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• W2024145410 abstract "Perilipins regulate triacylglycerol storage and hydrolysis in adipocytes. The central 25% of the perilipin A sequence, including three hydrophobic sequences (H1, H2, and H3) and an acidic region, targets and anchors perilipins to lipid droplets. Thus, we hypothesized that H1, H2, and H3 are targeting and anchoring motifs. We now show that deletion of any single hydrophobic sequence or combinations of H1 and H3 or H2 and H3 does not prevent targeting of the mutated perilipin to lipid droplets. In contrast, mutated perilipin lacking H1 and H2 showed reduced targeting, whereas perilipin lacking H1, H2, and H3 targeted poorly to lipid droplets; thus, H3 is a weak targeting signal and either H1 or H2 is required for optimal targeting. Complete elimination of perilipin targeting was observed only when all three hydrophobic sequences were deleted in combination with either the acidic region or N-terminal sequences predicted to form amphipathic β-strands. Unlike intact perilipin A, mutated perilipin lacking either H1 and H2 or H1, H2, and H3 was released from lipid droplets after alkaline carbonate treatment, suggesting that these forms are loosely associated with lipid droplets.The three hydrophobic sequences play a major role in targeting and anchoring perilipins to lipid droplets. Perilipins regulate triacylglycerol storage and hydrolysis in adipocytes. The central 25% of the perilipin A sequence, including three hydrophobic sequences (H1, H2, and H3) and an acidic region, targets and anchors perilipins to lipid droplets. Thus, we hypothesized that H1, H2, and H3 are targeting and anchoring motifs. We now show that deletion of any single hydrophobic sequence or combinations of H1 and H3 or H2 and H3 does not prevent targeting of the mutated perilipin to lipid droplets. In contrast, mutated perilipin lacking H1 and H2 showed reduced targeting, whereas perilipin lacking H1, H2, and H3 targeted poorly to lipid droplets; thus, H3 is a weak targeting signal and either H1 or H2 is required for optimal targeting. Complete elimination of perilipin targeting was observed only when all three hydrophobic sequences were deleted in combination with either the acidic region or N-terminal sequences predicted to form amphipathic β-strands. Unlike intact perilipin A, mutated perilipin lacking either H1 and H2 or H1, H2, and H3 was released from lipid droplets after alkaline carbonate treatment, suggesting that these forms are loosely associated with lipid droplets. The three hydrophobic sequences play a major role in targeting and anchoring perilipins to lipid droplets. Perilipins are lipid droplet-associated phosphoproteins that function as key regulators of triacylglycerol storage and hydrolysis in adipocytes (1Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H. Quast M.J. Gorenstein D. Chen K.H. Chan L. Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice.Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (488) Google Scholar, 2Souza S.C. de Vargas L.M. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. Overexpression of perilipin A and B blocks the ability of tumor necrosis factor alpha to increase lipolysis in 3T3-L1 adipocytes.J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 3Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. Londos C. Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity.Proc. Natl. Acad. Sci. USA. 2001; 98: 6494-6499Crossref PubMed Scopus (601) Google Scholar, 4Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis.J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 5Souza S.C. Muliro K.V. Liscum L. Lien P. Yamamoto M.T. Schaffer J.E. Dallal G.E. Wang X. Kraemer F.B. Obin M. Greenberg A.S. Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.J. Biol. Chem. 2002; 277: 8267-8272Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 6Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation.J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (415) Google Scholar, 7Tansey J.T. Huml A.M. Vogt R. Davis K.E. Jones J.M. Fraser K.A. Brasaemle D.L. Kimmel A.R. Londos C. Functional studies on native and mutated forms of perilipins. A role in protein kinase A-mediated lipolysis of triacylglycerols.J. Biol. Chem. 2003; 278: 8401-8406Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 8Zhang H.H. Souza S.C. Muliro K.V. Kraemer F.B. Obin M.S. Greenberg A.S. Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis.J. Biol. Chem. 2003; 278: 51535-51542Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Perilipins are members of the PAT family of lipid droplet-associated proteins (9Miura S. Gan J.W. Brzostowski J. Parisi M.J. Schultz C.J. Londos C. Oliver B. Kimmel A.R. Functional conservation for lipid storage droplet association among perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium.J. Biol. Chem. 2002; 277: 32253-32257Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), which includes adipophilin (also called adipose differentiation-related protein) (10Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar, 11Heid H.W. Moll R. Schwetlick I. Rackwitz H.R. Keenan T.W. Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.Cell Tissue Res. 1998; 294: 309-321Crossref PubMed Scopus (356) Google Scholar), TIP47 (9Miura S. Gan J.W. Brzostowski J. Parisi M.J. Schultz C.J. Londos C. Oliver B. Kimmel A.R. Functional conservation for lipid storage droplet association among perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium.J. Biol. Chem. 2002; 277: 32253-32257Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 12Wolins N.E. Rubin B. Brasaemle D.L. TIP47 associates with lipid droplets.J. Biol. Chem. 2001; 276: 5101-5108Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), and S3-12 (13Scherer P.E. Bickel P.E. Kotler M. Lodish H.F. Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.Nat. Biotechnol. 1998; 16: 581-586Crossref PubMed Scopus (107) Google Scholar, 14Wolins N.E. Skinner J.R. Schoenfish M.J. Tzekov A. Bensch K.G. Bickel P.E. Adipocyte protein S3-12 coats nascent lipid droplets.J. Biol. Chem. 2003; 278: 37713-37721Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) as well as proteins in species as diverse as Drosophila (Lsd1 and Lsd2) and Dictyostelium (9Miura S. Gan J.W. Brzostowski J. Parisi M.J. Schultz C.J. Londos C. Oliver B. Kimmel A.R. Functional conservation for lipid storage droplet association among perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium.J. Biol. Chem. 2002; 277: 32253-32257Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Three perilipin isoforms, perilipins A, B, and C, are encoded by alternatively spliced forms of mRNA transcribed from a single gene (15Greenberg A.S. Egan J.J. Wek S.A. Moos Jr., M.C. Londos C. Kimmel A.R. Isolation of cDNAs for perilipins A and B: sequence and expression of lipid droplet-associated proteins of adipocytes.Proc. Natl. Acad. Sci. USA. 1993; 90: 12035-12039Crossref PubMed Scopus (211) Google Scholar, 16Lu X. Gruia-Gray J. Copeland N.G. Gilbert D.J. Jenkins N.A. Londos C. Kimmel A.R. The murine perilipin gene: the lipid droplet-associated perilipins derive from tissue-specific, mRNA splice variants and define a gene family of ancient origin.Mamm. Genome. 2001; 12: 741-749Crossref PubMed Scopus (182) Google Scholar). Perilipins A, B, and C share a common N terminus but have distinct C termini. Perilipin A is the longest isoform with a unique C-terminal sequence of 112 amino acids (aa) and is the most abundant lipid droplet-associated protein in adipocytes. The current model for lipid droplet assembly hypothesizes the nucleation of a lens of neutral lipids within the membrane bilayer of the endoplasmic reticulum, which upon accumulating a critical mass of neutral lipid is released into the cytoplasm (17Murphy D.J. Vance J. Mechanisms of lipid-body formation.Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 18Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms.Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (755) Google Scholar). Thus, lipid droplet-associated proteins may be synthesized on membrane-bound ribosomes and localize cotranslationally to nascent lipid droplets, as has been demonstrated for oleosins in the seeds of plants (18Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms.Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (755) Google Scholar, 19Beaudoin F. Napier J.A. Targeting and membrane-insertion of a sunflower oleosin in vitro and in Saccharomyces cerevisiae: the central hydrophobic domain contains more than one signal sequence, and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism.Planta. 2002; 215: 293-303Crossref PubMed Scopus (37) Google Scholar, 20Hills M.J. Watson M.D. Murphy D.J. Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.).Planta. 1993; 189: 24-29Crossref PubMed Scopus (55) Google Scholar, 21Huang A.H. Oleosins and oil bodies in seeds and other organs.Plant Physiol. 1996; 110: 1055-1061Crossref PubMed Scopus (422) Google Scholar, 22Loer D.S. Herman E.M. Cotranslational integration of soybean (Glycine max) oil body membrane protein oleosin into microsomal membranes.Plant Physiol. 