Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer

Füllbrunn, Nadia; Li, Zehao; Jorde, Lara; Richter, Christian; Kurre, Rainer; Langemeyer, Lars; Chen, Yu; Meyer, Carola; Enderlein, Joerg; Ungermann, Christian; Piehler, Jacob; You, Changjiang

doi:10.7554/elife.62501

Cited by 21 publications

(34 citation statements)

References 74 publications

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“…As a peptide in an extended conformation may span ∼3 A ˚per peptide bond, Ypt7 can easily accommodate the proposed distance from the membrane. This model is also consistent with experimental data obtained by grapheneinduced energy transfer measurements (28). Here, Ypt7 was observed 38 A ˚away from the membrane surface in the presence of Mon1-Ccz1.…”

Section: Resultssupporting

confidence: 92%

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation

Klink

Herrmann

Antoni

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate–binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

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Section: Resultssupporting

confidence: 92%

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation

Klink

Herrmann

Antoni

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…These lipids likely contribute directly to bilayer rearrangements. HOPS has been proposed to have an elongated and somewhat unstructured conformation (Chou et al, 2016), though recent studies have shown that its association with Ypt7 can induce a major conformational change (Fuellbrunn et al, 2021) which is likely related to the functional activation reported here. Its binding to three co-receptors on the vacuole, PI, PI3P, and Ypt7, may orient and activate it for productive engagement with SNAREs.…”

Section: Discussionmentioning

confidence: 73%

“…HOPS has been proposed to have an elongated and somewhat unstructured conformation ( Chou et al, 2016 ), though recent studies have shown that its association with Ypt7 can induce a major conformational change ( Fuellbrunn et al. , 2021 ), which is likely related to the functional activation reported here.…”

Section: Discussionmentioning

confidence: 80%

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion

Torng

Wickner

2021

MBoC

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Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the 8 major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.

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“…MIET allows for nanoscale axial localization of fluorescent probes above the conducting substrate [43,44]. In particular, using graphene as the conducting substrate results in significant dependence of the fluorescence lifetime to sub-nm changes in the axial direction [72,79], making this method highly relevant for the study of membrane proteins. Applying optical techniques such as FRET and MIET utilizing the platform described in this work can allow for novel experiments that probe voltage-induced molecular dynamics of membrane proteins.…”

Section: Future Workoptical Investigation Of Voltage-dependent Protein Dynamics and Biologically Relevant Model Membranesmentioning

confidence: 99%

Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers

Yudovich

Marzouqe

Kantorovitsch

et al. 2021

Preprint

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Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipids bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of the equivalent electrical circuit. We also describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.

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Nanoscopic anatomy of dynamic multi-protein complexes at membranes resolved by graphene-induced energy transfer

Cited by 21 publications

References 74 publications

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation

Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion

Electrically Controlling and Optically Observing the Membrane Potential of Supported Lipid Bilayers

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