Jack of all trades – the lipid droplet organization (LDO) proteins are multifunctional organelle surface receptors

1 Introduction: organelle surfaces as communication platforms

Eukaryotic cells contain a set of organelles, spatially defined structures that offer cellular sub-compartments with distinct biochemical features. Every organelle maintains a certain degree of independence within its cellular environment. Most organelles are composed of a central aqueous compartment that is delimited from the surrounding cytosol by an outer phospholipid bilayer membrane, but there are exceptions to this basic organelle construction plan. Some organelles, most notably mitochondria and chloroplasts, have more than one membrane. Lipid droplets on the other hand, the cellular lipid storage organelles, are delimited by a phospholipid monolayer instead of a bilayer membrane, which shields a central hydrophobic compartment composed of neutral storage lipids. And membrane-less organelles maintain their structure without the help of phospholipids, via liquid-liquid phase separation. These different modes of organelle organization all have in common that they create spatial organelle identity and enable the maintenance of a unique molecular composition that in turn defines the organelle’s functional profile. Organelle-based compartmentalization thus broadens the spectrum of cellular functions and promotes physiological complexity through division of labor.

For a cell to work in coordination, structural organelle delimitation must be finely balanced with processes that ensure a defined level of organelle communication with the rest of the cell. Organelle surfaces therefore act as communication platforms that coordinate the exchange of material and information with the cytosol and with neighboring organelles, the spatial organization toward other cellular structures, signaling processes, and the integration of organelle functions with adaptations of the physiological cell state. These communication processes depend on the recruitment of numerous functional proteins to the outer surfaces of each type of organelle.

For some organelles, special organelle surface proteins have been identified that are involved in not only one, but multiple, distinct processes related to the interplay of the organelle with its environment. These proteins all have in common that they mediate the recruitment of different effector proteins to the respective organelle surface. In this review, we will refer to this functional class of proteins with the term multifunctional organelle surface receptors.

A prime example is Vac8, a resident of the vacuole membrane (lysosome-like organelle in yeast) that coordinates diverse vacuole functions. Vac8 contains an armadillo repeat domain that acts as interaction interface for multiple partner proteins involved in actomyosin-based vacuole motility and inheritance, vacuole fusion, autophagy, and the formation of vacuole contact sites to the nuclear ER and to lipid droplets (Hönscher and Ungermann 2014; Diep et al. 2024; Álvarez-Guerra et al. 2024; Rao et al. 2025).

On the mitochondrial surface, Tom70 proteins (Tom70 and Tom71 in yeast; TOM70 in human) may serve a similar role. These proteins are subunits of the translocase of the outer membrane TOM, and act as receptors for incoming precursor proteins destined for import into mitochondria. They consist of an outer mitochondrial membrane anchor and a tetratricopeptide repeat domain that recognizes chaperone-bound mitochondrial precursors. Besides their roles in protein translocation, Tom70 proteins recruit mitochondrial morphology factors, act as mediators of antiviral signaling, interact with multiple partner proteins for formation of mitochondria-ER contact sites and are involved in the physical interplay between mitochondria and parasite vacuoles during Toxoplasma infection (Kreimendahl and Rassow 2020; Li et al. 2022).

The ER membrane houses members of the vesicle associated membrane protein (VAMP) associated protein (VAP) family. VAP proteins are evolutionary conserved, comprising for example Scs2 and Scs22 in yeast and VAPA, VAPB and the VAP-like MOSPD proteins in human. VAP family proteins are characterized by a major sperm protein domain and an ER membrane anchor. The major sperm protein domain recruits proteins containing an FFAT (two phenylalanines in an acidic tract) motif or an FFAT-like motif to the ER surface. Dozens of VAP interactors have been identified, a large fraction of which are involved in formation and function of contact sites between the ER and the plasma membrane, mitochondria, and endosomes (Murphy and Levine 2016). Additionally, VAP interactors fulfill roles related to ER morphology, nuclear pore complexes, and organization of the cytoskeleton.

