Monotopic topology is required for lipid droplet targeting of ancient ubiquitous protein 1
2012; Elsevier BV; Volume: 54; Issue: 2 Linguagem: Inglês
10.1194/jlr.m033852
ISSN1539-7262
AutoresAna Stevanović, Christoph Thiele,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoAncient ubiquitous protein 1 (AUP1) is a multifunctional protein, which acts on both lipid droplets (LDs) and the endoplasmic reticulum (ER) membrane. Double localization to these two organelles, featuring very different membrane characteristics, was observed also for several other integral proteins, but little is known about the signals and mechanisms behind dual protein targeting to ER and LDs. Here we dissect the AUP1 targeting signals by analyses of localization and topology of several deletion and point mutants. We found that AUP1 is inserted into the membrane of the ER in a monotopic hairpin fashion, and subsequently transported to the hemi-membrane of LDs. A single domain localized in the N-terminal part of AUP1 enables its ER residence, the monotopic insertion, and the LD localization. Different specific residues within this multifunctional domain are responsible for achieving the complex spatial distribution pattern. A mutation of three amino acids, which changes AUP1 topology from hairpin to transmembrane, abolishes LD localization. These findings suggest that the cell is able to target a protein to multiple intracellular locations using a single domain. Ancient ubiquitous protein 1 (AUP1) is a multifunctional protein, which acts on both lipid droplets (LDs) and the endoplasmic reticulum (ER) membrane. Double localization to these two organelles, featuring very different membrane characteristics, was observed also for several other integral proteins, but little is known about the signals and mechanisms behind dual protein targeting to ER and LDs. Here we dissect the AUP1 targeting signals by analyses of localization and topology of several deletion and point mutants. We found that AUP1 is inserted into the membrane of the ER in a monotopic hairpin fashion, and subsequently transported to the hemi-membrane of LDs. A single domain localized in the N-terminal part of AUP1 enables its ER residence, the monotopic insertion, and the LD localization. Different specific residues within this multifunctional domain are responsible for achieving the complex spatial distribution pattern. A mutation of three amino acids, which changes AUP1 topology from hairpin to transmembrane, abolishes LD localization. These findings suggest that the cell is able to target a protein to multiple intracellular locations using a single domain. The intracellular localization of proteins is achieved by the complex interplay of multiple transport processes using combinations of targeting and retention signals on the cargo proteins that are recognized by the transport machinery. For several localizations, e.g., to the nucleus, mitochondrium, peroxisome, or lysosome, both signals and machinery have been identified and extensively studied (1Matlin K.S. Spatial expression of the genome: the signal hypothesis at forty.Nat. Rev. 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The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake.J. Lipid Res. 2012; 53: 888-900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). One such protein is ancient ubiquitous protein 1 (AUP1) that specifically localizes to ER and LDs (35Mueller B. Klemm E.J. Spooner E. Claessen J.H. Ploegh H.L. SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins.Proc. Natl. Acad. Sci. USA. 2008; 105: 12325-12330Crossref PubMed Scopus (187) Google Scholar, 36Spandl J. Lohmann D. Kuerschner L. Moessinger C. Thiele C. Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.J. Biol. Chem. 2011; 286: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 37Klemm E.J. Spooner E. Ploegh H.L. Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.J. Biol. Chem. 2011; 286: 37602-37614Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). AUP1 is a 410 amino acids long, integral protein with a single predicted membrane domain (36Spandl J. Lohmann D. Kuerschner L. Moessinger C. Thiele C. Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.J. Biol. Chem. 2011; 286: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 37Klemm E.J. Spooner E. Ploegh H.L. Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.J. Biol. Chem. 2011; 286: 37602-37614Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The membrane domain of AUP1 is located internally and both the N and C termini of AUP1 are facing the cytoplasm, resulting in monotopic/hairpin topology (36Spandl J. Lohmann D. Kuerschner L. Moessinger C. Thiele C. Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.J. Biol. Chem. 2011; 286: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). AUP1 has multiple functional domains and is reported to facilitate ER-associated protein degradation, inside-out signaling in platelets, and neutral lipid storage (35Mueller B. Klemm E.J. Spooner E. Claessen J.H. Ploegh H.L. SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins.Proc. Natl. Acad. Sci. USA. 2008; 105: 12325-12330Crossref PubMed Scopus (187) Google Scholar, 36Spandl J. Lohmann D. Kuerschner L. Moessinger C. Thiele C. Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.J. Biol. Chem. 2011; 286: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 37Klemm E.J. Spooner E. Ploegh H.L. Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.J. Biol. Chem. 2011; 286: 37602-37614Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 38Kato A. Kawamata N. Tamayose K. Egashira M. Miura R. Fujimura T. Murayama K. Oshimi K. Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3.J. Biol. Chem. 2002; 277: 28934-28941Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 39Kato A. Oshimi K. Ancient ubiquitous protein 1 and Syk link cytoplasmic tails of the integrin alpha(IIb)beta(3).Platelets. 2009; 20: 105-110Crossref PubMed Scopus (7) Google Scholar). Domains important for a function of AUP1 in ER-associated degradation are found in its C-terminal region. The CUE (coupling of ubiquitin to ER degradation) domain binds dislocation substrates and components of the ER quality control machinery (37Klemm E.J. Spooner E. Ploegh H.L. Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.J. Biol. Chem. 2011; 286: 37602-37614Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and the G2 binding region (G2BR) domain recruits a specific E2 ubiquitin conjugase, Ube2g2 (36Spandl J. Lohmann D. Kuerschner L. Moessinger C. Thiele C. Ancient ubiquitous protein 1 (AUP1) localizes to lipid droplets and binds the E2 ubiquitin conjugase G2 (Ube2g2) via its G2 binding region.J. Biol. Chem. 2011; 286: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The function of the N-terminal region of AUP1 is less-well understood. It contains the membrane domain and a predicted acyltransferase domain, which was proposed to function in neutral lipid storage (37Klemm E.J. Spooner E. Ploegh H.L. Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control.J. Biol. Chem. 2011; 286: 37602-37614Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Domains or motifs involved in AUP1 intracellular targeting are not known. Here we dissect the localization signals of AUP1 and find that the N-terminal region including the membrane domain mediates its ER targeting, LD localization, and monotopic membrane insertion. The following antibodies were used: rabbit polyclonal anti-long-chain acyl-CoA synthetase 3 (ACSL3) (32Moessinger C. Kuerschner L. Spandl J. Shevchenko A. Thiele C. Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine.J. Biol. Chem. 2011; 286: 21330-21339Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), rabbit polyclonal anti-calnexin, and rabbit polyclonal anti-protein-disulfide isomerase (PDI) (Enzo Life Sciences; Loerrach, Germany), mouse monoclonal anti-GAPDH (Novus Biologicals; Littleton, CO), mouse monoclonal anti-hemagglutinin (clone F-7; Santa Cruz Biotechnology, Santa Cruz, CA), Alexa555- and Alexa488-conjugated secondary antibodies (Invitrogen; Darmstadt, Germany), and HRP-coupled secondary antibodies (Jackson Immuno Research; West Grove, PA). Truncation mutants of AUP1 were generated by PCR amplification and inserted into pCDNA3.1 vector with inserted C-terminal 3xHA tag. Site-directed mutagenesis was performed either using the QuikChange® site-directed mutagenesis kit (Stratagene; Amsterdam, The Netherlands) according to the manufacturer's instructions, or using a PCR-based site-directed mutagenesis approach with overlap extensions (40Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction.Gene. 1989; 77: 51-59Crossref PubMed Scopus (6830) Google Scholar). DNA fragments encoding AUP1 point mutants were inserted into pCDNA3.1 vector with inserted C-terminal 3xHA tag or pN3HA vector with N-terminal 3xHA tag. Supplementary Tables I–III contain detailed information about expression constructs, including primers and restriction sites used. All constructs were verified by sequencing. The monkey fibroblast cell line, COS-7, was grown at 37°C, 5% CO2 in Dulbecco's modified medium supplemented with 10% fetal calf serum (FCS) (GIBCO; Darmstadt, Germany). When indicated, cells were incubated in medium supplemented with 150 μM oleic acid (Sigma-Aldrich; Taufkirchen, Germany). DNA transfection was performed using Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's instructions or the transfection reagent (N4, N9-dioleoyl spermine) (41Ahmed O.A. Adjimatera N. Pourzand C. Blagbrough I.S. N4,N9-dioleoyl