1993; 101: 993-998Crossref PubMed Scopus (59) Google Scholar). Alternatively, proteins may be synthesized on cytoplasmic ribosomes and then assembled onto lipid droplets. Because perilipins traffic posttranslationally to lipid droplets (23Brasaemle D.L. Barber T. Kimmel A.R. Londos C. Post-translational regulation of perilipin expression. Stabilization by stored intracellular neutral lipids.J. Biol. Chem. 1997; 272: 9378-9387Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), one or more signals within the primary amino acid sequence directs the targeting of nascent perilipin to lipid droplets. We recently reported that the central 25% of the perilipin A sequence, which includes three moderately hydrophobic sequences of 16–23 aa at positions 242–260 (H1), 320–342 (H2), and 349–364 (H3) and an acidic sequence from aa 291–318 (Fig. 1), contains all of the information necessary to target and anchor nascent perilipin A to lipid droplets (24Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Deletion of the entire central region eliminated targeting of the mutated perilipin to lipid droplets; conversely, fusion of this central region to Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria targeted the GFP chimera to the surfaces of lipid droplets (24Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, although it was clear from our study that the central region of the perilipin sequence contains partially redundant targeting signals, the exact identity of these signals, and their relative importance, were unresolved. In the current study, we tested the hypothesis that the central sequences of hydrophobic amino acids are the critical determinants for targeting perilipin A to lipid droplets. We have stably expressed forms of perilipin with internal deletion mutations in 3T3-L1 fibroblasts and characterized the localization of the mutated perilipins using immunofluorescence microscopy and immunoblotting of subcellular fractions. We have found that multiple partially redundant targeting signals include not only H1, H2, and H3 but also the central acidic region and N-terminal sequences predicted to form amphipathic β-strands. Because the anchoring of perilipins into lipid droplets is resistant to alkaline carbonate treatments that disrupt electrostatic interactions (24Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and is disrupted by detergents such as SDS (25Londos C. Brasaemle D.L. Gruia-Gray J. Servetnick D.A. Schultz C.J. Levin D.M. Kimmel A.R. Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells.Biochem. Soc. Trans. 1995; 23: 611-615Crossref PubMed Scopus (101) Google Scholar), we further tested the hypothesis that perilipins anchor into lipid droplets by embedding the three hydrophobic sequences into the triacylglycerol core. Pfu DNA polymerase was purchased from Stratagene. Alexa Fluor 488-conjugated goat anti-rabbit IgG and Bodipy 493/503 were obtained from Molecular Probes, Inc. (Eugene, OR). Lissamine rhodamine-conjugated goat anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Geneticin was purchased from Mediatech, Inc. (Herndon, VA). 3T3-L1 preadipocytes and 293T cells were cultured as described previously (4Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis.J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). The coding sequence of mouse perilipin A cDNA subcloned into the pSRαMSVtkneo retroviral expression vector was reported previously (4Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis.J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). To generate mutated forms of perilipin A, nucleotide sequences encoding H1, H2, and H3 were replaced with sequences for the restriction sites Sal I, XbaI, and Bgl II, respectively; the resulting perilipin A cDNA was amplified using Pfu DNA polymerase and 5′ and 3′ oligonucleotide primers with added Hin dIII sites and subcloned into pSRαMSVtkneo (26Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau N.R. Littman D.R. Witte O.N. BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias.Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (354) Google Scholar). Perilipin cDNAs lacking the nucleotide sequences for H1, H2, or H3 were then used as templates to generate additional constructs lacking all combinations of two hydrophobic sequences. The perilipin cDNA lacking H1 and H3 was then used as a template to generate constructs lacking the nucleotide sequences for 1) H1, H2, and H3 and 2) H1, H2, H3, and the acidic region from aa 291 to 318 by replacing the nucleotides encoding aa 291–342 (including the acidic region and H2) with an XbaI restriction site. Mutated perilipin cDNA lacking the sequences for H1, H2, and H3 was also used as a template to replace the nucleotide sequence encoding aa 111–182, which is predicted to form five amphipathic β-strands with an SpeI restriction site. A summary of all constructs is shown in Table 1. The amplified intact and mutated perilipin cDNAs were ligated into the unique Hin dIII site of the pSRαMSVtkneo retroviral expression vector (26Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau N.R. Littman D.R. Witte O.N. BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias.Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (354) Google Scholar). All mutated forms of perilipin cDNA were sequenced to confirm the fidelity of amplification. The procedures used to assemble the retrovirus in 293T cells, transduce 3T3-L1 fibroblasts, and select cells stably expressing the various cDNAs were described previously (4Brasaemle D.L. Rubin B. Harten I.A. Gruia-Gray J. Kimmel A.R. Londos C. Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis.J. Biol. Chem. 2000; 275: 38486-38493Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Control cells were selected to stably express the retroviral vector lacking a cDNA insert. All experiments were conducted on cells stably expressing the various mutated forms of perilipin from two or more transduction procedures.TABLE 1Summary of mutated perilipin constructsName of ConstructAmino Acids DeletedIntact perilipin A0Δ1H242–260Δ2H320–342Δ3H349–364Δ1,2H242–260320–342Δ2,3H320–342349–364Δ1,3H242–260349–364Δ1,2,3H242–260320–342349–364AΔ242–260291–342349–364βΔ111–182242–260320–342349–364 Open table in a new tab Cells were grown on glass coverslips in culture media without additions or media supplemented with 400 μM oleic acid (Sigma) complexed to fatty acid-free BSA (Biocell Laboratories, Inc.) at a 6:1 molar ratio for 24 h before fixation to increase triacylglycerol synthesis and storage and, hence, increase protein levels of perilipin by increasing its stability (23Brasaemle D.L. Barber T. Kimmel A.R. Londos C. Post-translational regulation of perilipin expression. Stabilization by stored intracellular neutral lipids.J. Biol. Chem. 1997; 272: 9378-9387Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Cells were fixed with 3% paraformaldehyde in PBS and prepared for indirect immunofluorescence microscopy, as described previously (27Blanchette-Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. Perilipin is located on the surface layer of intracellular lipid droplets in adipocytes.J. Lipid Res. 1995; 36: 1211-1226Abstract Full Text PDF PubMed Google Scholar). Cells were stained with polyclonal antisera raised against a recombinant N-terminal peptide of perilipin A (10Brasaemle D.L. Barber T. Wolins N.E. Serrero G. Blanchette-Mackie E.J. Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.J. Lipid Res. 1997; 38: 2249-2263Abstract Full Text PDF PubMed Google Scholar) diluted 1:1,500. Secondary antibodies used were either Alexa Fluor 488-conjugated goat anti-rabbit IgG or Lissamine rhodamine-conjugated goat anti-rabbit IgG when costaining for neutral lipids with Bodipy 493/503. Cells were viewed with a Nikon Eclipse E800 fluorescence microscope equipped with a Hamamatsu Orca digital camera interfaced with a Power Macintosh G4. Images were captured in monochrome and processed using Improvision Openlab software; for aesthetic reasons, doubly stained cells are depicted in the opposite colors to those observed. The intracellular localization of ectopically expressed forms of mutated perilipin or intact perilipin A was quantified by scoring at least 100 cells per transduction experiment; classification of the mutated perilipins as clearly targeting to lipid droplets, poorly targeting to lipid droplets, or failing to target to lipid droplets was compared with values acquired for intact perilipin A. Confluent monolayers of 3T3-L1 fibroblasts stably expressing intact perilipin A or mutated forms of perilipin were incubated with 600 μM oleic acid complexed to fatty acid-free BSA for 24 h. Cells were harvested, homogenized, and fractionated to collect lipid droplets, as described previously (24Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Lipid droplet fractions recovered from subcellular fractionation were adjusted to 100 mM sodium carbonate, pH 11.5, with 10 μg/ml leupeptin, 1 mM benzamidine, 100 μM [4-(2-aminoethyl)-benzenesulfonylfluoride] hydrochloride, and 20% sucrose (final concentration) and layered beneath 100 mM sodium carbonate, pH 11.5, with protease inhibitors in centrifuge tubes. The tubes were centrifuged for 30 min at 26,000 g at 4°C in a Sorvall TH660 rotor, and the floating lipid droplets were recovered by slicing off the tops of the tubes with a Beckman tube slicer. Isolated lipid droplets were delipidated by precipitation with cold acetone overnight at −20°C; precipitated proteins were solubilized in 2× concentrated Laemmli's sample buffer (28Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar). Proteins were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes. Membranes were probed with anti-perilipin polyclonal antisera and a horseradish peroxidase-conjugated secondary antibody (Sigma); protein bands were