Biochemical and functional analysis of a mammalian ER/Golgi SNARE complex.

Intracellular membrane fusion is catalyzed by membrane anchored proteins called SNAREs (SNAP receptors). In addition to promoting fusion, SNAREs are also thought to play a role in maintaining the functional compartmentalization of the cell by determining the specificity of various fusion events. Mammalian ER/Golgi transport is mediated by a set of four SNARE proteins: syntaxin5, rbet1, membrin and sec22b which form a quaternary complex. Using biochemical substitution experiments with recombinant proteins, I demonstrate the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b and also show that the ER/Golgi SNARE complex is structurally super-imposable on the synaptic SNARE complex. I also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics and show that although the rbet1 SNARE motif was essential for the proper targeting of the protein, heteromeric SNARE interactions were dispensable. The helical SNARE motif is unstructured when not bound in a SNARE complex. Repetitive cycles of membrane fusion involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, very little is known about the conformational regulation of SNARE complex assembly. I show that hsc70 molecular chaperones, possibly in conjunction with unidentified cofactors in rat brain cytosol, render the SNARE motif competent for SNARE assembly allowing SNARE complex formation under conditions in which assembly is normally inhibited in vitro. These results are supported by the data from cross-linking and immunoprecipitation experiments showing the association of hsc70 with ER/Golgi SNAREs in vivo and that excess hsc70 specifically inhibits ER/Golgi transport in permeabilized cells. Lastly, I also present evidence for the regulation by calcium of ER/Golgi transport, specifically, the fusion of COPII vesicles. I show that calcium from intracellular pools may play an essential role in ER/Golgi transport and that calmodulin is the likely calcium effector for this transport step. Furthermore, an in vitro assay for the fusion of COPII vesicles indicates that, unlike all the fusion events studied so far, calcium is certainly not required for the fusion of COPII vesicles and more interestingly, may in fact be inhibitory.Ph.D.Biological SciencesCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125109/2/3186657.pd