The "Dsl1p complex" in Saccharomyces cerevisiae, consisting of Dsl1p and Tip20p, is involved in Golgi-ER retrograde transport and it is functionally conserved from yeast to mammalian cells. To further characterize this complex, we analyzed the function of Dsl3p, a protein that interacts with Dsl1p in yeast two hybrids screens. DSL3, recently identified in a genome wide analysis of essential genes as SEC39, encodes a cytosolic protein of 82 kDa that is peripherally associated with membranes derived from the ER. There is strong genetic interaction between DSL3 and other factors required for Golgi-ER retrograde transport. Size exclusion chromatography and affinity purification approaches confirmed that Dsl3p is associated with subunits of the "Dsl1p complex." The complex also includes the Q/t-SNARE proteins, Use1p, Sec20p, and Ufe1p, integral membrane proteins that constitute the trimeric acceptor for R/v-SNAREs on Golgi-derived vesicles at the ER. Using mutants, we performed a detailed analysis of interactions between subunits of the Dsl1p complex and the ER-localized SNARE proteins. This analysis showed that both Dsl1p and Dsl3p are required for the stable interaction of the SNARE Use1p with a central subcomplex consisting of Tip20p and the SNARE proteins Ufe1p and Sec20p. INTRODUCTIONThe protein trafficking pathway in the yeast S. cerevisiae is composed of several distinct membrane-bounded compartments, which interact via a bidirectional flow of membranebounded vesicles in a process referred to as vesicular transport (Kaiser and Schekman, 1990;Rothman and Orci, 1992;Ferro-Novick and Jahn, 1994;Rothman, 1994;Waters and Hughson, 2000). Briefly, secretory proteins destined for transport must be sorted away from resident proteins, packaged into the proper cargo vesicles, and subsequently, delivered to the correct target membrane (Palade, 1975). Each step of this process must be tightly regulated to ensure efficient secretion and maintenance of the distinct cellular compartments. Transport in the retrograde direction ensures further rounds of anterograde transport by recycling components of the transport machinery, recovering wayward proteins and maintaining the balance of lipids between the distinct compartments of the pathway.The cell makes use of a variety of coated vesicles to transport proteins, whose formation is nucleated by the action of small GTP-binding proteins. Specifically, vesicle budding in the retrograde direction from the Golgi to the ER involves a heptameric coat protein complex called COPI (Waters et al., 1991;Stenbeck et al., 1993;Letourneur et al., 1994;Barlowe, 2000). The COPI coat consists of coatomer, an ϳ700 -800 kDa protein complex comprised of an equimolar assembly of ␣-, ␤-, ␤'-, ␥-, ␦-, ⑀-, and -COP (Cop1[Ret1]p, Sec26p, Sec27p, Sec21p, Ret2p, Sec28p, and Ret3p, respectively, in yeast) and a small ras-like GTPase termed Arf (encoded by ARF1 or ARF2).Current models of vesicular transport propose that the vesicle coat is removed soon after vesicles are formed (although this has not been di...

RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary singlestrand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2¢-O-methyl (2¢-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2¢-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2¢-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.

DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of approximately 80 and approximately 55 kDa. The approximately 80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is approximately 50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the delta-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction.

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The nimO predicted protein of Aspergillus nidulans is related structurally and functionally to Dbf4p, the regulatory subunit of Cdc7p kinase in budding yeast. nimOp and Dbf4p are most similar in their C-termini, which contain a PEST motif and a novel, short-looped Cys2-His2 zinc finger-like motif. DNA labelling and reciprocal shift assays using ts-lethal nimO18 mutants showed that nimO is required for initiation of DNA synthesis and for efficient progression through S phase. nimO18 mutants abrogated a cell cycle checkpoint linking S and M phases by segregating their unreplicated chromatin. This checkpoint defect did not interfere with other checkpoints monitoring spindle assembly and DNA damage (dimer lesions), but did prevent activation of a DNA replication checkpoint. The division of unreplicated chromatin was accelerated in cells lacking a component of the anaphase-promoting complex (bimEAPC1), consistent with the involvement of nimO and APC/C in separate checkpoint pathways. A nimO deletion conferred DNA synthesis and checkpoint defects similar to nimO18. Inducible nimO alleles lacking as many as 244 C-terminal amino acids supported hyphal growth, but not asexual development, when overexpressed in a ts-lethal nimO18 strain. However, the truncated alleles could not rescue a nimO deletion, indicating that the C terminus is essential and suggesting some type of interaction among nimO polypeptides.