3,481 research outputs found

    Recognizing Co-Creators in Four Configurations: Critical Questions for Web Archiving

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    Four categories of co-creator shape web archivists\u27 practice and influence the development of web archives: social forces, users and uses, subjects of web archives, and technical agents. This paper illustrates how these categories of co-creator overlap and interact in four specific web archiving contexts. It recommends that web archivists acknowledge this complex array of contributors as a way to imagine web archives differently. A critical approach to web archiving recognizes relationships and blended roles among stakeholders; seeks opportunities for non-extractive archival activity; and acknowledges the value of creative reuse as an important aspect of preservation

    DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein.

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    Both an N-Terminal 65-Kda Domain and a C-Terminal 30-Kda Domain of Seca Cycle into the Membrane at Secyeg During Translocation

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    SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overex- pressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the mem- brane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835–843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites and is rapidly followed by deinsertion. SecA also asso- ciates nonsaturably and unproductively with lipid. In the presence of ATP, yet without involvement of preprotein or SecYEG, lipid-bound SecA forms domains that are protease- resistant and that remain so even upon subsequent membrane disruption. Unlike the [35S]SecA that inserts into the mem- brane at SecYEGDFyajC as it promotes preprotein translo- cation, lipid-associated [35S]SecA does not chase from its protease-resistant state upon the addition of excess SecA. The finding that two domains of SecA (which together represent most regions of the polypeptide chain) cycle into the mem- brane during preprotein translocation, as well as the distinc- tion between the membrane association of SecA at transloca- tion sites of SecYEGDFyajC and at nonproductive lipid sites, are fundamental to the study of the role of SecA in preprotein movement

    The Tethering Complex HOPS Catalyzes Assembly of the Soluble SNARE Vam7 into Fusogenic Trans-SNARE Complexes

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    The fusion of yeast vacuolar membranes depends on the disassembly of cis–soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes and the subsequent reassembly of new SNARE complexes in trans. The disassembly of cis-SNARE complexes by Sec17/Sec18p releases the soluble SNARE Vam7p from vacuolar membranes. Consequently, Vam7p needs to be recruited to the membrane at future sites of fusion to allow the formation of trans-SNARE complexes. The multisubunit tethering homotypic fusion and vacuole protein sorting (HOPS) complex, which is essential for the fusion of vacuolar membranes, was previously shown to have direct affinity for Vam7p. The functional significance of this interaction, however, has been unclear. Using a fully reconstituted in vitro fusion reaction, we now show that HOPS facilitates membrane fusion by recruiting Vam7p for fusion. In the presence of HOPS, unlike with other tethering agents, very low levels of added Vam7p suffice to induce vigorous fusion. This is a specific recruitment of Vam7p rather than an indirect stimulation of SNARE complex formation through tethering, as HOPS does not facilitate fusion with a low amount of a soluble form of another vacuolar SNARE, Vti1p. Our findings establish yet another function among the multiple tasks that HOPS performs to catalyze the fusion of yeast vacuoles

    A Cascade of Multiple Proteins and Lipids Catalyzes Membrane Fusion

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    Recent studies suggest revisions to the SNARE paradigm of membrane fusion. Membrane tethers and/or SNAREs recruit proteins of the Sec 1/Munc18 family to catalyze SNARE assembly into trans-complexes. SNARE-domain zippering draws the bilayers into immediate apposition and provides a platform to position fusion triggers such as Sec 17/α-SNAP and/or synaptotagmin, which insert their apolar wedge domains into the bilayers, initiating the lipid rearrangements of fusion

    N-Terminal Domain of Vacuolar SNARE Vam7p Promotes Trans-SNARE Complex Assembly

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    SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R- and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R- and Q-SNAREs. This function of the Vam7p N-domain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain

    LMA1 Binds to Vacuoles at Sec18p (NSF), Transfers upon ATP Hydrolysis to a t-SNARE (Vam3p) Complex, and Is Released during Fusion

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    AbstractVacuole fusion requires Sec18p (NSF), Sec17p (α-SNAP), Ypt7p (GTP binding protein), Vam3p (t-SNARE), Nyv1p (v-SNARE), and LMA1 (l ow Mr a ctivity 1, a heterodimer of thioredoxin and IB2). LMA1 requires Sec18p for saturable, high-affinity binding to vacuoles, and Sec18p “priming” ATPase requires both Sec17p and LMA1. Either the sec18-1 mutation and deletion of IB2, or deletion of both IB2 and p13 (an IB2 homolog) causes a striking synthetic vacuole fragmentation phenotype. Upon Sec18p ATP hydrolysis, LMA1 transfers to (and stabilizes) a Vam3p complex. LMA1 is released from vacuoles in a phosphatase-regulated reaction. This LMA1 cycle explains how priming by Sec18p is coupled to t-SNARE stabilization and to fusion

    Trans-SNARE interactions elicit Ca2+ efflux from the yeast vacuole lumen

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    Ca2+ transients trigger many SNARE-dependent membrane fusion events. The homotypic fusion of yeast vacuoles occurs after a release of lumenal Ca2+. Here, we show that trans-SNARE interactions promote the release of Ca2+ from the vacuole lumen. Ypt7p–GTP, the Sec1p/Munc18-protein Vps33p, and Rho GTPases, all of which function during docking, are required for Ca2+ release. Inhibitors of SNARE function prevent Ca2+ release. Recombinant Vam7p, a soluble Q-SNARE, stimulates Ca2+ release. Vacuoles lacking either of two complementary SNAREs, Vam3p or Nyv1p, fail to release Ca2+ upon tethering. Mixing these two vacuole populations together allows Vam3p and Nyv1p to interact in trans and rescues Ca2+ release. Sec17/18p promote sustained Ca2+ release by recycling SNAREs (and perhaps other limiting factors), but are not required at the release step itself. We conclude that trans-SNARE assembly events during docking promote Ca2+ release from the vacuole lumen
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