An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif.

No abstract

Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.Vesicles transport biosynthetic cargo and lipids between different compartments of the endomembrane system. Formation of the transport vesicles requires adaptors, coat proteins, and regulatory GTPases of the Arf1/Sar1 family. The initial contact between a vesicle and its target membrane requires Rab GTPases and tethers, which are in most cases multisubunit complexes. Rab GTPases, which cycle between an inactive GDP-and active GTP-bound state, and tethers coordinate the assembly of SNARE proteins on vesicle and target membrane into a four-helix bundle, which ultimately drives bilayer fusion (1).The Dsl1 tethering complex functions in fusion of Golgiderived vesicles at the ER 4 membrane and consists of the three subunits Dsl1, Dsl3/Sec39, and Tip20. It forms a stable complex with the ER SNAREs Sec20, Ufe1, and Use1 (2, 3). In addition, Dsl1 and Tip20 are linked to the coatomer, which implies a role in the recognition and/or uncoating of the COPI vesicle (4 -7). In agreement with this, Dsl1 depletion leads to a massive accumulation of COPI-coated vesicles (8).The Dsl1 complex is closely linked to the SNAREs Ufe1, Use1/Slt1, Sec20, and Sec22, which are required for fusion at the ER membrane (9 -12). The R-SNARE Sec22 is generally accepted as the v-SNARE on COPI vesicles. However, Sec22 has not been previously identified as part of the Dsl1 complex and is dispensable for yeast survival. In addition, it can be functionally replaced by the R-SNARE Ykt6 in anterograde (13) and potentially also in retrograde transport. Ykt6, which lacks a transmembrane domain and thus is unlikely to function as the sole v-SNARE, is found in multiple SNARE complexes at the Golgi, endosomes, and the vacuole (10, 14).Here, we present additional insights into the interactions and functions of the Dsl1 complex. We show that the two R-SNAREs Sec22 and Ykt6 are associated with the Dsl1 complex, with Sec22 being the preferred subunit. Only Ykt6 is sensitive to Sec18/NSF, whereas the remaining interaction between SNAREs and the Dsl1 complex is unaffe...

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD(+) or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD(+) and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD(+) inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD(+)-dependent xanthine oxidation indicates that both NAD(+) and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.