detected using enhanced chemiluminescence reagents from Amersham Biosciences. Total RNA was extracted from cells stably expressing intact perilipin A and mutated perilipins using RNAeasy (Qiagen) and separated by electrophoresis on 1% agarose gels, as described previously (24Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). RNA was transferred electrophoretically to MagnaCharge nylon membranes (Osmonics) and probed with 32P-labeled cDNA for the coding sequence of perilipin A. Membranes were reprobed for β-actin to correct for variations in RNA load. The mass of perilipin mRNA relative to β-actin mRNA was determined by densitometric scanning of exposed X-ray films using a Personal Densitometer from Molecular Dynamics. When using immunofluorescence microscopy to observe the localization of mutated forms of perilipin, we consistently noted three patterns of fluorescence in transduced 3T3-L1 fibroblasts (Fig. 2). Intact perilipin A and some mutated forms of perilipin clearly targeted to lipid droplets and were detected as bright, uninterrupted rings of fluorescence around the lipid droplets when cells were stained with polyclonal antisera raised against perilipin A (Fig. 2A). Cells expressing some of the mutated forms of perilipin displayed weaker, interrupted rings of fluorescence around lipid droplets (Fig. 2B); we interpreted this pattern to represent less efficient targeting of the mutated perilipins to lipid droplets. Control cells lacking perilipins and cells expressing some of the mutated forms of perilipin lacked a specific staining pattern around lipid droplets and displayed only diffuse background fluorescence (Fig. 2C). Using this classification scheme, 95% of 3T3-L1 fibroblasts expressing intact ectopic perilipin A showed strong localization, 4% of cells showed weak localization, and 1% showed no specific staining on lipid droplets (Figs. 2, 3, Table 2). In contrast, 100% of cells stably expressing neomycin resistance conferred by the retroviral vector lacking an inserted cDNA sequence showed no specific staining on lipid droplets (Figs. 2, 3, Table 2).Fig. 3Targeting of mutated perilipins to lipid droplets in 3T3-L1 fibroblasts. A: 3T3-L1 fibroblasts stably expressing intact perilipin A (PeriA), control retroviral vector lacking a cDNA insert (Control), mutated perilipins lacking individual hydrophobic sequences (Δ1H, Δ2H, Δ3H), combinations of two hydrophobic sequences (Δ1,2H, Δ1,3H, Δ2,3H), all three hydrophobic sequences (Δ1,2,3H), or all three hydrophobic sequences and either the acidic region (AΔ) or N-terminal sequences predicted to form amphipathic β-strands (βΔ) were stained with anti-perilipin polyclonal antisera and an Alexa Fluor 488-tagged secondary antibody. Each panel shows a single representative cell from more than 100 observed in each of two to three transduction experiments. Bar = 10 μm. B: Schematic diagrams of the expressed regions of intact perilipin A and the mutated forms of perilipin. Δs above dotted lines indicate the positions of the deleted sequences. See legend to Fig. 1 for details of the depicted structural motifs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Summary of the targeting of mutated forms of perilipin to lipid dropletsPercentage of Cells (Mean ± SD)Name of ConstructStrong LocalizationWeak LocalizationNo Specific StainingPerilipin A (n = 7)95 ± 44 ± 31 ± 2Control cells lacking perilipins (n = 7)00100Δ1H (n = 1)9613Δ2H (n = 1)9235Δ3H (n = 1)9730Δ1,2H (n = 4)9 ± 881 ± 310 ± 9Δ2,3H (n = 3)89 ± 910 ± 81 ± 1Δ1,3H (n = 3)73 ± 1126 ± 111 ± 1Δ1,2,3H (n = 3)036 ± 264 ± 2AΔ (n = 3)00100βΔ (n = 3)00100n, number of repetitions of 100 cell counts. Open table in a new tab n, number of repetitions of 100 cell counts. Based on our previous findings, we hypothesized that the hydrophobic sequences of perilipin A are important for the targeting of nascent perilipins to lipid droplets. To systematically dissect the functional roles of these hydrophobic sequences, we first deleted H1, H2, or H3 individually from otherwise intact perilipin A; mutated perilipins Δ1H (lacking H1; aa 242–260), Δ2H (lacking H2; aa 320–342), and Δ3H (lacking H3; aa 349–364) clearly localized to lipid droplets by immunofluorescence microscopy of fixed cells (Fig. 3) and by immunoblotting of proteins from isolated lipid droplets (Fig. 4). Ninety-six percent of cells expressing mutated perilipin Δ1H, 92% of cells expressing mutated perilipin Δ2H, and 97% of cells expressing mutated perilipin Δ3H showed strong staining of perilipin on lipid droplets (Table 2). Furthermore, the mass of mutated perilipins Δ1H, Δ2H, and Δ3H detected on immunoblots of lipid droplet fractions was similar to that of intact perilipin A (Fig. 4A). Thus, perilipin A lacking any one of the three