Autoantigen Golgin-97, an Effector of Arl1 GTPase, Participates in Traffic from the Endosome to theTrans-Golgi Network

Abstract

The precise cellular function of Arl1 and its effectors, the GRIP domain Golgins, is not resolved, despite our recent understanding that Arl1 regulates the membrane recruitment of these Golgins. In this report, we describe our functional study of Golgin-97. Using a Shiga toxin B fragment (STxB)-based in vitro transport assay, we demonstrated that Golgin-97 plays a role in transport from the endosome to the trans-Golgi network (TGN). The recombinant GRIP domain of Golgin-97 as well as antibodies against Golgin-97 inhibited the transport of STxB in vitro. Membrane-associated Golgin-97, but not its cytosolic pool, was required in the in vitro transport assay. The kinetic characterization of inhibition by anti-Golgin-97 antibody in comparison with anti-Syntaxin 16 antibody established that Golgin-97 acts before Syntaxin 16 in endosome-to-TGN transport. Knock down of Golgin-97 or Arl1 by their respective small interference RNAs (siRNAs) also significantly inhibited the transport of STxB to the Golgi in vivo. In siRNA-treated cells with reduced levels of Arl1, internalized STxB was instead distributed peripherally. Microinjection of Golgin-97 antibody led to the fragmentation of Golgi apparatus and the arrested transport to the Golgi of internalized Cholera toxin B fragment. We suggest that Golgin-97 may function as a tethering molecule in endosome-to-TGN retrograde traffic. INTRODUCTIONIn eukaryotic cells, different types of cargo (solutes, lipids, and membrane proteins, including receptors and their ligands) internalized from the plasma membrane follow several routes upon reaching the early/sorting endosome. They could recycle back to the plasma membrane directly via early/sorting endosome or indirectly via the recycling endosome, or travel further to the lysosome via the late endosome. Recently, there was evidence suggesting the existence of two retrograde transport pathways from endosomes to the trans-Golgi network (TGN) (Ghosh et al., 1998;Mallard et al., 1998;Mallet and Maxfield, 1999). One pathway, used by furin (Mallet and Maxfield, 1999) and mannose-6-phosphate receptor (M6PR) (Diaz and Pfeffer, 1998;Sincock et al., 2003), is from the late endosome to the TGN. The other one, used by TGN38 (Ghosh et al., 1998), Shiga Toxin B fragment (STxB) (Mallard et al., 1998) and likely GLUT4 (Shewan et al., 2003), proceeds via the early endosome (EE) and/or the recycling endosome (RE), without passing through late endosomes. In addition, the large cation-independent M6PR and the small cation-dependent MPR46 are recently shown to also use this EE/RE-TGN pathway, in addition to its well characterized late endosome-TGN route (Medigeshi and Schu, 2003;Lin et al., 2004). These endosome-TGN pathways could be used by other proteins such as P-selectin (Straley and Green, 2000), membrane-type matrix metalloproteinases (Kang et al., 2002;Zucker et al., 2002;Remacle et al., 2003; Wang et al., 2004a,b), copper transporters (Petris and Mercer, 1999;Petris et al., 2002), and VAMP4, a TGN SNARE (unpublished observations). In addition, ...

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