PE17 Associates with Lipid Droplets via its C-terminal domain
Lipid droplets (LDs) are monolayered organelles involved in nutrient storage, trafficking, and toxicity prevention. Recent investigations into the interaction between host lipid droplets and infection with Mtb highlight the importance of these for the intracellular survival of the bacterium (Lovewell et al. , 2016). Lipid droplets can translocate to the bacteria-laden vacuole after infection, possibly serving as an important nutrient source (Daniel et al. , 2011). Due to the circular shape of lipid droplets, we wondered if the PE17-positive clusters we observed might be localizing to the LD surface. Post-transfection with PE17-myc, A549 cells were labelled with anti-myc antibodies to visualize PE17 and BODIPY 493/503, which stains neutral lipids (lipid droplets). The large spherical structures observed in the light micrographs were positive for both BODIPY and PE17 (Figure 5A), suggesting these large accumulations contain neutral lipids and are therefore likely lipid droplets. On closer inspection, the staining for PE17 forms a ring surrounding the LDs, with the central portion of the structure staining for a neutral lipid core (Figure 5B).
Lipid droplets have a unique membrane structure in comparison to other organelles, consisting of a phospholipid monolayer surrounding a core of neutral lipids, comprised primarily of steryl esters and triacylglycerol. Proteins can associate with the cytosolic face of the lipid droplet but are not found in the inner core (Olzmann & Carvalho, 2019). To determine which portion of PE17 is important for LD association, a truncated form of PE17 was generated lacking the C-terminal 77 amino acids, PE171-233-Myc (Figure 5C). The PE171-233 truncation did not localize to or cause redistribution of LDs (Figure 5D), rather it was distributed throughout the cell. In addition, it did not elicit the changes in Golgi stack morphology compared to the full-length protein (Figure 5E) but did result in alterations of mitochondrial morphology (Figure 5F). These findings indicate that the C-terminus of PE17 is not only important for localization to LDs, but also for its effects on organelle morphology and distribution.
In addition to perturbing organelle integrity, PE17-myc-his expression appeared to diminish cell size in most of our micrographs. To confirm this finding, we found that A549 cells expressing PE17-myc-his were indeed smaller, overall, than a control strain (Figure 6A). Therefore, we investigated whether PE17 was also inducing cell death upon expression. As our protocol is based on transient transfections making analysis of cell death in the population more difficult, we utilized a yeast-based system to address this question. This approach is relevant due to the general conservation of lipid droplet synthesis pathways across eukaryotes. Upon high-level expression of PE17 driven by a galactose-inducible promoter (using β-estradiol for induction), we noted strong inhibition of growth when compared to the empty vector control (Figure 6B). Therefore, ectopic expression of PE17 in eukaryotic cells appears to be inhibitory to cellular viability.
In order to localize PE17 in yeast cells and compare this localization to what we previously observed in A549 cells, we fused the fluorescent protein, mRuby2, to the C-terminus of PE17 in the same galactose-inducible vector; high level expression of this construct inhibited yeast cell growth to similar levels as the untagged protein (Figure 6C) and full-length expression of PE17-mRuby2 in yeast was observed via immunoblot (Figure 6D). We then moved this construct into yeast strains harboring a plasmid constitutively expressing GFP-Erg6, the yeast ∆24-sterol methyltransferase known to strongly associate with the surface of lipid droplets (Leber et al. , 1994). GFP-Erg6 was observed to localize to punctate structures defining lipid droplets, as expected (Figure 6E). PE17-mRuby also localized to punctate structures which strongly overlapped with GFP-Erg6 localization (Figure 6E), suggesting that PE17-mRuby also bound lipid droplets in yeast.
Yeast strains lacking the two sterol acetyltransferases, Are1p and Are2p, and two enzymes with diacylglycerol O-acyltransferase activity, Lro1p and Dga1p, are unable to accumulate lipid droplets (Joshi et al. , 2018); expression of only a single one of these genes allows the formation of lipid droplets in vivo (Jacquier et al. , 2011). As lipid droplets are synthesized at the ER, lipid droplet-binding proteins are known to redistribute to the ER in the absence of free lipid droplets. Therefore, to confirm that PE17 is specifically recruited to lipid droplets, we expressed GFP-Erg6 and the galactose-inducible PE17-mRuby in either the are1∆ are2∆ lro1∆ dga1∆ (3∆dga1∆ ) background, or an isogenic genetic background that contains a galactose-inducible DGA1 gene (3∆ GAL-DGA1 ) (Jacquier et al. , 2011). Under glucose growth conditions for both yeast strains, the constitutively-expressed GFP-Erg6 protein accumulates on both the perinuclear and cortical ER instead of punctate lipid droplets (Figure 6F), as expected. After induction of PE17-mRuby under galactose conditions, PE17-mRuby localizes to cortical membrane regions in the 3∆ dga1∆ strain, similar to the localization of GFP-Erg6 (Figure 6F). In contrast, however, the 3∆ GAL-DGA1strain under galactose conditions now begins to accumulate cytosolic lipid droplets marked by GFP-Erg6, which also recruit PE17-mRuby (Figure 6F). Finally, we isolated membrane fractions highly enriched for lipid droplets from strains expressing GFP-Erg6 and either a vector control or PE17-mRuby. Direct observation of these lipid droplets shows distinct labeling with GFP-Erg6, as anticipated (Figure 6G). Lipid droplets isolated from the PE17-mRuby strains show near-perfect colocalization of GFP-Erg6 and PE17-mRuby on the lipid droplet surface membrane (Figure 6G). Taken together, the localization patterns of PE17-mRuby strongly support the hypothesis that PE17 specifically binds free lipid droplets in both A549 and yeast cells, and likely the lipid droplets contained within host cells during an infection.