Identification of a Major Lipid Droplet Protein in a Marine DiatomPhaeodactylum tricornutum

Abstract

Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Expression levels of J43469, diacylglycerol acyltransferase 2 (DGAT2), which completes the final step in TAG biosynthesis, although previously reported as strongly upregulated in N-stress conditions (Mus et al, 2013;Levitan et al, 2015), were only slightly higher in NR-KO14 cells (18 h) relative to the wild type ( Figure 10F). At 18 h, expression of the gene (J48859), a homolog in P. tricornutum of the major lipid droplet protein (MLDP), was ;5 times more highly expressed in the NR-KO14 transcriptome compared with the wild type ( Figure 10G); MLDP has been reported in other microalgae to regulate lipid droplet size (Yoneda et al, 2016).…”

“…Expression was significantly different in the paired t tests for five of six genes shown at the 1-and 18-h time points (FDR < 6.7 3 10 24 ), but the sixth, DGAT2, was not. (G) A putative major lipid droplet protein (MLDP, J48859) has recently been identified (Yoneda et al, 2016). It regulates lipid droplet size.…”

Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa

“…The proteins that have been identified as LBPs in other algae so far can be grouped into highly abundant structural proteins [3, 10, 38, 40, 41, 66, 67] and enzymes that play a role in lipid metabolism or trafficking [38, 57]. Confirmation of the subcellular localization is essential in investigating the LB proteome and tobacco pollen tubes are a useful tool to do this [51].…”

“…Members of the MLDP family have been characterized in the microalgae Haematococcus pluvialis [10] and Dunaliella salina [3], while homologous genes can be found in the genomes of further members of the Volvocales and Chlorellales order [3]. The diatom Phaeodactylum tricornutum [40] and the heterokont microalga Nannochloropsis [41] each contain unique structural LB proteins, which, in contrast to MLDPs, contain prominent hydrophobic domains similar to oleosins.…”

Plant Lipid Droplets and Their Associated Proteins: Potential for Rapid Advances

“…Three LD structural proteins in different microalgae have 218 been identified (Table I). The first, LD Surface Protein, is present in Nanochloropsis (Vieler et 219 al., 2012), diatoms and several other heterokont algae (Yoneda et al, 2016). This is the only LD 220 protein other than oleosin in all organisms that has a long hydrophobic hairpin (~56 residues with conserved proline residues at the center) ( Table I).…”