spermine is a novel nonviral lipopolyamine vector for plasmid DNA formulation.Pharm. Res. 2005; 22: 972-980Crossref PubMed Scopus (22) Google Scholar) using the same protocol but with 20% higher concentration of the transfection reagent. Cells were fixed with 3% (v/v) formaldehyde in PBS for 30 min, washed with PBS, blocked, and permeabilized for 30 min in PBS containing 0.5% BSA and 0.1% saponin (blocking buffer, BB). If indicated, saponin in the BB was replaced by 0.004% digitonin. Cells were incubated with primary antibodies in BB for 1 h, washed three times with BB, incubated with secondary antibody in BB, washed three times in BB and three times in PBS, and counterstained with LD540 (42Spandl J. White D.J. Peychl J. Thiele C. Live cell multicolor imaging of lipid droplets with a new dye, LD540.Traffic. 2009; 10: 1579-1584Crossref PubMed Scopus (182) Google Scholar) in PBS, followed by three washes in PBS. After rinsing in water, coverslips were mounted in Mowiol 4-88 (Merck; Darmstadt, Germany) containing 2.5% DABCO (1,4-diazabicyclo[2.2.2]octane) (Carl Roth; Karlsruhe, Germany). Images were acquired with a ZeissAxio Observer.Z1 microscope (Carl Zeiss; Oberkochen, Germany) equipped with a 63×/NA1.4 objective and a Photometrics Coolsnap K4 camera. Light source was a Polychrome V 150 W xenon lamp (TillPhotonics; Graeffeling, Germany). Images were processed using ImageJ (National Institutes of Health) or Adobe Illustrator software (Adobe). Live-cell imaging was performed in HEPES-buffered medium (Invitrogen) supplemented with 10% FCS, at 37°C and 5% CO2. Cycloheximide was from Applichem (Darmstadt, Germany). Cells were grown in 10-cm dishes in Dulbecco's modified medium supplemented with 10% FCS and 150 μM oleic acid for 36 h. In experiments with three fractions, four dishes of cells were used per gradient. Cells were washed once in PBS and once with Buffer A (0.2 M sucrose, 20 mM HEPES/NaOH, pH 7.5, protease inhibitor cocktail Complete (Roche; Grenzach, Germany) 1 tablet/50 ml). Subsequently, cells were scraped in Buffer A, passed through a 0.7 × 30 mm needle five times, and homogenized in a European Molecular Biology Laboratory cell cracker (HGM; Heidelberg, Germany) (inner diameter 8.020 mm, ball diameter 8.004 mm, with nine strokes). Nuclei and cell debris were pelleted by centrifugation (1,000 g, 4°C). Postnuclear supernatant was adjusted to 2 ml volume with Buffer A and gently mixed with 1 ml Buffer B (2 M sucrose, 20 mM HEPES/NaOH, pH 7.5, protease inhibitor cocktail 1 tablet/50 ml). The lysate was loaded to the bottom of the tube and overlaid with Buffer A. LDs were floated by ultracentrifugation (SW40Ti rotor at 100,000 g, at 4°C for 3 h). Fractions were collected as follows: LDs (upper 2 ml), intermediate fraction (2 ml), bottom fraction (remaining volume). In the figures, fraction LDs correspond to the LD fraction, fraction II to the intermediate fraction, and fraction III to the bottom fraction. Fractions were generally stored at −20°C prior to further analysis (SDS-PAGE and immunoblotting). In experiments with four fractions, ultracentrifugation was performed using a different rotor (SW55Ti rotor; 100,000 g, 4°C, 3 h), and fractions were collected as follows: LDs (upper 2 ml), intermediate fraction (1 ml), upper bottom fraction (2 ml), and bottom fraction (remaining volume). In the figure panels, fraction LDs correspond to the LD fraction, fraction II to the intermediate fraction, fraction III to the upper bottom fraction, and fraction IV to the bottom fraction. For N-glycosidase F treatment, COS-7 cells were grown in 10-cm dishes and transfected with appropriate constructs. After 24 h of expression, cells were washed with PBS and harvested by scraping in cold PBS. Cell lysis was performed using ball bearing EMBL cell cracker (HGM; 10 strokes, inner diameter 8.020 mm, ball diameter 8.004 mm). The lysate was centrifuged to remove nuclei and cellular debris (1,431 g, 10 min, 4°C). The supernatant (containing 800 μg total protein) was subjected to chloroform-methanol protein precipitation. Protein precipitate was resuspended in 20 μl solubilization buffer (20 mM sodium phosphate, pH 7.4, 2% SDS) and 380 μl reaction buffer [20 mM sodium phosphate, pH 7.4, 1% n-octyl-β-d-glucopyranosid (Applichem)]. The sample was divided into two equal parts and either treated with 4–16 U of N-glycosidase F (Roche; Grenzach, Germany) or left untreated. Samples were incubated at 20°C overnight. The reaction was stopped by addition of 5× Laemmli buffer and heating to 95°C for 10 min. Proteins were separated by SDS-PAGE and analyzed by immunoblotting. To reveal the pathway of AUP1 targeting, we transfected COS-7 cells with green fluorescent protein-tagged AUP1 and grew them under lipid-depleted conditions to deprive them of LDs. After recording an initial micrograph, cells were supplemented with oleate and cycloheximide to induce LD formation and to stop protein biosynthesis, and microscopic imaging was continued to observe LD formation and GFP-AUP1 redistribution. As shown in the supplementary movie, GFP-tagged AUP1 initially localized to a reticular structure, presumably the ER. After 24 min, a progressive redistribution to punctate structures became visible. After 72 min, the punctate structures were identified as LDs by in situ staining with the LD-specific dye LD540 (42Spandl J. White D.J. Peychl J. Thiele C. Live cell multicolor imaging of lipid droplets with a new dye, LD540.Traffic. 2009; 10: 1579-1584Crossref PubMed Scopus (182) Google Scholar), demonstrating sequential targeting of the protein from the ER to LDs (see the supplementary Movie and supplementary Fig. I). Therefore, in the following, we first study the ER localization of AUP1, followed by analysis of its LD localization. To identify the ER-targeting domain of AUP1, COS-7 cells were transfected with HA-tagged C-terminal truncation constructs (Fig. 1A), and their localization was examined by fluorescence microscopy (Fig. 1B). Full-length AUP1 partially localized to the ER, identified by a marker protein, PDI, as described previously for endogenous AUP1 (35Mueller B. Klemm E.J. Spooner E. Claessen J.H. Ploegh H.L. SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins.Proc. Natl. Acad. Sci. USA. 2008; 105: 12325-12330Crossref PubMed Scopus (187) Google Scholar), indicating no apparent influence of the HA tag on the ER localization of AUP1. AUP1 protein lacking either the G2BR domain or both the CUE and the G2BR domains colocalized with the ER marker (Fig. 1B). The shortest truncation construct also colocalized with the ER marker, although an additional perinuclear distribution pattern became visible (Fig. 1B). We conclude that the N-terminal 93 amino acids of AUP1 are sufficient for ER localization of AUP1. To determine which part of the AUP1 sequence is required for its localization to LDs, we expressed the constructs described above and examined their localization to LDs by fluorescence microscopy (Fig. 2A). Although full-length AUP1 and the two longer truncations displayed continuous rings around LDs, the shortest truncation, containing only 93 N-terminal amino acids of AUP1, formed discrete patches around LDs. The observed staining pattern could represent localization to either LDs or to neighboring organelles, especially ER structures that frequently surround LDs (32Moessinger C. Kuerschner L. Spandl J. Shevchenko A. Thiele C. Human lysophosphatidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the formation of phosphatidylcholine.J. Biol. Chem. 2011; 286: 21330-21339Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 43Blanchette-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). To address this question, localization of full-length AUP1 and the truncation mutants was examined by sucrose density gradient centrifugation and subsequent analysis of the organelle fractions by immunoblotting. A significant fraction of the full-length AUP1 construct and the truncated forms floated with the LD fraction (Fig. 2B), as identified by the LD marker protein, ACSL3 (34Poppelreuther M. Rudolph B. Du C. Grossmann R. Becker M. Thiele C. Ehehalt R. Fuellekrug J. The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake.J. Lipid Res. 2012; 53: 888-900Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The LD fraction was devoid of ER contamination, as demonstrated by the absence of calnexin, an ER marker protein. We conclude that the N-terminal 93 amino acids of AUP1 are also sufficient for LD localization. The LD-targeting domain contains a continuous hydrophobic region disrupted by a single arginine residue at position 42 (Fig. 3A). Mutation of this arginine to isoleucine (R42I), within the full-length AUP1, abolished LD targeting of both the N-terminally (Fig. 3B, top panel) and C-terminally (Fig. 3C, top panel) HA-tagged constructs, which was confirmed by subcellular fractionation analysis (see supplementary Fig. II, lanes 7–9). To examine whether charged residues downstream of the hydrophobic domain are also important for LD localization, we generated the C-terminally HA-tagged double mutant of full-length AUP1 (R62F/R63F_HA). This mutant did not accumulate on LDs (Fig. 3C, bottom panel), which was confirmed by the N-terminal tagging of the protein (Fig. 3B, bottom panel) and biochemical examination of the localization of both the N- and C-terminally-tagged constructs (see supplementary Fig. II, lanes 13–15 and 28–31). In contrast, substitution of aspartic acid at position 5
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