scholarly journals The lipid transfer function of RDGB at ER-PM contact sites is regulated by multiple interdomain interactions

2019 ◽  
Author(s):  
Bishal Basak ◽  
Harini Krishnan ◽  
Padinjat Raghu
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Homeostasis ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Accurate Localization ◽  
Membrane Contact ◽  
Phosphatidylinositol Transfer ◽  
Interdomain Interactions

SummaryIn Drosophila photoreceptors, following Phospholipase C-β activation, the phosphatidylinositol transfer protein (PITP) RDGB, is required to maintain lipid homeostasis at endoplasmic reticulum (ER) plasma membrane (PM) membrane contact sites (MCS). Depletion or mis-localization of RDGB results in multiple defects in photoreceptors. Previously, interaction between the FFAT motif of RDGB with the integral ER protein dVAP-A was shown to be important for its localization at ER-PM MCS. Here, we report that in addition to FFAT motif, a large unstructured region (USR1) of RDGB is required to support the RDGB/dVAP-A interaction. However, interaction with dVAP-A alone is insufficient for accurate localization of RDGB: this also requires association of RDGB with apical PM, through its C-terminal LNS2 domain. Deletion of LNS2 domain results in complete mis-localisation of RDGB and also induces large mis-regulated interdomain movements abrogating RDGB function. Thus, multiple independent interactions between individual domains of RDGB supports its function at ER-PM MCS.

scholarly journals Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

2020 ◽  
Author(s):  
Bishal Basak ◽  
Harini Krishnan ◽  
Padinjat Raghu
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Transfer Protein ◽  
Intramolecular Interaction ◽  
Lipid Transfer ◽  
Loss Of Function ◽  
Transfer Protein ◽  
Contact Sites ◽  
Accurate Localization ◽  
Phosphatidylinositol Transfer ◽  
Interdomain Interactions

Abstract During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.

scholarly journals Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites

Biology Open ◽  
2021 ◽  
Vol 10 (3) ◽  
Author(s):  
Bishal Basak ◽  
Harini Krishnan ◽  
Padinjat Raghu
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Transfer Protein ◽  
Intramolecular Interaction ◽  
Lipid Transfer ◽  
Loss Of Function ◽  
Transfer Protein ◽  
Contact Sites ◽  
Accurate Localization ◽  
Phosphatidylinositol Transfer ◽  
Interdomain Interactions

ABSTRACT During phospholipase C-β (PLC-β) signalling in Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER–PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER–PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function. This article has an associated First Person interview with the first author of the paper.

scholarly journals Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. eaaz7714 ◽  
Author(s):  
Justyna Sawa-Makarska ◽  
Verena Baumann ◽  
Nicolas Coudevylle ◽  
Sören von Bülow ◽  
Veronika Nogellova ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Lipid Transfer ◽  
Related Protein ◽  
Membrane Formation ◽  
Selective Autophagy ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Phosphatidylinositol 3

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)–containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12–Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.

scholarly journals Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

2019 ◽  
Author(s):  
RS D’Souza ◽  
JY Lim ◽  
A Turgut ◽  
K Servage ◽  
J Zhang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Lipid Transfer Protein ◽  
Calcium Influx ◽  
Focal Adhesions ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Lipid Exchange ◽  
Membrane Contact

AbstractCoordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.

scholarly journals SAC1 Degrades its Lipid Substrate PtdIns4P in the Endoplasmic Reticulum to Maintain a Steep Chemical Gradient with Donor Membranes

2018 ◽  
Author(s):  
James P. Zewe ◽  
Rachel C. Wills ◽  
Sahana Sangappa ◽  
Brady D. Goulden ◽  
Gerald R. V. Hammond
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Homeostasis ◽  
Chemical Gradient ◽  
Vesicular Traffic ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Catalytic Domains ◽  
In Trans ◽  
Membrane Contact ◽  
And Function

AbstractGradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a “cis” configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in “trans” at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in “trans”, unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate “cis” activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.

scholarly journals VPS13: A lipid transfer protein making contacts at multiple cellular locations

2018 ◽  
Vol 217 (10) ◽  
pp. 3322-3324 ◽  
Author(s):  
Mingming Gao ◽  
Hongyuan Yang
Keyword(s):  
Lipid Transfer Protein ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Cell Biol ◽  
Evolutionarily Conserved ◽  
Membrane Contact

The evolutionarily conserved VPS13 proteins localize to multiple membrane contact sites though their function and regulation has been elusive. Bean et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201804111) found that competitive adaptors control the different localizations of yeast Vps13p, while Kumar et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201807019) provide biochemical and structural evidence for VPS13 proteins in the nonvesicular transport of phospholipids.

scholarly journals Endoplasmic Reticulum–Plasma Membrane Contact Sites: Regulators, Mechanisms, and Physiological Functions

2021 ◽  
Vol 9 ◽  
Author(s):  
Chenlu Li ◽  
Tiantian Qian ◽  
Ruyue He ◽  
Chun Wan ◽  
Yinghui Liu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Lipid Homeostasis ◽  
Ion Dynamics ◽  
Cellular Processes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Intracellular Signals ◽  
Membrane Contact ◽  
External Stimuli

The endoplasmic reticulum (ER) forms direct membrane contact sites with the plasma membrane (PM) in eukaryotic cells. These ER-PM contact sites play essential roles in lipid homeostasis, ion dynamics, and cell signaling, which are carried out by protein-protein or protein-lipid interactions. Distinct tethering factors dynamically control the architecture of ER-PM junctions in response to intracellular signals or external stimuli. The physiological roles of ER-PM contact sites are dependent on a variety of regulators that individually or cooperatively perform functions in diverse cellular processes. This review focuses on proteins functioning at ER-PM contact sites and highlights the recent progress in their mechanisms and physiological roles.

scholarly journals ORP5 AND ORP8 ORCHESTRATE LIPID DROPLET BIOGENESIS AND MAINTENANCE AT ER-MITOCHONDRIA CONTACT SITES

2021 ◽  
Author(s):  
Valentin Guyard ◽  
Vera F Monteiro-Cardoso ◽  
Mohyeddine Omrane ◽  
Cecile Sauvanet ◽  
Audrey Houcine ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Nucleation And Growth ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the Endoplasmic Reticulum (ER). The ER-protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at Mitochondria-Associated ER Membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulate seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for the ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at membrane contact sites.

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