The Peters lab is involved in the following areas of research:
Mutual Control of Membrane Fusion and Membrane Fission Components in Vesicular Trafficking
Like numerous other eukaryotic organelles the vacuole of the yeast Saccharomyces cerevisiae undergoes coordinated cycles of membrane fission and fusion in the course of the cell cycle and in adaptation to environmental conditions. The aim of our research is to investigate the relationships of the protein machineries involved in membrane fusion and membrane fission, using the fragmentation and fusion of yeast vacuoles as a model.
Mutagenesis of the Dynamin-like GTPase Vps1
The dynamin-like GTPase Vps1p is required for the fragmentation of yeast vacuoles that occurs e.g. upon shifting the cells into hypertonic media. Vps1 interacts physically with the vacuolar t-SNARE Vam3p. In addition, dynamin-like GTPases can polymerize in a fashion influenced by the nucleotide cycle. Polymerization is believed to be required for the function of dynamins in fission processes. In order to investigate the influence of the nucleotide cycle of Vps1 on the fusion and fission of yeast vacuoles we have generated a series of point mutations in the GTPase and GED domains of Vps1.
For a start, we chose mutations of residues homologous to those that had been shown to influence the polymerization and activity of mammalian dynamins. Our results suggest that mutations known to reduce GTP binding to mammalian dynamins, mimicking the nucleotide-free form, interfere with fission as well as fusion of vacuoles. Nucleotide binding alone is not sufficient to support fission because a T63A mutation, shown to permit GTP binding but not hydrolysis in mammalian dynamins, behaves like a Δvps1 knockout cell in this respect. Interestingly, the T63A mutant showed still approx. 2/3 of the wildtype fusion activity, indicating that GTP binding may suffice to at least largely fulfill the role of Vps1 in fusion but that GTP hydrolysis is not essential.
Thus, fission may require GTP hydrolysis whereas fusion may only require GTP binding but not hydrolysis. Future work will have to verify that the point mutants tested have indeed the deduced effect on GTP binding and hydrolysis. To this end we will have to purify the proteins and determine their binding constants and kcat values.
Topology and Function of SNARE Complexes
In an effort to reinvestigate the SNARE activation cycle during fusion, we generated a large collection of SNAREs labeled at their C-termini (facing the organelle lumen) with different tags. We intend to use these tags to systematically map the protein-protein interactions between SNAREs and between SNAREs and Vps1 during membrane fusion and fission.
As an additional series of experiments on the side we use this collection of strains with different combinations of tagged SNAREs to re-analyze the formation of trans-SNARE complexes during fusion. Data from the use of purified SNAREs reconstituted in liposomes have led to the current belief that trans-SNARE complexes originate by pairing of an R-SNARE from one membrane with 3 Q-SNARE subunits (Qa/b/c) from the other fusion partner (Qa/b/c-R trans-complexes).
Our analysis of trans-SNARE complex assembly yielded a fundamentally different result: Using all possible combinations of differentially tagged SNAREs in one or the other fusion partner we could show that the R-SNARE forms a cis-complex with the Qb and Qc-SNARE subunit on the same membrane. This complex then recruits the Qa-SNARE from the opposite membrane (Qa-RQbQc trans-complexes). This physical distribution of subunits in a trans-SNARE complex was tested and confirmed by functional analyses: We generated strains producing vacuoles carrying SNAREs with conditional or knockout mutations in various combinations.
We investigated the fusion activities of most possible combinations of these SNAREs and could identify: 1) Several combinations of Q- and R-SNARE mutations that provided good fusion activity although they should not be functional for fusion according to the well-accepted topology proposed by the SNAREpin hypothesis (Qa/b/c-R); 2) Several combinations of Q- and R-SNARE mutations that did not yield fusion although they should be functional according to the SNAREpin hypothesis.
Thus, we have obtained both evidence that the currently accepted mode of assembly of trans-SNARE complexes is wrong - or at least not general - and we have evidence for an alternative assembly pathway of SNAREs that leads to trans-SNARE complexes with different topologies (and possibly even stoichiometries - this is currently under investigation).