Fluorescent Tools to Analyze Peroxisome–Endoplasmic Reticulum Interactions in Mammalian Cells

Bishop, Alexa; Kamoshita, Maki; Passmore, Josiah B.; Hacker, Christian; Schrader, Tina A.; Waterham, Hans R.; Costello, Joseph L.; Schrader, Michael

doi:10.1177/2515256419848641

Cited by 12 publications

(16 citation statements)

References 49 publications

(86 reference statements)

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“…4 C, mean ER contact; Costello et al, 2017b). We observed that expression of Myc-ACBD5 WT restored peroxisome-ER associations in ACBD5 KO cells to a level comparable to control HeLa cells (Bishop et al, 2019; Fig. 4, A-C).…”

Section: Korsmentioning

confidence: 64%

Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Kors

Hacker

Bolton

et al. 2022

Journal of Cell Biology

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Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.

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Section: Korsmentioning

confidence: 64%

Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Kors

Hacker

Bolton

et al. 2022

Journal of Cell Biology

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“…Previous studies have shown that split-fluorescent proteins are effective in visualizing inter-organelle-contact sites (Cieri et al, 2018;Kakimoto et al, 2018;Shai et al, 2018;Yang et al, 2018). However, a drawback of this method is the irreversible association of the split-fluorescent proteins, which induces artificial contact sites between different organelles and leads to an abnormality in the structure and function of the organelles (Figure 1F; Bishop et al, 2019). In this study, we found that the incorporation of the split-GFP genes into the yeast genome resulted in a decrease in the expression levels of the split-GFP proteins (Figure 4).…”

Section: Discussionmentioning

confidence: 97%

“…However, since split-GFP hardly dissociates once it gets associated, it has a disadvantage of inducing artificial inter-organellar contacts, especially when overexpressed. Indeed, a previous study reported that the split-GFP probes expressed on peroxisome and the ER could act as an artificial tether and increase their organelle contacts (Bishop et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells

Tashiro

Kakimoto

Shinmyo

et al. 2020

Front. Cell Dev. Biol.

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Inter-organelle contact sites have attracted a lot of attention as functionally specialized regions that mediate the exchange of metabolites, including lipids and ions, between distinct organelles. However, studies on inter-organelle contact sites are at an early stage and it remains enigmatic what directly mediates the organelle-organelle interactions and how the number and degree of the contacts are regulated. As a first step to answer these questions, we previously developed split-GFP probes that could visualize and quantify multiple inter-organelle contact sites in the yeast and human cultured cells. However, the split-GFP probes possessed a disadvantage of inducing artificial connections between two different organelle membranes, especially when overexpressed. In the present study, we developed a way to express the split-GFP probes whose expressions remained at low levels, with minimal variations between different yeast cells. Besides, we constructed a HeLa cell line in which the expression of the split-GFP probes could be induced by the addition of doxycycline to minimize the artificial effects. The improved split-GFP systems may be faithful tools to quantify organelle contact sites and screen new factors involved in organelle-organelle tethering in yeast and mammalian cells.

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“…4C, mean ER contact) (Costello et al, 2017b). We observed that expression of Myc-ACBD5 WT restored peroxisome-ER associations in ACBD5 KO cells to a level comparable to control HeLa cells (Bishop et al, 2019) (Fig. 4A-C).…”

Section: Phosphosites Within the Acbd5 Ffat-like Motif Influence Peroxisome-er Contactsmentioning

confidence: 60%

“…4A-C). We have recently shown that ACBD5 KO HeLa cells have reduced peroxisome-ER associations (Bishop et al, 2019). Restoration of peroxisome-ER contacts upon ACBD5 expression was quantified by determining the mean population of peroxisomes in close contact (<15 nm) with the ER (Fig.…”

Section: Phosphosites Within the Acbd5 Ffat-like Motif Influence Peroxisome-er Contactsmentioning

confidence: 99%

Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Kors

Hacker

Bolton

et al. 2021

Preprint

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Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 [acyl-coenzyme A-binding domain protein 5] and the ER-resident protein VAPB [vesicle-associated membrane protein-associated protein B]. ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like [two phenylalanines (FF) in an acidic tract] motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB and thus, peroxisome-ER contact sites differently. Moreover, we demonstrate that GSK3β [glycogen synthase kinase-3 beta] regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.SUMMARYKors et al. reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation and GSK3β in mammalian cells. Phosphorylation sites in the FFAT-like motif of ACBD5 affect the binding to VAPB and thus, peroxisome-ER contact sites, differently.

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Fluorescent Tools to Analyze Peroxisome–Endoplasmic Reticulum Interactions in Mammalian Cells

Cited by 12 publications

References 49 publications

Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells

Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

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