University of California, Berkeley
A Genetic Strategy to Identify Lipid Droplet Protein Degradation Pathways
Melissa Roberts*1; Dara Leto2; Julian Stevenson1; Kartoosh Heydari1; Lawrence Bacudio1; Ron Kopito2; Michael Bassik2 and James Olzmann1, (1)University of California, Berkeley, (2)Stanford University
Nearly all cells store lipids in endoplasmic reticulum (ER)-derived organelles called lipid droplets (LDs), which consist of a hydrophobic core of lipids encircled by a phospholipid monolayer with a unique proteome of regulatory proteins and enzymes. Although LD-resident proteins have essential roles in maintaining cellular lipid homeostasis, their regulation – in particular, the degradative pathways that control their abundance is poorly understood. Previous studies report that LD proteins are degraded by the ubiquitin-proteasome system; however, the speciﬁc machinery required (e.g. E3 ligases and E2 conjugating enzymes) remains unclear. To address this question, we performed a genome-wide, ﬂuorescence-based CRISPR/Cas9 screen to identify the degradation pathway of the LD protein perilipin-2 (PLIN2) in human hepatoma cells. We genomically tagged PLIN2 with GFP and conﬁrmed that PLIN2-GFP localizes to LDs and undergoes proteasomal degradation. We then pharmacologically induced lipolytic clearance of LDs to promote PLIN2-GFP degradation and screened reporter cells with a whole-genome sgRNA library. Cells with PLIN2-GFP degradation defects were isolated by ﬂuorescence-activated cell sorting and deep sequencing was used to identify genes required for PLIN2 degradation. Our preliminary data identiﬁed the E2 conjugating enzyme Ube2j2 and E3 ligase MARCH6, which have been implicated in the degradation of proteins from the early secretory pathway via a process known as ER-associated degradation. This suggests an intriguing model in which ER protein quality control machinery regulates LD protein abundance and lipid homeostasis. Future studies will elucidate how aberrations in LD protein degradation contribute to dysfunctions in cellular lipid metabolism and metabolic disease pathogenesis.
Honolulu, October 31 - November 2, 2019
- Roberts, MA, Olzmann, JA. Protein Quality Control and Lipid Droplet Metabolism. Annu Rev Cell Dev Biol 2020; 36 : 115-139. PubMed PMID:33021827 PubMed Central PMC7593838.
- Roberts, MA, Segura-Roman, A, Olzmann, JA. Organelle Biogenesis: ER Shape Influences Lipid Droplet Nucleation. Curr Biol 2020; 30 (13): R770-R773. PubMed PMID:32634419 .
- Bersuker, K, Hendricks, JM, Li, Z, Magtanong, L, Ford, B, Tang, PH, Roberts, MA, Tong, B, Maimone, TJ, Zoncu, R et al.. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 2019; 575 (7784): 688-692. PubMed PMID:31634900 PubMed Central PMC6883167.
- To, M, Peterson, CW, Roberts, MA, Counihan, JL, Wu, TT, Forster, MS, Nomura, DK, Olzmann, JA. Lipid disequilibrium disrupts ER proteostasis by impairing ERAD substrate glycan trimming and dislocation. Mol Biol Cell 2017; 28 (2): 270-284. PubMed PMID:27881664 PubMed Central PMC5231896.