The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.

It is proposed here that the delayed cytotoxicity of thioguanine involves the postreplicative DNA mismatch repair system. After incorporation into DNA, the thioguanine is chemically methylated by S-adenosylmethionine to form S6-methylthioguanine. During DNA replication, the S6-methylthioguanine directs incorporation of either thymine or cytosine into the growing DNA strand, and the resultant S6-methylthioguanine-thymine pairs are recognized by the postreplicative mismatch repair system. Azathioprine, an immunosuppressant used in organ transplantation, is partly converted to thioguanine. Because the carcinogenicity of N-nitrosamines depends on formation of O6-alkylguanine in DNA, the formation of the analog S6-methylthioguanine during azathioprine treatment may partly explain the high incidence of cancer after transplantation.

The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.

PRL-1, -2, and -3 represent a novel class of proteintyrosine phosphatase with a C-terminal prenylation motif. Although PRL-1 has been suggested to be associated with the nucleus, the presence of three highly homologous members and the existence of a prenylation motif call for a more detailed examination of their subcellular localization. In the present study, we first demonstrate that mouse PRL-1, -2, and -3 are indeed prenylated. Examination of N-terminal epitope-tagged PRL-1, -2, and -3 expressed in transiently transfected cells suggests that PRL-1, -2, and -3 are present on the plasma membrane and intracellular punctate structures. Stable Chinese hamster ovary cells expressing PRL-1 and -3 in an inducible manner were established. When cells were treated with brefeldin A, PRL-1 and -3 accumulated in a collapsed compact structure around the microtubuleorganizing center. Furthermore, PRL-1 and -3 redistributed into swollen vacuole-like structures when cells were treated with wortmannin. These characteristics of PRL-1 and -3 are typical for endosomal proteins. Electron microscope immunogold labeling reveals that PRL-1 and -3 are indeed associated with the plasma membrane and the early endosomal compartment. Expression of PRL-3 is detected in the epithelial cells of the small intestine, where PRL-3 is present in punctate structures in the cytoplasm. When cells are treated with FTI-277, a selective farnesyltransferase inhibitor, PRL-1, -2, and -3 shifted into the nucleus. Furthermore, a mutant form of PRL-2 lacking the C-terminal prenylation signal is associated with the nucleus. These results establish that the primary association of PRL-1, -2, and -3 with the membrane of the cell surface and the early endosome is dependent on their prenylation and that nuclear localization of these proteins may be triggered by a regulatory event that inhibits their prenylation.

The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. In our present study, it is revealed that unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15°C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis-and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus.

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant ␣-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway. INTRODUCTIONProtein trafficking along the exocytotic and endocytotic pathways is a vital and fundamental cellular process. Proteins destined for the exocytotic pathway are initially targeted to the endoplasmic reticulum and transported through the Golgi apparatus. At the transGolgi network (TGN), proteins are sorted to distinct post-Golgi structures such as the plasma membrane (or its subdomains) and the endosomal and lysosomal compartments (Palade, 1975;Mellman and Simons, 1992;Hong and Tang, 1993;Rothman and Wieland, 1996;Schekman and Orci, 1996). The endosomal compartment plays a central role in cellular physiology (Gruenberg and Maxfield, 1995;Mellman, 1996;Robinson et al., 1996). Endocytosed proteins are internalized from the plasma membrane via coated vesicles and then delivered to the early endosomal compartment, from which proteins can be either recycled to the plasma membrane or delivered to the late endosomal compartment and subsequently to the lysosome or the TGN.Intracellular trafficking is primarily mediated by various types of transport vesicles that bud from a donor membrane and then fuse with a specific cognate target membrane. To achieve such specificity, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) hypothesis proposes that specific docking and fusion of vesicles with the cognate membrane compartment is mediated by specific interaction between the vesicle-associated SNAREs with the cognate target SNAREs on the target mem- ‡ Corresponding author. E-mail address: mcbhwj@imcb.nus. edu.sg.© 1998 by The American Society for Cell Biology 1549brane (Rothman and Warren, 1994;Bennett, 1995;Whiteheart and Kuba...

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Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.