Golgins transport

Functional Analysis of Aril and Golgin-97 in Endosome-to-TGN Transport Using Recombinant Shiga Toxin B Fragment... 

Inhibition of In Vitro STxB Transport by Recombinant GRIP Domain of Golgin-97... 

The in vitro transport assay is routinely characterized to ensure that the assay has indeed faithfully reconstituted the EE/RE-to-TGN transport in vitro (Tai et ah, 2004). First, the assay must be cytosol-dependent. The basal level of transport in the absence of cytosol is usually 5-10% of that in the presence of 1.2 mg/ml of rat liver cytosol. Second, the assay is tested with some commonly used inhibitors such as Brefeldin A and guanosine 5 -0-(3-thio) triphosphate (GTP7S), as their inhibitory role in EE/RE-to-TGN traffic is well documented. Finally, the assay is tested using antibodies (such as autibody agaiust syutaxiulb) or recombinant proteins (such as GST-GRIP of golgin-97) that have been shown to inhibit in vitro EE/RE-to-TGN transport of STxB. 

Fig. 1. The recombinant GRIP domain of golgin-97 inhibits in vitro EE/RE-TGN transport. STxB transport assays are carried out in the absence (lanes 1 and 4) or presence of 133 g/ml of GST-GRIP domain proteins (lane 2, GST-GRIP lane 3, heat-denatured GST-GRIP lane 5, GST-GRIPA 697A). Extent of transport relative to controls (set as 100%) is calculated from two to three independent experiments. Error bars represent standard deviations. Bottom panel shows representative phosphor-imaging data. Reprinted from Molecular Biology of the Cell (Mol. Biol. Cell (2004). 15, 4426-4443.) with the permission of The American Society for Cell Biology.

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