Lipid droplets are organelles involved in lipid storage and lipid metabolism. In this review, we will highlight recent studies on the formation and function of lipid droplet contact sites to the ER and to the vacuole, and on the process of actomyosin-based lipid droplet motility. Collectively, these studies suggest that the lipid droplet organization proteins Ldo16 and Ldo45 act as multifunctional surface receptors for lipid droplets, which coordinate the different stages of the lipid droplet life cycle (Figure 1A–E).

Figure 1:

The yeast lipid droplet organization proteins Ldo16 and Ldo45 are multifunctional lipid droplet surface receptors that coordinate lipid droplet biogenesis, degradation and motility. (A) Ldo16 and Ldo45 are encoded by overlapping genes (left). Both LDO proteins comprise a hydrophobic lipid droplet binding domain and a C-terminal cytosolic domain. The longer Ldo45 protein has a second cytosolic domain at the N-terminus (right). (B) Three distinct functions of the LDO proteins have been described to date. They act as components of the seipin lipid droplet biogenesis machinery; as tethers at lipid droplet-vacuole contact sites; and as lipid droplet motility proteins. (C) Ldo45 is an integral component of the seipin complex. This complex comprises an outer oligomeric ring composed of 10 Sei1 monomers. Ldb16 has important roles in mediating neutral lipid nucleation for lipid droplet biogenesis within the seipin ring, and in coordinating positioning of Ldo45, likely in the center of the ring complex. Ldo16 may be more peripherally associated. (D) Ldo16 and Ldo45 are key players in the vacuole lipid droplet (vCLIP) contact site. Together with the vacuole surface protein Vac8, they act as organelle tethers. Ldo45 additionally recruits the phosphatidylinositol transfer protein Pdr16 to the contact site. In stationary growth phase, Ldo16/45-based lipid droplet tethering to the vacuole is a prerequisite for efficient lipid droplet uptake into the vacuole lumen for lipid droplet degradation in a microautophagy process termed lipophagy. (E) Ldo16 acts as lipid droplet receptor for Ldm1, a myosin adaptor protein that binds to the cargo binding domain of the type V myosin Myo2. Lipid droplets coupled to Myo2 are motile in an actin-dependent manner.

2 The lipid droplet organization (LDO) proteins and seipin: lipid droplet-ER interplay

Lipid droplets uniquely enable the cell to store variable amounts of lipid molecules, thereby acting as the cell’s lipid buffering system. This lipid droplet function is important for cells to cope with limited nutrient availability and to prevent the accumulation of toxic lipid species. Lipid droplets store esterified, neutral lipid variants, predominantly triglycerides and sterol esters. These storage lipids are initially synthesized by enzymes located in the ER membrane and released into the hydrophobic environment of the ER phospholipid bilayer where they remain dispersed at low concentrations. Above a critical concentration, neutral lipids phase-separate and form lenses between the ER bilayer leaflets. These structures grow by acquiring additional neutral lipid molecules and bud toward the cytoplasmic face of the ER membrane, ultimately resulting in formation of a mature lipid droplet (Klemm and Carvalho 2024; Walther et al. 2023).

