PAT family proteins pervade lipid droplet cores

The PAT family proteins, named after perilipin, adipophilin, and the tail-interacting protein of 47 kDa (TIP47), are implicated in intracellular lipid metabolism. They associate with lipid droplets, but how is completely unclear. From immunofluorescence studies, they are reported to be restricted to the outer membrane monolayer enveloping the lipid droplet and not to enter the core. Recently, we found another kind of lipid droplet-associated protein, caveolin-1, inside lipid droplets. Using freeze-fracture immunocytochemistry and electron microscopy, we now describe the distributions of perilipin and caveolin-1 and of adipophilin and TIP47 in lipid droplets of adipocytes and macrophages. All of these lipid droplet-associated proteins pervade the lipid droplet core and hence are not restricted to the droplet surface. Moreover, lipid droplets are surprisingly heterogeneous with respect to their complements and their distribution of lipid droplet-associated proteins. Whereas caveolin-1 is synthesized in the endoplasmic reticulum and is transferred to the lipid droplet core by inundating lipids during droplet budding, the PAT proteins, which are synthesized on free ribosomes in the cytoplasm, evidently target to the lipid droplet after it has formed. How the polar lipid droplet-associated proteins are accommodated among the essentially hydrophobic neutral lipids of the lipid droplet core remains to be determined.     

The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets

Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.     

Identification of the putative protein phosphatase gene PTC1 as a virulence-related gene using a silkworm model of Candida albicans infection

Protein phosphatases are critical for the regulation of many cellular processes. Null mutants of 21 putative protein phosphatases of Candida albicans were constructed by consecutive allele replacement using the URA3 and ARG4 marker genes. A simple silkworm model of C. albicans infection was used to screen the panel of mutants. Four null mutant (cmp1Δ, yvh1Δ, sit4Δ, and ptc1Δ) strains showed attenuated virulence in the silkworm model relative to that of control and parental strains. Three of the mutants, the cmp1Δ, yvh1Δ, and sit4Δ mutants, had previously been identified as affecting virulence in a conventional mouse model, indicating the validity of the silkworm model screen. Disruption of the putative protein phosphatase gene PTC1 of C. albicans, which has 52% identity to the Saccharomyces cerevisiae type 2C protein phosphatase PTC1, significantly reduced virulence in the silkworm model. The mutant was also avirulent in a mouse model of disseminated candidiasis. Reintroducing either of the C. albicans PTC1 alleles into the disruptant strain, using a cassette containing either allele under the control of a constitutive ACT1 promoter, restored virulence in both infection models. Characterization of ptc1Δ revealed other phenotypic traits, including reduced hyphal growth in vitro and in vivo, and reduced extracellular proteolytic activity. We conclude that PTC1 may contribute to pathogenicity in C. albicans.     

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.     

Ptc1 Protein Phosphatase 2C Contributes to Glucose Regulation of SNF1/AMP-activated Protein Kinase (AMPK) in Saccharomyces cerevisiae

The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr-210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1Δ and sit4Δ single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C also has a role in dephosphorylation of Snf1 Thr-210 in vivo. The sit4Δ ptc1Δ mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1Δ ptc1Δ mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr-210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1Δ sit4Δ mutant, indicates that although Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.     

Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets

Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.     

PAT proteins,an ancient family of lipid droplet proteins that regulate cellular lipid stores

The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3–12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms.     

Rab32 is important for autophagy and lipid storage in Drosophila

Lipids are essential components of all organisms. Within cells, lipids are mainly stored in a specific type of organelle, called the lipid droplet. The molecular mechanisms governing the dynamics of lipid droplets have been little explored. The protein composition of lipid droplets has been analyzed in numerous proteomic studies, and a large number of lipid droplet-associated proteins have been identified, including Rab small GTPases. Rab proteins are known to participate in many intracellular membranous events; however, their exact role in lipid droplets is largely unexplored. Here we systematically investigate the roles of Drosophila Rab family proteins in lipid storage in the larval adipose tissue, fat body. Rab32 and several other Rabs were found to affect the size of lipid droplets as well as lipid levels. Further studies showed that Rab32 and Rab32 GEF/Claret may be involved in autophagy, consequently affecting lipid storage. Loss-of-function mutants of several components in the autophagy pathway result in similar effects on lipid storage. These results highlight the potential functions of Rabs in regulating lipid metabolism.     

