AGAP2 regulates retrograde transport between early endosomes and the TGN

Abstract

SummaryThe retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are ~20 kDa GTP-binding proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early endosomes and the TGN.

Retromer: A Master Conductor of Endosome Sorting

“…Similarly, other, less well described sorting factors such as sorting nexin-17 (SNX17) (NPxY, NxxY, and NxxF sequences), EHD proteins (NPF motif ), and sorting nexin-27 (SNX27) (PDZ ligands) also contribute to plasma membrane recycling (Burden et al 2004;Joubert et al 2004;Braun et al 2005;van Kerkhof et al 2005). Endosome-to-TGN retrograde sorting is similarly complex, with contributions by clathrin in conjunction with AP1, PACS1, and epsinR (Meyer et al 2000;Crump et al 2001;Saint-Pol et al 2004;Johannes and Popoff 2008;Shiba et al 2010;Johannes and Wunder 2011). In addition, export of the mannose-6-phosphate receptor from the late endosome appears to be regulated by the TIP47/Rab9 assembly (Lombardi et al 1993;Díaz and Pfeffer 1998;Carroll et al 2001; also see Bulankina et al 2009).…”

GTP-binding Protein-like Domain of AGAP1 Is Protein Binding Site That Allosterically Regulates ArfGAP Protein Catalytic Activity

“…AGAP1-HA was subcloned into the NotI and EcoRI sites of pCDNA3.1(Ϫ) (Invitrogen). HA-AGAP2 and AGAP2-His 6 have been described (11). Briefly, HA-AGAP2 was constructed from the full-length MGC image clone (IMAGE id 5218187; GenBank accession number NM014770).…”

“…AGAP1 binds through its PH domain to the clathrin adaptor protein AP-3 (6), which is important for the biogenesis of lysosomes and lysosomal-related organelles (7,8). AGAP2, which has been implicated in glioblastoma (9), binds to the clathrin adaptor protein AP-1 and affects transferrin recycling (10) and retrograde transport from the early endosome to the trans-Golgi network (11). Both AGAP1 and AGAP2, as recombinant proteins, associate with endocytic structures containing AP-1 and AP-3.…”

ArfGAPs; Depletion or Overexpression?

“…The screen by 6 siRNAs of ArfGAPs determined AGAP2 as a positive regulator for transport of Shiga Toxin B subunit (STxB) from the early endosomes to the Golgi apparatus [41]. The screen by 25 siRNAs of ArfGAPs determined ArfGAP3 to be required for Mannose 6-phosphate receptor (MPR) transport from the early endosomes to the late endosomes [42].…”

“…One may still think that the depletion of ArfGAPs should increase Arf•GTP levels in cells, therefore intracellular traffic should be accelerated. Actually, I have often seen that the depletion of several different ArfGAPs resulted in the increase of the transport pathway of interest ( [41] data not shown). These phenomena could be explained by the increase of Arf•GTP levels generally in cells.…”