Figure 3.

How membrane vesicles can be used to study transport of substrates by ABC-transporters. When cells are disrupted, the plasma membrane forms two types of vesicles, inside-in vesicles in which the transporter has its normal orientation pumping substrates out; and inside-out vesicles in which the orientation is inverted, the transporter pumping substrates into the vesicle, if ATP is provided to energise the pump. Following the incubation of the vesicles with radioactive substrate and ATP, the vesicles are washed on filters and counted. The inside-in vesicles do not contribute to the reaction, as under these conditions no ATP is present inside the vesicles to support ATP hydrolysis at the ABCC6 nucleotide-binding domains.