Characterization of the lipid droplet proteome of a clonal insulin-producing β-cell line (INS-1 832/13)

Lipids are known to play a crucial role both in the normal control of insulin release and in the deterioration of β-cell function, as observed in type 2 diabetes. Despite this established dual role of lipids, little is known about lipid storage and handling in β-cells. Here, we isolated lipid droplets from oleate-incubated INS-1 832/13 cells and characterized the lipid droplet proteome. In a total of four rounds of droplet isolation and proteomic analysis by HPLC-MS/MS, we identified 96 proteins that were specific to droplets. The proteins fall into six categories based on function or previously observed localization: metabolism, endoplasmic reticulum/ribosomes, mitochondria, vesicle formation and transport, signaling, and miscellaneous. The protein profile reinforces the emerging picture of the lipid droplet as an active and dynamic organelle involved in lipid homeostasis and intracellular trafficking. Proteins belonging to the category mitochondria were highly represented, suggesting that the β-cell mitochondria and lipid droplets form a metabolic unit of potential relevance for insulin secretion.     

Phosphatase-associated protein MoTip41 interacts with the phosphatase MoPpe1 to mediate crosstalk between TOR and cell wall integrity signalling during infection by the rice blast fungus Magnaporthe oryzae

The type 2A (PP2A) and type 2A-like (PP4 and PP6) serine/threonine phosphatases participate in a variety of cellular processes, such as cell cycle progression, signal transduction and apoptosis. Previously, we reported that the PP6 catalytic subunit MoPpe1, which interacts with and is suppressed by type 2A associated protein of 42 kDa (MoTap42), an essential protein involved in the target of rapamycin (TOR) signalling pathway, has important roles in development, virulence and activation of the cell wall integrity (CWI) pathway in the rice blast fungus Magnaporthe oryzae. Here, we show that Tap42-interacting protein 41 (MoTip41) mediates crosstalk between the TOR and CWI signalling pathways; and participates in the TOR pathway via interaction with MoPpe1, but not MoTap42. The deletion of MoTIP41 resulted in disruption of CWI signalling, autophagy, vegetative growth, appressorium function and plant infection, as well as increased sensitivity to rapamycin. Further investigation revealed that MoTip41 modulates activation of the CWI pathway in response to infection by interfering with the interaction between MoTap42 and MoPpe1. These findings enhance our understanding of how crosstalk between TOR and CWI signalling modulates the development and pathogenicity of M. oryzae.     

Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation

Lipid droplet is a cellular organelle with a neutral lipid core surrounded by a phospholipid monolayer and coated with structural as well as functional proteins. The determination of these proteins, especially their functional regulations and dynamic movement on and off droplets, holds a key to resolving the biological functions of the cellular organelle. To address this, we carried out a comprehensive proteomic study that includes a complete proteomic, a phosphoprotein proteomic, and a comparative proteomic analysis using purified lipid droplets and mass spectrometry techniques. The complete proteome identified 125 proteins of which 70 proteins had not been identified on droplets of mammalian cells previously. In phosphoprotein proteomic analysis, 7 functional lipid droplet proteins were determined to be phosphorylated, including adipose differentiation related protein (ADRP/ADFP), two Rab proteins, and four lipid metabolism enzymes, including adipose triglyceride lipase (ATGL). To understand the dynamics of lipid droplets, GTP-dependent protein recruitment was analyzed by comparative proteomics. Arf1 and some of its coatomers, three other Arfs, several other small G-proteins including 3 Rabs, and several lipid synthetic enzymes were recruited from cytosol to purified droplets. Together, the present study suggests that lipid droplet is an active and dynamic cellular organelle that governs lipid homeostasis and intracellular trafficking through protein phosphorylation as well as GTP-regulated protein translocation.     

Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.     

