Sequential involvement of p115, SNAREs, and Rab proteins in intra-Golgi protein transport

Delivery of transport vesicles to their receptor compartment involves tethering, priming, and fusion. Soluble NSF attachment protein-alpha (alphaSNAP) mediates the disruption of SNAREs by N-ethylmaleimide sensitive factor (NSF) and was employed to determine the hierarchy of proteins responsible for intra-Golgi protein transport. The N-terminal 23 amino acids of alphaSNAP are necessary for SNARE binding. The antibody 2F10 recognizes this SNARE interaction domain of alphaSNAP and inhibits intra-Golgi protein transport reversibly. This antibody was applied to modify the transport assay to determine the protein requirements relative to the action of alphaSNAP and NSF. We found that 1) p115 acts independently of alphaSNAP and NSF, 2) SNAREs are required after tethering and interact selectively after activation by alphaSNAP and NSF, and 3) Rab proteins act after SNARE activation and before fusion.