Expression of oleosin and perilipins in yeast promote formation of lipid droplets from the endoplasmatic reticulum

Abstract

SummaryMost cells store neutral lipids in a dedicated compartment, the lipid droplet (LD). These LDs are structurally and functionally conserved across species. In higher eukaryotes, LDs are covered by abundant scaffolding proteins, such as the oleosins in plants and perilipins (PLINs) in animal cells. Saccharomyces cerevisiae, however, has no homologues of these scaffolding proteins. To analyze a possible function of these proteins in the biogenesis of LDs, oleosin and perilipin family members (PLIN1, ADRP/PLIN2 and TIP47/PLIN3) were expressed in yeast cells and their targeting to LDs, membrane association and function in neutral lipid homeostasis and LD biogenesis were analyzed. When expressed in wild-type cells, these proteins were properly targeted to LDs. However, when expressed in cells lacking LDs, oleosin was localized to the ER bilayer and was rapidly degraded. PLINs, on the other hand, did not localize to the ER membrane in the absence of LDs and lost their membrane association. Photobleaching experiments revealed that PLIN2 and PLIN3 rapidly exchanged their LD association, but PLINs did not move as quickly as integral membrane proteins, such as oleosin, over the LD surface. Interestingly, expression of these scaffolding LD proteins in mutant cells containing elevated levels of neutral lipids within the ER bilayer resulted in the formation of LDs. These results suggest that these LD scaffolding proteins promote the sequestration of neutral lipids from the ER bilayer and thereby induce LD formation. Consistent with this proposition, addition of a cell-permeable diacylglycerol (DAG) was sufficient to promote LD formation in cells expressing the LD scaffolding proteins but lacking the capacity to synthesize storage lipids.

Mature lipid droplets are accessible to ER luminal proteins

“…3E). We have previously shown that the cytosolic PLINs are greatly enriched on isolated lipid droplets when expressed in yeast (Jacquier et al, 2013). A similar result was found for the signal-sequence-containing PLIN1-PLIN3 proteins (Fig.…”

“…Expression of PLINs in cells lacking neutral lipids, profoundly affected their subcellular localization. Instead of showing a punctate lipid droplet localization as observed in wild-type cells, the cytosolic PLINs showed a diffuse cytosolic distribution (Jacquier et al, 2013) (Fig. 2B).…”

“…PLINs constitute a family of related proteins, sharing a common PAT domain, originally identified in the founding members of that family, PLIN1, PLIN2 (also known as adipophilin, ADRP) and PLIN3 (also known as TIP47) (Bickel et al, 2009;Miura et al, 2002). PLINs are properly targeted to lipid droplets when expressed in yeast, and they are soluble proteins in cells that lack the capacity to form lipid droplets (Jacquier et al, 2013). When fused to the signal sequence of Pry1, all three PLINs localized to Nile-Redpositive punctate structures, indicating that even soluble proteins when targeted into the ER lumen localize to lipid droplets ( Fig.…”

“…Cloning and expression of PLINs in yeast was performed as previously described (Jacquier et al, 2013). For mammalian expression, we used pLENTI6-mCherry-Plin2 (Eyre et al, 2014) and GFP-PLIN2 and mss-GFP-PLIN2 that were cloned into pSiCoR-GFP-EF1α (Jacob et al, 2014).…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…There is even less known about how this signal (singular or plural) is/are specifically recognized at the droplet, and whether this recognition has its origin in a classical receptor/docking complex or is a product of the physicochemical nature of the droplet phospholipid monolayer and underlying neutral lipid core. Whatever the rules, this process is conserved: mammalian and plant droplet proteins can target efficiently in yeast and even induce droplet formation (Jacquier et al, 2013). There is evidence supporting the role of phospholipid density and surface tension on droplets controlling trafficking of proteins (Thiam et al, 2013b).…”