Membrane trafficking proteins, enriched

The central limitation of the subtractive approach is that it identifies only proteins that are unique to the NE it will disregard proteins that normally occur in both of the fractions analyzed. One such protein is Seel 3, an ER trafficking protein that also has a demonstrated function in NPC mediated transport (Siniossoglou et al. 1996). Seel 3 thus appeared in both datasets and was accordingly discarded from the NE-unique in silico purified protein set. TorsinA also appeared in both datasets and was discarded, but it has since been shown to shuttle between the INM and the ER (Goodchild and Dauer 2004). As 41% of the 566 proteins identified in the membrane-enriched NE fraction were similarly discarded, there may be many other proteins listed in the pre-subtraction dataset that are truly NE proteins, though they are not unique to the NE. 

In the kiss-and-run mode exocytosis and endocytosis are directly coupled to each other, while in the case of classical complete vesicle fusion, exocytosis and slow clathrin-mediated endocytosis are timely and spatially separated. However, it appears that also in the latter case exocytosis and endocytosis occur coordinated, as both are stimulated by an increase of the cytoplasmic calcium concentration. It has been shown that after calcium entry the enzyme phospho-inositol-5 kinase Iy, which is enriched in the synapse, catalyzes the synthesis of phosphatidylinos-itol (4,5)-bisphosphate and that this mechanism is important for synaptic vesicle trafficking (Di Paolo et al. 2004). As many proteins involved in clathrin-mediated endocytosis are recruited to the plasma membrane by binding to phosphatidylinosi-tol (4,5)-bisphosphate (e.g., amphiphysin, dynamin, epsin, AP-180, and AP-2) it is attractive to speculate that elevated levels of calcium mediate the recruitment of en-docytic proteins to the plasma membrane by this mechanism. The increased level of phosphatidylinositol (4,5)-bisphosphate could be in part degraded by synaptojanin that thereby initiates the disassembly of the clathrin coat. Hence, calcium-induced transient increases in the level of phosphatidylinositol (4,5)-bisphosphate appear to play a central role for coupling exocytosis to clathrin-mediated endocytosis. In addition, it has been demonstrated that calcium also leads to the dephosphorylation of endocytic proteins as amphiphysin, dynamin, and synaptojanin, which in vitro is important for efficient coat assembly (Cousin and Robinson 2001). 

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