Developmental Cell
Volume 44, Issue 1, 8 January 2018, Pages 97-112.e7
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A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes

https://doi.org/10.1016/j.devcel.2017.11.020Get rights and content
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Highlights

  • Selective enzymatic tagging of lipid droplet proteins using APEX2 fusion proteins

  • Mapping of high-confidence lipid droplet proteomes in two human cell lines

  • Inhibition of the AAA ATPase VCP stabilizes c18orf32, an ER-lipid droplet protein

  • C18orf32 levels on lipid droplets are regulated by a gp78 and derlin-1 ERAD pathway

Summary

Lipid droplet (LD) functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to two different cell types identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins. Moreover, quantitative analysis of LD proteome dynamics uncovered a role for endoplasmic reticulum-associated degradation in controlling the composition of the LD proteome. These data provide an important resource for future LD studies and demonstrate the utility of proximity labeling to study the regulation of LD proteomes.

Keywords

lipid droplet
proximity labeling
proteome
endoplasmic reticulum
ERAD
ubiquitin
proteasome
APEX
APEX2
biotinylation

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