Abstract
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.
Highlights
Vesicles mediate the transport between membrane-bound compartments of eukaryotic cells [1]
In interpreting our data and the data obtained in the yeast system, we propose that the function of ␣SNAP and NSF is to continuously activate the SNAREs on all membranes involved in constitutive transport
The model systems studied suggest a role for ␣SNAP and NSF before and after vesicle fusion; in neuroendocrine cells, a readily releasable pool of vesicles is docked to the plasma membrane and fuses after the influx of calcium ions from the extracellular medium [67, 68]
Summary
Recombinant Material—Bovine His6-␣SNAP was purified from Escherichia coli lysate according to Ref. 12, and GST-VAMP was purified as described in Ref. 44. To bacterially express the head domain of p115 from rat [45], a DNA fragment coding for amino acids 1– 651 was amplified by polymerase chain reaction introducing a BamHI site at the 5Ј end and an EcoRI site at the 3Ј end. Intra-Golgi protein transport assays were carried out at 37 °C for 60 min as described [53, 54]. The reactions were placed on ice, and the cytoplasmic domains of GOS28, GDI, and anti-Rab were added, respectively. The two-stage assays containing K-Golgi membranes were inhibited with 500 ng of 2F10 as described above, and anti-p115 antibody and 400 ng of His6␣SNAP were added. Error bars indicate standard deviations of experiments carried out as triplicates (see Fig. 4) or as duplicates (see Figs. 5 and 6)
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