hydrophobic sequences targets to lipid droplets. We next asked whether deletion of more than one hydrophobic sequence alters targeting of the mutated perilipin to lipid droplets. Mutated forms of perilipin lacking H1 and H3 (Δ1,3H) or H2 and H3 (Δ2,3H) targeted efficiently to lipid droplets, as assessed by immunofluorescence microscopy (Fig. 3); 99% of cells showed either strong or weak staining of perilipin on lipid droplets (Table 2). In contrast, mutated perilipin lacking H1 and H2 (Δ1,2H) targeted less efficiently to lipid droplets (Fig. 3); only 9% of cells showed strong staining of perilipin on lipid droplets, 81% of cells showed weak staining, and 10% of cells displayed only background staining (Table 2). Correspondingly, levels of Δ2,3H and Δ1,3H mutated perilipins isolated in lipid droplet fractions were comparable to that of intact ectopic perilipin A, whereas the mass of Δ1,2H mutated perilipin on lipid droplets was significantly reduced (Fig. 4B). Thus, perilipin A lacking the combination of H1 and H2 targets less efficiently to lipid droplets, indicating that H3 is a relatively weak targeting signal. Cells expressing mutated perilipin lacking H1, H2, and H3 (Δ1,2,3H) displayed either weak staining of perilipin on lipid droplets (36% of cells) or no specific staining pattern (64% of cells; Table 2, Fig. 3). Correspondingly, immunoblots showed only a trace amount of Δ1,2,3H mutated perilipin on lipid droplets (Fig. 4C). Surprisingly, the deletion of H1, H2, and H3 did not completely eliminate targeting of the mutated perilipin to lipid droplets, suggesting that additional sequences may play a role in targeting. Thus, we further deleted the N-terminal sequences from aa 111 to 182 that are predicted to form amphipathic β-strands (βΔ) and, separately, the acidic region from aa 291 to 318 (AΔ) from mutated perilipin that lacks H1, H2, and H3. Both the βΔ and AΔ mutated forms of perilipin failed to target to lipid droplets when assessed by immunofluorescence microscopy or immunoblotting of lipid droplet fractions (Figs. 3, 4C, Table 2). Furthermore, these nontargeting forms of perilipin were not detected in supernatant or membrane fractions (data not shown), suggesting that the proteins were rapidly degraded. Interestingly, deletion of either the sequence from aa 111 to 182 (our unpublished observation) or from aa 291 to 318 (24Garcia A. Sekowski A. Subramanian V. Brasaemle D.L. The central domain is required to target and anchor perilipin A to lipid droplets.J. Biol. Chem. 2003; 278: 625-635Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) from otherwise intact perilipin di" @default.
• W2024145410 created "2016-06-24" @default.
• W2024145410 creator A5035449295 @default.
• W2024145410 creator A5067668919 @default.
• W2024145410 creator A5075961421 @default.
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• W2024145410 date "2004-11-01" @default.
• W2024145410 modified "2023-10-16" @default.
• W2024145410 title "Hydrophobic sequences target and anchor perilipin A to lipid droplets" @default.
• W2024145410 cites W1504753880 @default.
• W2024145410 cites W1895078941 @default.
• W2024145410 cites W1945140151 @default.
• W2024145410 cites W1969741764 @default.
• W2024145410 cites W1975514965 @default.
• W2024145410 cites W1977684071 @default.
• W2024145410 cites W1981540523 @default.
• W2024145410 cites W1986318660 @default.
• W2024145410 cites W1987703403 @default.
• W2024145410 cites W1999898913 @default.
• W2024145410 cites W2020543398 @default.
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• W2024145410 cites W2055443182 @default.
• W2024145410 cites W2063083457 @default.
• W2024145410 cites W2069583796 @default.
• W2024145410 cites W2085733039 @default.
• W2024145410 cites W2094744133 @default.
• W2024145410 cites W2095231162 @default.
• W2024145410 cites W2096519088 @default.
• W2024145410 cites W2097521595 @default.
• W2024145410 cites W2098681671 @default.
• W2024145410 cites W2100837269 @default.
• W2024145410 cites W2101543956 @default.
• W2024145410 cites W2103973399 @default.
• W2024145410 cites W2107940734 @default.
• W2024145410 cites W2109813770 @default.
• W2024145410 cites W2116803744 @default.
• W2024145410 cites W2119598335 @default.
• W2024145410 cites W2124366383 @default.
• W2024145410 cites W2128693388 @default.
• W2024145410 cites W2132721964 @default.
• W2024145410 cites W2138377765 @default.
• W2024145410 cites W2147963733 @default.
• W2024145410 cites W2148584142 @default.
• W2024145410 cites W2157295729 @default.
• W2024145410 cites W2160107586 @default.
• W2024145410 cites W2293412032 @default.
• W2024145410 cites W2305216237 @default.
• W2024145410 cites W4229881459 @default.
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