The biophysical process of lipid droplet biogenesis is modulated by proteins. The conserved lipid droplet biogenesis factor seipin is a central player in this process (Fei et al. 2008; Szymanski et al. 2007). Seipin safeguards lipid droplets at different stages of their life cycle. The protein fulfills important roles in the early stages of lipid droplet formation (Klemm and Carvalho 2024; Walther et al. 2023) but also remains positioned at lipid droplet-ER contact sites upon lipid droplet maturation, supporting maintenance of functional lipid droplets (Salo et al. 2019; Grippa et al. 2015). On an organismal level, seipin mutations cause a severe congenital form of the lipid storage disease lipodystrophy (Magré et al. 2001). On a cellular level and across species, loss of seipin function results in lipid droplet defects, presenting with striking alterations in lipid droplet morphology, size, number, and surface proteome (Szymanski et al. 2007; Fei et al. 2008; Wang et al. 2014; Grippa et al. 2015; Salo et al. 2016; Wang et al. 2016; Wolinski et al. 2015). Structural studies have in the past years shaped our understanding of the molecular role of seipin. Cryo-electron microscopy has revealed that seipin forms ring-shaped oligomeric complexes in human, fly and yeast cells, pointing toward a common mechanism for seipin function (Sui et al. 2018; Yan et al. 2018; Klug et al. 2021; Arlt et al. 2022; Li et al. 2024). Single seipin proteins comprise two transmembrane domains embedded in the ER membrane, and an ER luminal β-sandwich-like domain. The luminal domain has similarities to known lipid binding domains, and indeed binds to anionic phospholipids in vitro (Yan et al. 2018). In the oligomeric seipin complex, the luminal domains of the monomers interact with each other, forming a ring structure comprising 10 (yeast), 11 (human) or 12 (fly) subunits. Mutational studies and molecular dynamics simulations have revealed that seipin ring complexes act as catalyzers for neutral lipid nucleation even below the critical concentration for spontaneous phase separation. Human and fly seipin both comprise a hydrophobic α-helix located close to the center of the oligomeric β-sandwich ring (Sui et al. 2018; Yan et al. 2018). This helix binds to LDs in vitro and in vivo (Sui et al. 2018) and is important for mediating concentration of triglycerides and sterol esters in the center of seipin rings according to simulations (Prasanna et al. 2021; Zoni et al. 2021; Renne et al. 2022). The hydroxyl groups of serine residues in this seipin domain are involved in triglyceride binding (Zoni et al. 2021). Yeast has two non-redundant seipin proteins, Sei1 and Ldb16 (Wang et al. 2014; Grippa et al. 2015). sei1Δ and ldb16Δ mutants have similar phenotypes, and can both be rescued by expression of human seipin, indicating that the two yeast proteins, together, fulfill the molecular role of seipin (Wang et al. 2014; Renne et al. 2022). Oligomeric Sei1 rings have been structurally resolved, which however lack the characteristic luminal hydrophobic helices described as central factors for neutral lipid nucleation in human and fly seipin complexes (Arlt et al. 2022; Klug et al. 2021). Instead, Ldb16 is positioned within the Sei1 ring, which in turn mediates triglyceride concentration via a hydroxyl-rich α-helix (Klug et al. 2021). Both in yeast and human seipin complexes, the seipin transmembrane segments contribute to triglyceride concentration (Arlt et al. 2022; Klug et al. 2021; Kim et al. 2022).

In 2018, two additional partner proteins of the yeast Sei1/Ldb16 complex were identified by proteomic and genetic approaches (Eisenberg-Bord et al. 2018; Teixeira et al. 2018) (Figure 1A–C). These seipin partners were termed lipid droplet organization proteins of 16 (Ldo16) and 45 (Ldo45) kilodalton. The two LDO proteins are structurally closely related as they are derived from overlapping genes (Figure 1A, left). Ldo45 comprises a central hydrophobic region suitable for binding to lipid droplets or membranes, which is flanked by two soluble domains. The amino acid sequence of the shorter Ldo16 variant is identical to the C-terminal third of Ldo45. Ldo16 therefore comprises the same C-terminal soluble domain as Ldo45 and the majority of the hydrophobic domain but lacks the N-terminal soluble domain as well as the most N-terminal amino acids of the hydrophobic region (Figure 1A, right) (Eisenberg-Bord et al. 2018; Teixeira et al. 2018). Protein abundance of the two LDO variants responds differentially to metabolic alterations (Teixeira et al. 2018; Diep et al. 2024; Álvarez-Guerra et al. 2024). While it has not been ultimately clarified how LDO protein levels are determined, regulation on the transcriptional level appears to play a role. The two genes are controlled by distinct promoters, with part of the LDO16 promoter serving at the same time as LDO45 coding region (Figure 1A, left) (Miura et al. 2006; Eisenberg-Bord et al. 2018; Teixeira et al. 2018). It has been found that a characteristic drop in Ldo45 protein levels typically observed upon entry into stationary growth phase can be blocked by replacing the native LDO45 promoter (Teixeira et al. 2018).

