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

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

Correction: Membranes Linked by Trans-Snare Complexes Require Lipids Prone to Non-Bilayer Structure for Progression to Fusion

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans -SNARE pairing but cannot rearrange their lipids for fusion

Sec17 Can Trigger Fusion of Trans-SNARE Paired Membranes without Sec18

Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17(L291A,L292A) did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17(F21S,M22S), with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17(F21S,M22S), interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion

Yeast Vacuolar HOPS, Regulated by its Kinase, Exploits Affinities for Acidic Lipids and Rab:GTP for Membrane Binding and to Catalyze Tethering and Fusion

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide–sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion

Multidimensionaler Erinnerungsmonitor (MEMO) I/2018

Rees J, Papendick M, Zick A, Wäschle F. Multidimensionaler Erinnerungsmonitor (MEMO) I/2018. Forschungsbericht IKG. Institut für interdisziplinäre Konflikt- und Gewaltforschung (IKG), Universität Bielefeld; 2018

Multidimensionaler Erinnerungsmonitor (MEMO) II/2019

Rees J, Zick A, Papendick M, Wäschle F. Multidimensionaler Erinnerungsmonitor (MEMO) II/2019. Forschungsbericht IKG. Bielefeld: Institut für interdisziplinäre Konflikt- und Gewaltforschung (IKG), Universität Bielefeld; 2019

Bielefeld protestiert. Ergebnisse einer Online-Umfrage zu den Ereignissen rund um die Demonstrationen am 10. November 2018

Papendick M, Rees J, Rees Y, Wäschle F, Zick A. Bielefeld protestiert. Ergebnisse einer Online-Umfrage zu den Ereignissen rund um die Demonstrationen am 10. November 2018. IKG Forschungsbericht. Bielefeld: Universität Bielefeld, Institut für interdisziplinäre Konflikt- und Gewaltforschung; 2018

The MOSDEF Survey: Untangling the Emission-line Properties of z ∼ 2.3 Star-forming Galaxies

We analyze the rest-optical emission-line spectra of z∼2.3 star-forming galaxies in the complete MOSFIRE Deep Evolution Field (MOSDEF) survey. In investigating the origin of the well-known offset between the sequences of high-redshift and local galaxies in the [O III]5008/Hβ vs. [N II]6585/Hα ("[N II] BPT") diagram, we define two populations of z∼2.3 MOSDEF galaxies. These include the "high" population that is offset towards higher [O III]5008/Hβ and/or [N II]6585/Hα with respect to the local SDSS sequence and the "low" population that overlaps the SDSS sequence. These two groups are also segregated within the [O III]5008/Hβ vs. [S II]6718,6733/Hα and the [O III]4960,5008/[O II]3727,3730 (O32) vs. ([O III]4960,5008+[O II]3727,3730)/Hβ (R23) diagram, which suggests qualitatively that star-forming regions in the more offset galaxies are characterized by harder ionizing spectra at fixed nebular oxygen abundance. We also investigate many galaxy properties of the split sample and find that the "high" sample is on average smaller in size and less massive, but has higher specific star-formation rate and star-formation-rate surface density values and is slightly younger compared to the "low" population. From Cloudy+BPASS photoionization models, we estimate that the "high" population has a lower stellar metallicity (i.e., harder ionizing spectrum) but slightly higher nebular metallicity and higher ionization parameter compared to the "low" population. While the "high" population is more α-enhanced (i.e., higher α/Fe) than the "low" population, both samples are significantly more α-enhanced compared to local star-forming galaxies with similar rest-optical line ratios. These differences must be accounted for in all high-redshift star-forming galaxies -- not only those "offset" from local excitation sequences