Expression of perilipin 5 promotes lipid droplet formation in yeast

Abstract

Neutral lipids are packed into dedicated intracellular compartments termed lipid droplets (LDs). LDs are spherical structures delineated by an unusual lipid monolayer and they harbor a specific set of proteins, many of which function in lipid synthesis and lipid turnover. In mammals, LDs are covered by abundant scaffolding proteins, the perilipins (PLIN1-5). LDs in yeast are functionally similar to that of mammalian cells, but they lack the perilipins. We have previously shown that perilipins (PLIN1-3) are properly targeted to LDs when expressed in yeast and that they promote LD formation from the ER membrane enriched in neutral lipids. Here we address the question whether PLIN5 (OXPAT) has a similar function. Both human and murine PLIN5 were properly targeted to yeast LDs, but the protein localized to the cytosol and its steady-state level was reduced when expressed in yeast mutants lacking the capacity to synthesize storage lipids. When expressed in cells containing high levels of neutral lipids within the membrane of the endoplasmatic reticulum, PLIN5 promoted the formation of LDs. Interestingly, PLIN5 was properly targeted to LDs, irrespective of whether these LDs were filled with triacylglycerol or steryl esters, indicating that PLIN5 did not exhibit targeting specificity for a particular subtypes of LDs as was reported for mammalian cells.

Keywords: PLIN5 (OXPAT); Saccharomyces cerevisiae; endoplasmatic reticulum; lipid droplets; organelle biogenesis; perilipins; steryl esters; triacylglycerol.

Figure 1.

PLIN5 localizes to LDs when expressed in yeast cells. (A) Wild-type cells and cells lacking LDs (GAL-LRO1 are1Δ are2Δ dga1Δ) expressing either human or murine GFP-tagged PLIN5 were cultivated overnight in synthetic media (SC) media at 24°C. Cells were collected and stained with Nile Red and living cells were analyzed by confocal microscopy. Arrows in the merge indicate colocalization. Bar, 5 μm. (B) LD clustering in cells expressing PLIN5. LD morphology was analyzed by confocal microscopy in wild-type cells expressing either Erg6-GFP or GFP-PLIN5. The LDs in the boxed region are enlarged 2.5-fold in the panel to the right. Bar, 7 µm and 3 µm, respectively. (C) Total cell proteins from wild-type or cells lacking LDs were TCA precipitated and expression of GFP-tagged PLIN5 was analyzed by western blotting. Wbp1, a subunit of the oligosaccharyl transferase complex served as a loading control. (D) Cells were treated with cycloheximide (CHX, 50 µg/ml) and samples were removed at the indicated time points. Proteins were TCA precipitated, resolved by SDS-PAGE and probed with antibodies against GFP and against Pgk1, phosphoglycerate kinase, which served as a loading control.

Figure 2.

PLIN5 expression promotes formation of LDs in a sensitized strain. (A) PLIN5 expression results in induction of LDs in cells lacking Pah1. Cells of the indicated genotype were cultivated, stained with Nile Red and analyzed by confocal microscopy, as described in Figure 1. (B) PLIN5 colocalizes with Erg6. Pah1 mutant cells expressing human GFP-PLIN5 and Erg6-RFP were cultivated as described above and colocalization was assessed by imaging of living cells. Arrows in the merge indicate colocalization. Bar, 5 μm.

Figure 3.

PLIN5 localizes to LDs irrespective of whether they are filled with either TAG or STE. GFP-PLIN5 was expressed in are1Δ are2Δ or in dga1Δ lro1Δ double mutant strains, cells were stained with Nile Red and analyzed by confocal microscopy as described for Figure 1. Arrows in the merge indicate colocalization. Bar, 5 μm.

Figure 4.

PLIN5 co-localizes with the LD marker protein Erg6 on LDs filled with either TAG or STE. GFP-PLIN5 and Erg6-RFP were co-expressed in are1Δ are2Δ or in dga1Δ lro1Δ double mutant strains and cells were analyzed by confocal microscopy. Arrows in the merge indicate colocalization. Bar, 5 μm.