The reticulon homology domain of Pex30 generates membrane curvature at ER subdomains for lipid droplet biogenesis
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Abstract
Lipid droplets (LDs) are dynamic organelles that store neutral lipids and form in the endoplasmic reticulum (ER) membrane. Formation of new LDs is a controlled process and requires proteins with specific functions to form and grow from the ER membrane without any defect. In vitro studies have suggested a role for membrane curvature in LD emergence from the ER. Here, we use the membrane-shaping protein Pex30 to investigate the impact of ER membrane curvature on LD biogenesis and morphology. We modified the reticulon homology domain (RHD) of Pex30, which is responsible for tubulating the ER membrane, by extending the short hairpin transmembrane domains (TMD). The Pex30 (TMD) mutants cannot tubulate the ER membrane and generate less local membrane curvature that WT Pex30. Additionally, these mutants are unable to restore delayed LD biogenesis observed in cells devoid of Pex30. Our results indicate that Pex30 RHD generates local membrane curvature at ER subdomains that drives formation of new LDs.
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Moreover, unlike the cells devoid of Pex30, cells expressing Pex30 TMD mutants exhibit a dominant negative effect on LD biogenesis as they show decreased LD formation at 5-hour timepoint
Do you expect these mutations are strong enough to create a dominant negative effect in a wild-type Pex30 background?
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To do this, we overexpressed Pex30-GFP, Pex30 (TMD1)-GFP, Pex30 (TMD2)-GFP and Pex30 (TMD1.TMD2)-GFP in are1are2dga1pex30Δ with LRO1 expressed under GAL1 promoter and labelled with Erg6-mCherry
I would love to see some of these images in figure 5.
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are1are2dga1pex30Δ
Could you introduce the significance of this background for this experiment?
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Our data indicates that the short hairpin TMDs are important for Pex30 localization and ER shaping function.
It would help to add some quantification here to show the extent of this phenotype
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(Figure 1D and 1E).
I am having trouble seeing the strength of the phenotype, especially for the TMD2 mutant. Could you add some arrows to highlight WT vs abnormal cortical ER morphology?
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Identifying proteins and lipids enriched at Pex30 subdomains would shed light on mechanistic details of LD biogenesis.
Great findings on the origins of LDs. Do you expect Pex30 levels to change depending on lipid availability/organismal stress? It would be interesting to see if it's upregulated in response to high PA levels
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