RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal

Hussain, Safdar; Tran, Tuyet-Minh; Ware, Timothy B.; Luse, Melissa A; Prevost, Christopher T; Ferguson, Ashley N.; Kashatus, Jennifer A.; Hsu, Ku‐Lung; Kashatus, David F.

doi:10.1016/j.celrep.2021.109451

Cited by 20 publications

(11 citation statements)

References 110 publications

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“…Our data showed that the distribution pattern of PLIN3 did change slightly in cells expressing Sar1a, but not in cells expressing Sar1b ( Figure 3 and Figure 5 A), indicating PLIN3 could compensate for the PLIN2reduction on LD surface. Recently, PLIN3 was shown to localize to LDs in RalA-GTPase-dependent manner [ 41 ]. Thus, there may be several GTPases that regulate the localization of LD components to LDs in cells.…”

Section: Discussionmentioning

confidence: 99%

Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets

Makiyama

Obama

Watanabe

et al. 2022

IJMS

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Lipid droplets (LDs) are intracellular organelles that are ubiquitous in many types of cells. The LD core consists of triacylglycerols (TGs) surrounded by a phospholipid monolayer and surface proteins such as perilipin 2 (PLIN2). Although TGs accumulate in the phospholipid bilayer of the endoplasmic reticulum (ER) and subsequently nascent LDs buds from ER, the mechanism by which LD proteins are transported to LD particles is not fully understood. Sar1 is a GTPase known as a regulator of coat protein complex Ⅱ (COPⅡ) vesicle budding, and its role in LD formation was investigated in this study. HuH7 human hepatoma cells were infected with adenoviral particles containing genes coding GFP fused with wild-type Sar1 (Sar1 WT) or a GTPase mutant form (Sar1 H79G). When HuH7 cells were treated with oleic acid, Sar1 WT formed a ring-like structure around the LDs. The transient expression of Sar1 did not significantly alter the levels of TG and PLIN2 in the cells. However, the localization of PLIN2 to the LDs decreased in the cells expressing Sar1 H79G. Furthermore, the effects of Sar1 on PLIN2 localization to the LDs were verified by the suppression of endogenous Sar1 using the short hairpin RNA technique. In conclusion, it was found that Sar1 has some roles in the intracellular distribution of PLIN2 to LDs in liver cells.

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Section: Discussionmentioning

confidence: 99%

Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets

Makiyama

Obama

Watanabe

et al. 2022

IJMS

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“…[10] NMR experiments were used to compare binding of non-lipidated peptides corresponding to the RalA and RalB HVR [10] (Figure 1A). The peptides were titrated into 15 N labelled CaM, and HSQC experiments were recorded at each titration point. These spectra report on the change in the chemical environment in the presence of the HVR peptides, which can be quantified as a chemical shift perturbation (CSP) at each CaM backbone amide.…”

Section: Rala Versus Ralb Binding To Cammentioning

confidence: 99%

“…Both Ral isoforms play a role in the cellular starvation response. [6,15] Upon nutrient withdrawal, RalA can cause the formation of lipid droplets by interacting with phospholipase D1 (PLD1). [15] This interaction does not involve the nucleotide sensitive effector binding site but is suggested to utilise the N-terminal 11 residues of RalA, [16] outside the canonical G domain.…”

Section: Functional Consequences For Mitochondrial Dynamicsmentioning

confidence: 99%

“…[6,15] Upon nutrient withdrawal, RalA can cause the formation of lipid droplets by interacting with phospholipase D1 (PLD1). [15] This interaction does not involve the nucleotide sensitive effector binding site but is suggested to utilise the N-terminal 11 residues of RalA, [16] outside the canonical G domain. In contrast, RalB has not been reported to bind PLD1, and instead during nutrient scarcity RalB and the Ral-effector Exo84 drive autophagosome formation.…”

Section: Functional Consequences For Mitochondrial Dynamicsmentioning

confidence: 99%

“…[2][3][4] RalA and RalB have a central, conserved, Ras-like G-domain and are almost identical in their effector-binding regions, but they mediate markedly different cellular processes, especially in the context of ABBREVIATIONS: CaM, calmodulin; CSP, chemical shift perturbation; GAP, GTPase activating protein; GDI, GDP dissociation inhibitor; GEF, guanine nucleotide exchange factor; GSF, GDI-like solubilisation factor; HVR, hypervariable region; PDE-δ, phosphodiesterase δ; PLD1, phospholipase D1; TBK1, TANK-binding kinase 1 F I G U R E 1 NMR data comparing RalA and RalB interaction with CaM. (A) RalA and RalB HVR peptides were titrated into 15 N-labelled CaM and HSQC experiments performed as reported previously. [10] The RalA titration is shown on the left while the RalB titration is on the right.…”

Section: Introductionmentioning

confidence: 99%

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Membrane extraction by calmodulin underpins the disparate signalling of RalA and RalB

2022

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Both RalA and RalB interact with the ubiquitous calcium sensor, calmodulin (CaM). New structural and biophysical characterisation of these interactions strongly suggests that, in the native membrane‐associated state, only RalA can be extracted from the membrane by CaM and this non‐canonical interaction could underpin the divergent signalling roles of these closely related GTPases. The isoform specificity for RalA exhibited by CaM is hypothesised to contribute to the disparate signalling roles of RalA and RalB in mitochondrial dynamics. This would lead to CaM shuttling RalA to the mitochondrial membrane but leaving RalB localisation unperturbed, and in doing so triggering mitochondrial fission pathways rather than mitophagy.

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A pH‐Sensitive Double Chromophore Fluorescent Dye for Live‐Tracking of Lipophagy

Engelhardt,

Veronese,

Eryiğit

et al. 2024

Chemistry A European J

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Lipid droplet (LD) degradation provides metabolic energy and important building blocks for various cellular processes. The two major LD degradation pathways include autophagy (lipophagy), which involves delivery of LDs to autolysosomes, and lipolysis, which is mediated by lipases. While abnormalities in LD degradation are associated with various pathological disorders, our understanding of lipophagy is still rudimentary. In this study, we describe the development of a lipophilic dye containing two fluorophores, one of which is pH‐sensitive and the other pH‐stable. We further demonstrate that this “Lipo‐Fluddy” can be used to visualize and quantify lipophagy in living cells, in an easily applicable and protein label‐free approach. After estimating the ability of compound candidates to penetrate LDs, we synthesized several BODIPY and (pH‐switchable) rhodol dyes, whose fluorescence properties (incl. their photophysical compatibility) were analyzed. Of three Lipo‐Fluddy dyes synthesized, one exhibited the desired properties and allowed observation of lipophagy by fluorescence microscopy. Also, this dye proved to be non‐toxic and suitable for the examination of various cell lines. A method was developed to quantify the lipophagy process using flow cytometry, which could be applied in the future in the identification of lipophagy‐related genes or in the screening of potential drugs against lipophagy‐related diseases.

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RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal

Cited by 20 publications

References 110 publications

Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets

Sar1 Affects the Localization of Perilipin 2 to Lipid Droplets

Membrane extraction by calmodulin underpins the disparate signalling of RalA and RalB

A pH‐Sensitive Double Chromophore Fluorescent Dye for Live‐Tracking of Lipophagy

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