Dataset Information

Structural insights into the EGO-TC-mediated membrane tethering of the TORC1-regulatory Rag GTPases.

ABSTRACT: The Rag/Gtr GTPases serve as a central module in the nutrient-sensing signaling network upstream of TORC1. In yeast, the anchoring of Gtr1-Gtr2 to membranes depends on the Ego1-Ego2-Ego3 ternary complex (EGO-TC), resulting in an EGO-TC-Gtr1-Gtr2 complex (EGOC). EGO-TC and human Ragulator share no obvious sequence similarities and also differ in their composition with respect to the number of known subunits, which raises the question of how the EGO-TC fulfills its function in recruiting Gtr1-Gtr2. Here, we report the structure of EGOC, in which Ego1 wraps around Ego2, Ego3, and Gtr1-Gtr2. In addition, Ego3 interacts with Gtr1-Gtr2 to stabilize the complex. The functional roles of key residues involved in the assembly are validated by in vivo assays. Our structural and functional data combined demonstrate that EGOC and Ragulator-Rag complex are structurally conserved and that EGO-TC is essential and sufficient to recruit Gtr1-Gtr2 to membranes to ensure appropriate TORC1 signaling.

SUBMITTER: Zhang T

PROVIDER: S-EPMC6760929 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Structural insights into the EGO-TC-mediated membrane tethering of the TORC1-regulatory Rag GTPases.

Zhang Tianlong T Péli-Gulli Marie-Pierre MP Zhang Zhen Z Tang Xin X Ye Jie J De Virgilio Claudio C Ding Jianping J

Science advances 20190925 9

The Rag/Gtr GTPases serve as a central module in the nutrient-sensing signaling network upstream of TORC1. In yeast, the anchoring of Gtr1-Gtr2 to membranes depends on the Ego1-Ego2-Ego3 ternary complex (EGO-TC), resulting in an EGO-TC-Gtr1-Gtr2 complex (EGOC). EGO-TC and human Ragulator share no obvious sequence similarities and also differ in their composition with respect to the number of known subunits, which raises the question of how the EGO-TC fulfills its function in recruiting Gtr1-Gtr2 ...[more]

PMID: 31579828

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Project description: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.

Dataset Information

Structural insights into the EGO-TC-mediated membrane tethering of the TORC1-regulatory Rag GTPases.

Publications

Structural insights into the EGO-TC-mediated membrane tethering of the TORC1-regulatory Rag GTPases.

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