomal transport and cell proliferation [6]. Arl is involved in the transport of at least one GPI-anchored protein from the late Golgi to the plasma membrane [7]. Arl localizes to the trans-Golgi network (TGN) and functions in vesicular trafficking.
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