ARFRP1 and ARL1, which are both ARF-like small GTPases, are mammalian orthologs of yeast Arl3p and Arl1p, respectively. In yeast, Arl3p targeted to trans-Golgi network (TGN) membranes activates Arl1p, and the activated Arl1p in turn recruits a GRIP domain-containing protein; this complex regulates retrograde transport to the TGN and anterograde transport from the TGN. In the present study, using RNA interference-mediated knockdown of ARFRP1 and ARL1, we have examined whether the orthologs of Arl3p-Arl1p-GRIP story serve similar functions in mammalian cells. However, we have unexpectedly found differential roles of ARL1 and ARFRP1. Specifically, ARL1 and ARFRP1 regulate retrograde transport of Shiga toxin to the TGN and anterograde transport of VSVG from the TGN, respectively. Furthermore, we have obtained evidence suggesting that a SNARE complex containing Vti1a, syntaxin 6, and syntaxin 16 is involved in Shiga toxin transport downstream of ARL1.The ARF/ARL 3 family of small GTPases play crucial roles in membrane trafficking and in other cellular processes by interacting with various effector proteins (1, 2). The functions of ARF proteins in membrane traffic have been well established, whereas much less is known about the functions of ARL proteins. Accumulating lines of evidence in yeast, however, have suggested a cascade model in which N-acetylated Arl3p is targeted to the trans-Golgi network (TGN) membranes where it causes activation of Arl1p; activated Arl1p in turn recruits a GRIP (golgin-97/RanBP2/Imh1p/p230) domain-containing protein, Imh1p, and regulates retrograde transport from endosomes to the TGN via tethering of endosome-derived vesicles to the TGN and anterograde transport from the TGN to the plasma membrane (3-7).It has been generally accepted that the Arl3p-Arl1p-GRIP cascade also occurs in mammalian cells. The mammalian orthologs of yeast Arl3p and Arl1p are ARFRP1 and ARL1, respectively. ARL1 has been shown to recruit GRIP domaincontaining golgins, golgin-97 and golgin-245/p230/tGolgin-1, onto TGN membranes (8 -11); it also participates in retrograde transport from endosomes to the TGN (10). Our laboratory and other groups previously showed, by expressing a dominantnegative mutant of ARFRP1, that the small GTPase functions upstream of ARL1 and the golgins and regulates anterograde traffic from and retrograde traffic to the TGN (11, 13). However, our subsequent experiments using the dominant-negative mutant of both ARL1 and ARFRP1 have suggested that these mutants might nonspecifically affect the recruitment of TGNlocalizing proteins, and ARL1 might not necessarily act downstream of ARFRP1.In the present study, we have therefore re-evaluated the roles of ARL1 and ARFRP1 by RNAi-mediated knockdown of these small GTPases. We have found that ARL1 predominantly regulates retrograde transport of Shiga toxin B fragment (StxB) to the TGN, whereas ARFRP1 regulates anterograde transport of vesicular stomatitis virus G protein (VSVG) from the TGN. Furthermore, our data have suggested that Q-SNAREs (syntaxi...