Immunocytochemical studies on lipid droplet-surface proteins in adrenal cells

Perilipin and ADRP, located on the surface of intracellular lipid droplets, are proposed to be involved in adipocyte lipid metabolism. The aim of the present study was to investigate the effect of PKA and PKC activities on the distribution of perilipin and ADRP in primary cultured adrenal cells, and the role of ERK in PMA- and calphostin C-induced steroidogenesis. Immunofluorescence staining indicated that in addition to p160, a capsular protein of steroidogenic lipid droplets, perilipin and ADRP were localized on the lipid droplet surface. Stimuli such as activation of PKA by db cAMP or inhibition of PKC by calphostin C, which increase corticosterone synthesis in various magnitudes, caused detachment of p160 and perilipin, but not ADRP, from the lipid droplet surface. Activation of PKC by PMA induced increase in corticosterone synthesis, however, it did not affect the distribution of perilipin, p160, or ADRP on the lipid droplet surface, suggesting the presence of mechanisms for promoting sterodiogensis other than causing detachment of lipid droplet surface proteins. We further demonstrated that ERK pathway was involved in PMA-induced steroidogenesis, since PD98059, specific inhibitor of MEK, blocked the increases in steroidogenesis and phosphorylation of ERK caused by PMA, but not by cAMP-PKA. These data indicate that p160, perilipin, and ADRP were all located on the lipid droplet surface in rat adrenal cells. On the basis of its non-responsiveness to lipolytic stimulation, ADRP may be a structural protein of the lipid droplet surface, whereas their immediate response to lipolytic stimuli suggest that perilipin and p160 are functional proteins. PKC regulates adrenal steroidogenesis through ERK cascade, whereas PKA pathway does not involve ERK.     

Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H₂O₂, ZnCl₂, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.     

Vacuole Fusion Regulated by Protein Phosphatase 2C in Fission Yeast

The gene ptc4+ encodes one of four type 2C protein phosphatases (PP2C) in the fission yeast Schizosaccharomyces pombe. Deletion of ptc4+ is not lethal; however, Deltaptc4 cells grow slowly in defined minimal medium and undergo premature growth arrest in response to nitrogen starvation. Interestingly, Deltaptc4 cells are unable to fuse vacuoles in response to hypotonic stress or nutrient starvation. Conversely, Ptc4 overexpression appears to induce vacuole fusion. These findings reveal a hitherto unrecognized function of type 2C protein phosphatases: regulation of vacuole fusion. Ptc4 localizes in vacuole membranes, which suggests that Ptc4 regulates vacuole fusion by dephosphorylation of one or more proteins in the vacuole membrane. Vacuole function is required for the process of autophagy that is induced by nutrient starvation; thus, the vacuole defect of Deltaptc4 cells might explain why these cells undergo premature growth arrest in response to nitrogen starvation.     

Lipid droplets throughout the evolutionary tree

Intracellular lipid droplets are utilized for lipid storage and metabolism in organisms as evolutionarily diverse as animals, fungi, plants, bacteria, and archaea. These lipid droplets demonstrate great diversity in biological functions and protein and lipid compositions, yet fundamentally share common molecular and ultrastructural characteristics. Lipid droplet research has been largely fragmented across the diversity of lipid droplet classes and sub-classes. However, we suggest that there is great potential benefit to the lipid community in better integrating the lipid droplet research fields. To facilitate such integration, we survey the protein and lipid compositions, functional roles, and mechanisms of biogenesis across the breadth of lipid droplets studied throughout the natural world. We depict the big picture of lipid droplet biology, emphasizing shared characteristics and unique differences seen between different classes. In presenting the known diversity of lipid droplets side-by-side it becomes necessary to offer for the first time a consistent system of categorization and nomenclature. We propose a division into three primary classes that reflect their sub-cellular location: i) cytoplasmic lipid droplets (CYTO-LDs), that are present in the eukaryotic cytoplasm, ii) prokaryotic lipid droplets (PRO-LDs), that exist in the prokaryotic cytoplasm, and iii) plastid lipid droplets (PL-LDs), that are found in plant plastids, organelles of photosynthetic eukaryotes. Within each class there is a remarkable array of sub-classes displaying various sizes, shapes and compositions. A more integrated lipid droplet research field will provide opportunities to better build on discoveries and accelerate the pace of research in ways that have not been possible.     

Activation of the lipid droplet controls the rate of lipolysis of triglycerides in the insect fat body

The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak approximately 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.     

Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system

Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains.