ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a small GTPase with significant similarity to the ARF family. However, little is known about the function of ARFRP1 in mammalian cells, although knockout mice of its gene are embryonic lethal. In the present study, we demonstrate that ARFRP1 is associated mainly with the trans-Golgi compartment and the trans-Golgi network (TGN) and is an essential regulatory factor for targeting of Arl1 and GRIP domain-containing proteins, golgin-97 and golgin-245, onto Golgi membranes. Furthermore, we show that, in concert with Arl1 and GRIP proteins, ARFRP1 is implicated in the Golgi-to-plasma membrane transport of the vesicular stomatitis virus G protein as well as in the retrograde transport of TGN38 and Shiga toxin from endosomes to the TGN.Supplementary material available online at

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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...

We have investigated the influence of the high centrifugal pressure caused by fast magic-angle spinning (MAS) on the molecular motion of styrene-butadiene rubbers (SBR) filled with SiO 2 (SBR/Si composite) though solid-state magic-angle spinning nuclear magnetic Resonance ( 1 H MAS NMR) measurements. Because the 1 H-1 H dipolar interaction of elastomers is weak compared with that of glassy polymers, a narrower 1 H linewidth is observed under fast MAS. The temperature dependence of the 1 H spin-lattice relaxation time (T 1 H ) revealed that the T 1 H minimum increases with the MAS rate. Furthermore, we observed a difference in the temperature dependence of T 1 H between end-chain-modified SBR and normal (unmodified) SBR in the SBR/Si composites. The temperature dependence of T 1 H is described by the Bloembergen-Purcell-Pound theory, with the assumption that the correlation time obeys the Williams-Landel-Ferry empirical theory. The fitting showed that the molecular motion does not change significantly until a MAS rate of 20 kHz, with the motional mode changing considerably at a MAS rate of 25 kHz. The motion of SBR in the unmodified SBR/Si composite was greatly affected by the fast MAS rates. Furthermore, the plot of the estimated centrifugal pressure versus the T 1 H minimum resembled the stress-strain curve. This result enables the detection of macroscopic physical deformation by the microscopic parameter INTRODUCTIONSolid-state NMR is useful for the investigation of the molecular motion of the functional groups of elastomers and polymers. In particular, the recent development of the magic-angle spinning (MAS) technique allows a sample rotor to be spun much faster than 20 kHz. Thus, we can detect the high-resolution 1 H signals of rubbers and elastomers under the fast rates that are possible in MAS because these fast MAS rates effectively reduce the signal broadening that arises from the relatively weak 1 H-1 H dipolar interaction of elastomers (in comparison with that of glassy solid polymers).Generally, spin-lattice relaxation time (T 1 ) is observed to study molecular motion. In the case of rare spins such as 13 C, however, it is time-consuming to observe the signals and measure an accurate T 1 with a good signal-to-noise ratio. In contrast, proton signals are intense enough to allow quick analysis of molecular motion through the T 1 measurement. However, for a glassy solid polymer, the strong 1 H-1 H dipolar interaction obscures the individual functional group signals, even with fast MAS. For rubbers and elastomers under fast MAS, in contrast, each peak assigned to a respective functional group can be detected because of the weak 1 H-1 H dipolar interaction. Furthermore, it is easy to detect 1 H-T 1 ( 1 H spin-lattice relaxation time (T 1 H )) accurately. In contrast, it is well known that MAS causes the temperature of the inner sample to increase because of friction between the MAS and the air. In addition, it has recently been reported that fast MAS causes