Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions.

Sanae Ueda,Joji Mima,Naoki Tamura

doi:10.3389/fcell.2020.577342

Abstract

Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

Highlights

All eukaryotic cells, from a unicellular yeast to human cells, organize the complex but highly-regulated endomembrane systems, in which diverse cellular components including proteins and lipids are selectively delivered to their correct destinations, such as subcellular organelles, the plasma membrane, or the extracellular space, through secretory and endocytic trafficking pathways (Bonifacino and Glick, 2004)
The process of membrane tethering is vital for determining the spatiotemporal specificity of intracellular membrane trafficking, before the irreversible final steps of membrane docking and fusion mediated by SNARE-family proteins (Jahn and Scheller, 2006), which are another critical layers to confer the fidelity of membrane trafficking (McNew et al, 2000; Parlati et al, 2002; Izawa et al, 2012; Furukawa and Mima, 2014)
In addition to these miscellaneous, sequentiallyand structurally-diverse classic tethering factors, our recent reconstitution studies have established the inherent tethering functions of human Rab-family small GTPases (Tamura and Mima, 2014; Inoshita and Mima, 2017; Mima, 2018; Segawa et al, 2019), following the pioneering work of Lo et al (2012) which reported for the first time the intrinsic tethering activity of endosomal Ypt/Rab-family proteins in the yeast Saccharomyces cerevisiae

Summary

Introduction

From a unicellular yeast to human cells, organize the complex but highly-regulated endomembrane systems, in which diverse cellular components including proteins and lipids are selectively delivered to their correct destinations, such as subcellular organelles, the plasma membrane, or the extracellular space, through secretory and endocytic trafficking pathways (Bonifacino and Glick, 2004). Using the chemically-defined reconstitution system with purified proteins of putative membrane tethers or tethering factors and synthetic liposomes for a model lipid membrane, which is known as the most valid experimental approach to investigating whether or not the protein components of interest act as a bona fide membrane tether (Brunet and Sacher, 2014; Mima, 2018), comprehensive analyses of human Rab-family GTPases demonstrated their intrinsic membrane tethering potency to physically link two distinct lipid bilayers by themselves, even in the absence of any other tethering factors previously identified (Tamura and Mima, 2014; Inoshita and Mima, 2017; Mima, 2018; Segawa et al, 2019). By quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering

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Conclusion