The functional roles of the Ldo16/45 proteins with respect to lipid droplet biogenesis are currently being explored. Sei1, Ldb16, Ldo16 and Ldo45 co-precipitate with each other in affinity chromatography experiments, suggesting that they form a protein complex (Eisenberg-Bord et al. 2018; Teixeira et al. 2018; Klug et al. 2021). This finding was corroborated by proximity ligation and yeast two hybrid experiments (Wang et al. 2024). Experimental manipulation of LDO protein levels results in altered lipid droplet size and surface proteome, however, the defects are overall milder than those observed in the absence of Sei1 or Ldb16. It has therefore been suggested that Ldo16/45 might fulfill regulatory roles at the seipin complex (Eisenberg-Bord et al. 2018; Teixeira et al. 2018; Klug and Carvalho 2025).

LDAF1/promethin has been identified as a human homolog of Ldo45 (Castro et al. 2019; Eisenberg-Bord et al. 2018; Chung et al. 2019), and dmLDAF1 as a homolog in Drosophila melanogaster (Chartschenko et al. 2021). Similar to the yeast proteins, human LDAF1 and seipin also co-precipitate with each other (Castro et al. 2019; Chung et al. 2019). Time-resolved microscopy shows that seipin and LDAF1 co-localize at mobile foci in the ER membrane. These foci are the preferred biogenesis sites for newly forming lipid droplets (Chung et al. 2019). Loss of LDAF1 results in a lipid droplet biogenesis defect. Knockout of seipin leads to a concomitant loss of LDAF1, suggesting that the alterations reported for loss of seipin function could partially stem from a secondary LDAF1 defect (Chung et al. 2019). Reconstitution of seipin alone or together with LDAF1 in membranes containing triglycerides showed that seipin-LDAF1 complexes promote lipid lens formation more efficiently than seipin alone (Malia et al. 2025). Cryo-electron microscopy combined with structural predictions reveal an LDAF1 ring assembly located in the center of the outer seipin ring. Molecular dynamics simulations and mutational analyses suggest that this assembly is important for efficient nucleation of triglycerides, which coalesce within the toroid-shaped cavity between the concentric LDAF1- and seipin-rings (Malia et al. 2025). Intriguingly, the luminal hydrophobic seipin α-helix that is important for triglyceride nucleation (Zoni et al. 2021; Sui et al. 2018; Prasanna et al. 2021) also appears to have a central role in the physical interaction of seipin with LDAF1 (Chung et al. 2019; Malia et al. 2025). Moreover, the spatial arrangement of this seipin domain toward the ER membrane responds to the presence of LDAF1 (Malia et al. 2025). These findings point toward an intricate dynamic interplay between seipin, LDAF1 and neutral lipids.

In yeast, the structure of the Sei1/Ldb16/LDO holocomplex has not been resolved yet, but a detailed exploration of the interplay between its different subunits by in vivo site-directed photo-crosslinking provides insights into its architecture (Klug et al. 2021; Klug and Carvalho 2025). While no direct interaction of Sei1 with the Ldo16/45 proteins was detected, Ldb16 appears to have a central role in complex architecture, as crosslinks of this protein to both Sei1 and Ldo45 were detected. The same hydroxyl-rich Ldb16 region involved in triglyceride nucleation also appears tightly linked to Ldo45 (Klug et al. 2021; Renne et al. 2022; Klug and Carvalho 2025). This structural link between neutral lipid nucleation and LDO interplay is reminiscent of the metazoan seipin complex, where overlapping seipin regions mediate neutral lipid nucleation and LDAF1 binding (Chung et al. 2019; Malia et al. 2025; Sui et al. 2018; Prasanna et al. 2021; Zoni et al. 2021; Renne et al. 2022). Based on the Ldb16 crosslinking map, a model was suggested where Ldb16 anchors Ldo45 at the very center of the Sei1 ring complex (Figure 1C) (Klug and Carvalho 2025). Ldo16 is also part of the seipin complex according to affinity chromatography, proximity ligation, and yeast-two hybrid assays (Eisenberg-Bord et al. 2018; Teixeira et al. 2018; Wang et al. 2024; Klug and Carvalho 2025). Although Ldo16 is more abundant than Ldo45, crosslinks with Sei1 or Ldb16 were not detected. Interestingly, efficient Ldo16 co-precipitation with Ldb16 depends on the presence of the cytosolic N-terminal region of Ldb16, consistent with a more peripheral position of Ldo16 in the complex (Figure 1C) (Klug and Carvalho 2025). The exact individual functional implications of the two LDO variants in the lipid droplet biogenesis process remain to be determined.

