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Abstract
ABSTRACTRab GTPases are master regulators of eukaryotic endomembrane systems, particularly functioning in membrane tethering to confer the directionality of intracellular membrane trafficking. However, how exactly Rab GTPases themselves act upon membrane tethering processes has remained enigmatic. Here, we thoroughly tested seven purified Rab GTPases in human, which localize at the various representative organelles, for their capacity to support membrane tethering in vitro. Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors. This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes. Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events.
Highlights
Eukaryotic cells organize and maintain the complex but highly specific secretory and endocytic trafficking pathways to deliver correct sets of cargo molecules towards their various subcellular organelles and plasma membranes (Bonifacino and Glick, 2004)
The first contact of organelles and transport vesicles before membrane docking and fusion, is a critical step to control the directionality of membrane traffic and has been proposed to be mediated by Rab GTPases and Rab-effector proteins (Yu and Hughson, 2010)
Received 30 June 2014; Accepted 8 October 2014 reconstitution studies have reported that yeast endosomal Rab GTPases and the HOPS complex, a Rab effector at yeast vacuoles, had the intrinsic capacity to tether liposomal membranes (Lo et al, 2012; Stroupe et al, 2009; Hickey and Wickner, 2010; Wickner, 2010)
Summary
Eukaryotic cells organize and maintain the complex but highly specific secretory and endocytic trafficking pathways to deliver correct sets of cargo molecules towards their various subcellular organelles and plasma membranes (Bonifacino and Glick, 2004). The current three in vitro analyses, including the streptavidin-bead assay (Fig. 3B–D), turbidity assay (Fig. 3E,F), and fluorescent microscopy (Fig. 3G–L), demonstrate that specific human Rab GTPases can mediate liposome aggregation, even when their specific Rab effectors and/or other tethering factors are not present.
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