Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides

Abstract

c Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers. Lipid droplets (LDs), intracellular organelles with deposits of neutral lipids and involved in many cellular activities, are widely present in both eukaryotic and prokaryotic cells (1-4). These organelles consist of a neutral lipid core surrounded by a phospholipid monolayer and associated proteins (3, 5). It has been known that LDs serve as the energy reservoir of cells, which may increase the adaptation by mobilization and degradation of lipids during nutrient deprivation, and also connect with other cellular processes, including lipid transport, membrane biogenesis, lipotoxicity relief, protein storage and degradation, pathogenicity, and autophagy (6-9). Because the biology of LDs is closely linked to some diseases, such as obesity, type 2 diabetes, and atherosclerosis, great progress has been made in elucidating the cellular trafficking, dynamics, and biogenesis of LDs in mammalian cells (2, 7, 10). However, there have been few studies on LDs in other species, especially naturally lipid-producing microorganisms (11-13). Analysis of these microorganisms is motivated by the fact that microbial lipid production holds a great promise to convert waste materials, including lignocellulosic biomass, into fatty acid-derived fuel molecules and chemicals in a scenario of biorefinery and sustainable development (14, 15).The major components of LDs are neutral lipids, including triacylglycerols (TAGs), sterol esters, and ether lipids (16). Neutral lipids constitute more than 90% of LDs by weight, but the ratio of TAGs to ste...

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species

“…Proteomic studies showed that it is a highly abundant protein during lipid accumulation phase [5]. Likewise, the α and β subunits of fatty acid synthase (Fas2 and Fas1, respectively), fatty acid transporter (Fat1), ATP:citrate lyase (Acl1) and urea carboxylase/allophanate hydrolase (Dur1/2) were also found in high abundance during lipid accumulation phase [14, 15]. Other known abundant targets are the p erilipin, a dipophilin and t ail-interacting (PAT) family proteins, which serve as dynamic scaffolds regulating the formation, growth and degradation of lipid bodies [15–19].…”

The why, when and how of lipid droplet diversity

“…In terms of the lipid-dropletassociated proteins, a core set of about 50 proteins has been found to decorate lipid droplets in various cellular systems (reviewed in Hodges and Wu, 2010). However, a diverse and large number of additional proteins has been identified across many studies (a selection of studies is given in Beilstein et al, 2013;Beller et al, 2006;Cermelli et al, 2006;Chen et al, 2016;Currie et al, 2014;Dahlhoff et al, 2015;Ivashov et al, 2013;Khan et al, 2015;Khor et al, 2014;Krahmer et al, 2013;Li et al, 2016;Na et al, 2013;Schmidt et al, 2013;Su et al, 2014;Zhang et al, 2011;Zhu et al, 2015), suggesting a significant amount of variability in terms of the lipid droplet protein composition between lipid droplets isolated from different cell types.…”

Integrative computational approach for genome-based study of microbial lipid-degrading enzymes

“…More recently, systematic studies on the poorly characterized organelles in oleaginous microorganisms [termed lipid droplets (LDs) or lipid particles (LPs)], have been demonstrated using proteome analysis. Studies in the lipid-producing yeast, Rhodosporidium toruloides, indicated that the LD proteome consists of 226 proteins, 11 of which were annotated as lipid-degrading enzymes (Zhu et al 2015). From that study, the orthologue of the bifunctional enzyme, Tgl5 (RHTO_03931), which has triacylglycerol lipase and lysophosphatidic acid acyltransferase activities, were identified, suggesting that the enzyme could promote LD fusion to generate larger LDs (Fei et al 2011;Yang et al 2012).…”

“…From that study, the orthologue of the bifunctional enzyme, Tgl5 (RHTO_03931), which has triacylglycerol lipase and lysophosphatidic acid acyltransferase activities, were identified, suggesting that the enzyme could promote LD fusion to generate larger LDs (Fei et al 2011;Yang et al 2012). However, the orthologue of the mammalian hormone-sensitive lipase (RHTO_02359) was absent in the LD proteome, suggesting that it was not recruited at the lipid accumulation stage (Zhu et al 2015). In addition, proteomic approaches could reveal an enzyme candidate missing in the target pathway.…”