In summary, the LDAF1/LDO proteins are tightly linked with the lipodystrophy factor seipin and are involved in the process of lipid droplet formation at the ER both in yeast and in metazoans.

3 The LDO proteins and Vac8: lipid droplet-vacuole interplay

The identification and subsequent characterization of Ldo16/45 and LDAF1 as seipin partners has been a key step in the development of our current molecular understanding of the lipid droplet formation process (Klemm and Carvalho 2024). However, already during the initial characterization of these proteins (Eisenberg-Bord et al. 2018; Teixeira et al. 2018), several LDO-related phenotypes were noted that are not easily explained by alterations in lipid droplet biogenesis. For instance, loss of the LDO proteins results in pronounced changes in a different organelle, the vacuole (yeast lysosome-like organelle), during stationary growth phase, when cells gradually run out of nutrients. ldo16/45Δ mutants display defects in lipophagy, a microautophagy process in which lipid droplets are normally internalized into the vacuole lumen during nutrient deprivation. Furthermore, ldo16/45Δ cells are impaired in formation of characteristic phase-separated vacuolar membrane domains that typically form in stationary phase (Teixeira et al. 2018). In logarithmic growth phase on the other hand, the Ldo16/45 proteins show a subcellular distribution that appears atypical for dedicated seipin partners: Ldo16/45 are strongly enriched on a subpopulation of lipid droplets at this condition, while other lipid droplets in the same cell carry little Ldo16/45 (Eisenberg-Bord et al. 2018). The Ldo16/45-enriched lipid droplets are not randomly distributed in the cell but are located adjacent to an organelle contact site between the nuclear ER and the vacuolar membrane, the nucleus vacuole junction (NVJ). Acute glucose starvation promotes accumulation of lipid droplets in this region of the cell, an effect that is blocked in ldo16/45Δ cells (Eisenberg-Bord et al. 2018). Finally, the longer LDO variant Ldo45 was found to be required for localization of the phosphatidylinositol transfer protein (PITP) Pdr16 to lipid droplets (Eisenberg-Bord et al. 2018; Teixeira et al. 2018), but how this PITP functionally relates to seipin was unclear.

In 2024, this conundrum found its resolution when a second, presumably seipin-independent function of Ldo16/45 at the vacuole membrane was uncovered. Two separate studies reported that the LDO proteins mediate formation of an organelle contact site between vacuoles and lipid droplets, termed vCLIP (Diep et al. 2024; Álvarez-Guerra et al. 2024) (Figure 1D). Mechanistically, Ldo16/45 act as tether proteins of this contact site. The C-terminal cytosolic domain that is present in both the longer and the shorter LDO variant has a central role in physical lipid droplet-vacuole tethering. Loss of this domain abolishes LDO-dependent vCLIP formation, while synthetic constructs composed of a generic lipid droplet anchor domain and the Ldo16/45 C-terminal domain induce vCLIPs. The vCLIP tether structure additionally comprises a vacuolar partner protein, Vac8. Physical organelle tethering is mediated by an interaction of an intrinsically disordered domain within the C-terminal soluble part of the LDO proteins with the armadillo repeat domain of Vac8. In contrast, Sei1 or Ldb16 are not required for vCLIP formation (Diep et al. 2024; Álvarez-Guerra et al. 2024).

Besides the two tether pairs Ldo16-Vac8 and Ldo45-Vac8, the PITP Pdr16 is a further vCLIP resident. Localization of Pdr16 to vCLIPs depends on an amphipathic α-helix in the N-terminal soluble domain of Ldo45 (Diep et al. 2024). The exact function of Pdr16 at the contact site is not clear yet, but multiple past studies have shown that the protein can act as a lipid transfer protein for sterols and phosphatidylinositol in vitro (Diep and Bohnert 2024), indicating that it may fulfill a similar role at vCLIP. The Ldo45 N-terminal region has therefore also been termed “PITP recruitment arm”, while the C-terminal domain present in both LDO variants has been termed “tether arm” (Diep et al. 2024).

