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...
A molecular dissection of contractile vacuole (CV) discharge shows that Rab8a is recruited to the CV a few seconds before the exocyst. Together they tether it to the plasma membrane and commit it to fusion. GTP hydrolysis is necessary for vacuole detethering, a process in which LvsA, a protein of the Chédiak–Higashi family, plays a crucial role.
SummaryChlamydia are obligate intracellular pathogens. Upon contact with the host, they use type III secretion to deliver proteins into the cell, thereby triggering actin-dependent entry and establishing the infection. We observed that Chlamydia caviae elicited a local and transient accumulation of ubiquitinated proteins at the entry sites, which disappeared within 20 min. We investigated the mechanism for the rapid clearance of ubiquitin. We showed that the OTU-like domain containing protein CCA00261, predicted to have deubiquitinase activity, was detected in infectious particles and was a type III secretion effector. This protein is present in several Chlamydia strains, including the human pathogen Chlamydia pneumoniae, and we further designate it as ChlaOTU. We demonstrated that ChlaOTU bound ubiquitin and NDP52, and we mapped these interactions to distinct domains. NDP52 was recruited to Chlamydia entry sites and was dispensable for infection and for bacterial growth. ChlaOTU functioned as a deubiquitinase in vitro. Heterologous expression of ChlaOTU reduced ubiquitin accumulation at the entry sites, while a catalytic mutant of the deubiquitinase activity had the opposite effect. Altogether, we have identified a novel secreted protein of chlamydiae. ChlaOTU targets both ubiquitin and NDP52 and likely participates in the clearance of ubiquitin at the invasion sites.
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