Next, we wondered whether the Wbp1-GFP-PLIN3, Sec61-GFP-PLIN3 and Pmt1-GFP-PLIN3 reporters can be targeted to mature pre-existing LDs. Therefore, we grew yeast cells expressing Erg6–mCherry in oleic acid-containing medium to induce large LDs, but repressed expression of the reporters by the presence of doxycycline. Cells were then switched to medium lacking doxycycline to induce expression of the reporter, and their localization was analyzed over time using fluorescence microscopy. Upon 2 h of induction, Wbp1–GFP–PLIN3 and Sec61–GFP–PLIN3 exhibited a crescent-like staining at the LD periphery (Fig. 5A). This crescent-shaped localization likely reflects the localization of the reporter at the ER–LD interface, as has previously been observed for Gat1, a yeast glycerol-3-phosphate acyltransferase, which localizes to crescent-like structures in the ER that are intimately associated with LDs (Marr et al., 2012). Similarly, the ER-luminal apolipoprotein B (ApoB) is localized to crescent-shaped areas adjacent to LDs in hepatoblastoma cells (Ohsaki et al., 2006). Upon overnight induction of the two membrane-anchored PLIN3 reporters, this crescent-like LD staining was superseded by a more uniform circular staining of the LD perimeter, as we previously observed in cells grown in the presence of oleic acid (Fig. 1D and Fig. 5A).

Induction of the reporter containing the ER-luminally oriented PLIN3, Pmt1–GFP–PLIN3, in contrast, did not result in its targeting to pre-existing LDs (Fig. 5A). Instead, Pmt1–GFP–PLIN3 appeared to localize to the cell periphery, probably at the cortical ER, and showed poor colocalization with Erg6–mCherry (5%). These results indicate that the targeting of these membrane-anchored reporters to pre-existing LDs differs depending on whether the LD-targeting signal provided by PLIN3 has a cytosolic or an ER-luminal orientation.

To analyze these differences in LD targeting in more detail, we examined the localization of the reporter constructs in FIT mutant cells. FIT proteins are required for the emergence of LDs towards the cytosol (Choudhary et al., 2015; Kadereit et al., 2008). The yeast genome encodes two FIT isoforms, SCS3 and YFT2. In the scs3Δ yft2Δ double mutant, Wbp1–GFP–PLIN3 and Sec61–GFP–PLIN3 initially labeled LDs only partially, in a crescent-like manner, before dispersing over the entire LD perimeter, following a similar time-dependence as observed in the wild-type background (Fig. 5A,B). Conversely, Pmt1–GFP–PLIN3 already localized to the circular perimeter of LDs after 2 h of induction of its expression, and it exhibited 81% colocalization with Erg6–mCherry in the FIT double mutant (Fig. 5B). These observations suggest that FIT proteins restrict the access of ER-luminal reporters to LDs. Thus, in the presence of functional FIT proteins, the membrane-anchored reporter containing an ER-luminal LD-targeting domain, Pmt1–GFP–PLIN3, failed to properly localize to pre-existing LDs. However, in the absence of FIT protein function, Pmt1–GFP–PLIN3 could distribute to the perimeter of pre-existing LDs. LD localization of Pmt1–GFP–PLIN3 was also observed in scs3Δ yft2Δ double-mutant cells cultivated without addition of oleic acid. Under these conditions, 80% colocalization between Pmt1–GFP–PLIN3 and Erg6–mCherry was observed in the double mutant (Fig. S2A). FIT-dependent access of Pmt1–GFP–PLIN3 to LDs was confirmed by isolating LDs from wild-type and scs3Δ yft2Δ double-mutant cells. Pmt1–GFP–PLIN3 was enriched on LDs from scs3Δ yft2Δ double-mutant cells, but not on those from wild-type cells (Fig. S2B). The function of FIT proteins in controlling access of Pmt1–GFP–PLIN3 to mature LDs is redundant, because the two single mutants, yft2Δ and scs3Δ, behaved as wild-type cells and prevented targeting of Pmt1–GFP–PLIN3 to pre-existing LDs (Fig. S2C,D). Although FIT function affected targeting of Pmt1–GFP–PLIN3 to the perimeter of pre-existing LDs, it did not affect the stability of the protein, as revealed by a cycloheximide chase western blot analysis (Fig. S2E). These observations support the hypothesis that FIT proteins affect the topology of LD budding (i.e., their emergence towards the cytosol or the ER lumen), as depicted in the models shown in Fig. 5C,D (Choudhary et al., 2015). Emergence of LDs towards the ER lumen would thus allow the reporter with the ER-luminal PLIN3 targeting domain, Pmt1–GFP–PLIN3, to gain access to pre-existing LDs.