The notion that one of the vCLIP tethers, Ldo45, can additionally recruit a putative lipid transfer protein to the contact site indicates that vCLIP comprises two functionally distinct tether modules. This is particularly interesting as protein abundance of the two LDO variants responds differentially to metabolic cues, suggesting that vCLIP tethering and PITP recruitment can be adjusted individually. vCLIPs are restricted to a small lipid droplet subpopulation at nutrient replete conditions (Diep et al. 2024) but expand in response to glucose restriction and when cells enter stationary growth phase (Diep et al. 2024; Álvarez-Guerra et al. 2024). However, protein levels of Ldo45 drop in stationary phase, suggesting that the expanded vCLIPs observed at this condition comprise little Pdr16 (Diep et al. 2024). Particularly excessive vCLIP formation has been observed in response to a nutrient stress protocol combining overall gradual nutrient deprivation with supplementation of lactate. In this condition, lipid droplets were observed simultaneously bound to the nuclear ER and, in a vCLIP-dependent manner, the vacuole, resulting in a unique organelle arrangement where cup-shaped vacuoles surrounded the majority of the nuclear surface, with tethered lipid droplets sandwiched between the two organelles (Qiu et al. 2025).

In late stationary phase, lipid droplets are ultimately internalized into the vacuole. This lipophagy process depends critically on vCLIP-mediated lipid droplet-vacuole tethering (Diep et al. 2024; Álvarez-Guerra et al. 2024; Teixeira et al. 2018). The C-terminal disordered LDO domain contains a phosphosite that is phosphorylated in logarithmic growth phase by cyclin dependent kinases, while it is gradually dephosphorylated when cells progress into stationary phase. Phosphomimetic LDO variants were found to display a lipophagy defect, while a non-phosphorylatable variant supported lipophagy efficiently, suggesting an implication of this posttranslational modification in the lipophagy process (Diep et al. 2024). Interestingly, a recent study suggests an additional layer of complexity in vCLIP biology. The lipid droplet protein Pln1 was identified as an additional vCLIP tether that is also required for lipophagy. ldo16/45Δ and pln1Δ mutations displayed differential effects on lipophagy, dependent on the specific type of underlying nutrient stress (Rao et al. 2025), suggesting a higher degree of metabolic regulation in this process.

In summary, the Ldo16/45 proteins fulfill an important role in lipid droplet-vacuole interplay via formation of vCLIP and in lipid droplet turnover via lipophagy, in addition to their link to the lipid droplet biogenesis machinery seipin.

4 The LDO proteins and Ldm1: lipid droplet motility

In 2025, the third and, so far, latest molecular role of the LDO machinery was uncovered in a systematic study aiming at characterizing the molecular machinery that mediates the process of lipid droplet motility (Zhao et al. 2025). Intracellular motility of lipid droplets has been observed in diverse cell types (Kilwein and Welte 2019). The molecular machineries underlying this dynamic behavior and the exact functional roles of lipid motility are less well defined. Functional implications in directional nutrient supply, clearance of toxic lipid species, regulation of lipid droplet formation or breakdown, and the re-organization of lipid droplet-organelle contact sites have been discussed (Kilwein and Welte 2019). In yeast cells, organelle motility has a special relevance as these cells undergo asymmetric cell divisions, necessitating motility-based delivery of organelles and material required for cell growth from mother to daughter cells. Organelle motility in yeast generally depends on the actin cytoskeleton, on type V myosin motor proteins, and on a group of myosin adaptor proteins that mediate specificity by coupling the myosin motors to the correct organelle surface. While lipid droplets were known to be motile in an actomyosin-dependent manner already for a decade (Knoblach and Rachubinski 2015), the molecular identity of the lipid droplet-specific motility factors was long enigmatic. This changed when a microscopy-based genome-wide screen identified the Lipid Droplet Motility protein 1 (Ldm1) as a lipid droplet-Myo2 adaptor (Zhao et al. 2025). A detailed characterization of Ldm1 revealed that this protein is in fact a bifunctional Myo2 adaptor, coupling Myo2 not only to lipid droplets, but also to mitochondria. Proteomic and genetic follow-up experiments then led to the identification of Ldo16 as a molecular player in Ldm1-dependent lipid droplet motility (Figure 1E). Ldo16 co-precipitates with Ldm1 in affinity chromatography assays, and loss of Ldo16 blocks Ldm1-driven motility of lipid droplets. LDO16 deletion enables a genetic separation of the Ldm1 effects on lipid droplets and mitochondria, as it abolishes lipid droplet motility selectively, while mitochondria remain unaffected. Together, these findings underpin a role of Ldo16 as lipid droplet surface receptor for Ldm1/Myo2 complexes. A structure-function analysis identified a minimal Ldo16 truncation variant sufficient for Ldm1 recruitment. This variant consists of the hydrophobic lipid droplet binding domain of the protein, and of approximately 20 adjacent amino acids that are exposed to the cytosol. Importantly, this minimal motility-competent LDO truncation variant lacks the intrinsically disordered LDO domain responsible for Vac8 binding, and cells expressing this variant display a vCLIP defect, showing that the functions of Ldo16 in vCLIP tethering and lipid droplet motility can be genetically separated (Figure 1D and E) (Zhao et al. 2025).

