scholarly journals Rational design and implementation of a chemically inducible hetero-trimerization system

2020 ◽  
Author(s):  
Helen D. Wu ◽  
Masaki Kikuchi ◽  
Onur Dagliyan ◽  
Adam K. Aragaki ◽  
Hideki Nakamura ◽  
...  

AbstractChemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools exemplified by the FKBP/FRB/rapamycin system have been widely employed to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) has not been developed, due to inherent challenges in designing or identifying a chemical that simultaneously binds three proteins with high affinity and target specificity. Nevertheless, by introducing a third recruitable component, CIT could enable versatile applications. Here, we devised the CIT by rationally splitting FRB and FKBP. Based on cellular and structural datasets, select split pairs of FRB or FKBP underwent efficient trimerization with full length FKBP or FRB, respectively, upon addition of rapamycin. We also demonstrated its potential for cellular applications by rapidly inducing tri-organellar plasma membrane-ER-mitochondria junctions, and by perturbing intended membrane lipids exclusively at the plasma membrane-ER membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells, to address cell biology questions otherwise intractable, and engineer cell functions for future synthetic biology applications.

Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 971
Author(s):  
Philipp Schlarmann ◽  
Atsuko Ikeda ◽  
Kouichi Funato

Sphingolipids are the most diverse class of membrane lipids, in terms of their structure and function. Structurally simple sphingolipid precursors, such as ceramides, act as intracellular signaling molecules in various processes, including apoptosis, whereas mature and complex forms of sphingolipids are important structural components of the plasma membrane. Supplying complex sphingolipids to the plasma membrane, according to need, while keeping pro-apoptotic ceramides in check is an intricate task for the cell and requires mechanisms that tightly control sphingolipid synthesis, breakdown, and storage. As each of these processes takes place in different organelles, recent studies, using the budding yeast Saccharomyces cerevisiae, have investigated the role of membrane contact sites as hubs that integrate inter-organellar sphingolipid transport and regulation. In this review, we provide a detailed overview of the findings of these studies and put them into the context of established regulatory mechanisms of sphingolipid homeostasis. We have focused on the role of membrane contact sites in sphingolipid metabolism and ceramide transport, as well as the mechanisms that prevent toxic ceramide accumulation.


2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.


2018 ◽  
Vol 115 (31) ◽  
pp. E7331-E7340 ◽  
Author(s):  
Ben Johnson ◽  
Ashley N. Leek ◽  
Laura Solé ◽  
Emily E. Maverick ◽  
Tim P. Levine ◽  
...  

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.


2018 ◽  
Vol 178 (2) ◽  
pp. 641-653 ◽  
Author(s):  
Kazuya Ishikawa ◽  
Kentaro Tamura ◽  
Haruko Ueda ◽  
Yoko Ito ◽  
Akihiko Nakano ◽  
...  

2017 ◽  
Vol 45 (2) ◽  
pp. 477-488 ◽  
Author(s):  
Yael Elbaz-Alon

Membrane contact sites (MCSs) are areas of close apposition between the membranes of two different organelles that enable non-vesicular transfer of ions and lipids. Recent studies reveal that mitochondria maintain contact sites with organelles other than the endoplasmic reticulum such as the vacuole, plasma membrane and peroxisomes. This review focuses on novel findings achieved mainly in yeast regarding tethers, function and regulation of mitochondria–organelle contact sites. The emerging network of MCSs linking virtually all cellular organelles is highly dynamic and integrated with cellular metabolism.


Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P. Haslam ◽  
Steffen Vanneste ◽  
...  

SUMMARYEndoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.


PLoS Biology ◽  
2018 ◽  
Vol 16 (5) ◽  
pp. e2003864 ◽  
Author(s):  
Evan Quon ◽  
Yves Y. Sere ◽  
Neha Chauhan ◽  
Jesper Johansen ◽  
David P. Sullivan ◽  
...  

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