Both types of protein participate in membrane tethering events that occur upstream of membrane fusion as well as contributing to the structural scaffold that defines Golgi architecture, referred to as the Golgi matrix.
Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events.
Taken together, the current biochemical analyses using purified human Rab proteins and synthetic liposomes have established that membrane-anchored Rab GTPases have the inherent potency to directly mediate reversible membrane tethering events (Figs 3– 6).
This may reflect that membrane-anchored Rab5a exclusively recognize and assemble in trans with Rab5a on opposing membranes destined to tether, not membrane-detached soluble Rab5a, thereby conferring specific membrane tethering events.
We then asked whether the liposome aggregation reactions mediated by membrane-anchored Rab proteins are indeed a reversible reaction, like physiological membrane tethering events (Ungermann et al., 1998).
For a negative control, we also purified the His12-tagged form of human HRas, which is a similar Ras-family GTPase with a C-terminal lipid anchor but not functionally related to membrane tethering events (Fig. 1A, lane 8).