In summary, Ldo16 acts as receptor for the Myo2 adaptor protein Ldm1, enabling actomyosin-based motility of lipid droplets.

5 Outlook

Studying the lipid droplet organization/LDO proteins has turned out to be a rollercoaster ride through seemingly unrelated lipid droplet biology topics (Figure 1A–E). Ldo16/45 and LDAF1 have entered the lipid droplet arena with a bang, when they were initially identified as partner proteins of the heavily studied lipid droplet biogenesis protein seipin. Functional implications of the LDO proteins in the birth of new lipid droplets were indeed subsequently uncovered, but also several molecular roles unrelated to lipid droplet formation. The second identified LDO function as a vCLIP tether and lipophagy factor highlights a link of these proteins to the opposite end of the lipid droplet life cycle, the process of lipid droplet degradation. Finally, the third function of LDO described so far is related to actomyosin-based lipid droplet motility, by acting as a lipid droplet receptor for the myosin adaptor protein Ldm1, fulfilling a crucial role in motor protein coupling to the lipid droplet surface.

These findings, collectively, imply that the molecular role of the LDO proteins might not be adequately described based on their individual functions as lipid droplet biogenesis proteins, as vacuole tethers, or as lipid droplet motility factors alone. Instead, Ldo16/45 appear to act as multifunctional handles on the lipid droplet surface that are optimized to coordinate lipid droplet binding of different effectors of the lipid droplet life cycle.

Understanding the functional benefits of coupling these diverse molecular roles in one protein machinery will be an important task for the future. Of note, multifunctional surface receptors of other organelles have been described that pool similar functional profiles, most notably the vacuolar surface receptor Vac8, which is involved in multiple organelle contact sites and acts as a membrane receptor for Vac17, a vacuole-Myo2 adaptor protein (Hönscher and Ungermann 2014). It is indeed conceivable that organelle motility and contact site-based organelle confinement must typically be counter-regulated, which can be achieved by shared organelle receptor proteins. In the case of the lipid droplet, the LDO proteins may additionally coordinate contact with either its parent organelle, the ER, or the degradative vacuole, to avoid futile cycles of lipid droplet biogenesis and breakdown.

A further task for the future will be to uncover how the multifunctionality of Ldo16/45 is mediated on a molecular basis. Can the LDO proteins bind to multiple partners simultaneously? Or do they switch from one effector to another, and if so, which molecular features underly such a switch? To which extent do the LDO proteins travel back and forth between the phospholipid bilayer of the ER membrane, for cooperation with seipin, and the lipid droplet monolayer, and how is this transition mediated mechanistically?

Finally, while both metazoan LDAF1 and yeast Ldo16/45 cooperate with seipin in lipid droplet biogenesis, only the yeast proteins have so far been linked to lipophagy and lipid droplet motility. It will be important to understand to which extent the multifunctional character of the yeast LDO machinery is conserved to metazoan LDAF1.