Investigation of SNARE function in the early endosomal compartment

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins catalyze organelle fusion in the secretory pathway. Di erent fusion steps are mediated by speci.c SNARE sets. By studying homotypic early endosomal fusion in neuroendocrine PC12 cells, I investigated how the speci.city of SNARE function is regulated. Even though early endosomal and exocytic SNAREs promote distinct fusion events (early endosomal fusion and exocytosis, respectively), they colocalize on the early endosomal membrane and associate in promiscuous cis-complexes, in a mass-action-dependent manner. I showed that two combinatorial mechanisms account for the speci.city in SNARE pairing and function: the enrichment of the necessary SNAREs at the prospective fusion sites, and preference for cognate SNARE associations in trans. To test the robustness of this highly regulated system under stress conditions, such as poor availability of its components, I downregulated by siRNA means the amount of single early endosomal or exocytic SNAREs in PC12 cells. Surprisingly, knock-down of early endosomal SNAREs, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. I found that the residual SNARE levels (typically about 10%) were sufficient for a substantial amount of SNARE-SNARE interactions and that in wild type cells most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the organelles derived from the knock-down cell lines recruited more of the tethering factor EAA1 and exhibited enhanced docking. I therefore conclude that, surprisingly, SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery, providing an additional example on the importance of cooperative functions for the regulation of intracellular fusion.