Abstract
Analysis of the cytosolic requirements for in vitro intra-Golgi transport led to the characterization of three proteins: N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment protein (SNAP), and p115, all involved in the docking and fusion of transport vesicles to their target membranes. In the course of determining the minimal cytosolic requirements for intra-Golgi transport in vitro, we identified three additional factors that are sufficient to replace crude cytosol. We describe here the purification and characterization of one of these factors, a novel 16-kDa protein, p16, an essential factor for intra-Golgi protein transport. Based on transport activity, this purification procedure resulted in approximately 1,400-fold enrichment of p16 to apparent homogeneity. The activity of p16 could be observed in the absence of vesicle formation, suggesting that it may participate in the docking and fusion processes.
Highlights
Several cytosolic transport factors were purified by the utilization of an in vitro intra-Golgi transport assay
Identification of Novel Cytosolic Factors Involved in IntraGolgi Transport—The cell-free system that reconstitutes intraGolgi transport has been used in recent years for the characterization and isolation of several factors involved in intracellular trafficking [2]
Proteins such as N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment protein (SNAP), and p115 were isolated by this transport assay in which the activity of each factor could be assessed
Summary
Several cytosolic transport factors were purified by the utilization of an in vitro intra-Golgi transport assay. P115 was isolated as a cytosolic factor required for intraGolgi transport in vitro [6] and was suggested to act together with NSF and SNAP in direct Golgi-Golgi fusion [17]. Uso1p, the yeast homolog of p115, is required for assembly of the endoplasmic reticulum-Golgi SNARE complex [20] Other cytosolic factors such as Rab proteins and their effectors were shown to be involved in this process [21,22,23]. That the amount of Rab proteins present on the membrane is sufficient to promote the transport reaction in vitro It was demonstrated originally by Clary and Rothman [24] that in addition to NSF and the SNAPs, several other cytosolic factors were required for reconstituting the SNAP-dependent transport assay. Our data suggest that p16 is a transport factor that participates in the docking/fusion reaction
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