Targeting of Tsc13p to nucleus-vacuole junctions:

TSC13 is required for the biosynthesis of very-long-chain fatty acids (VLCFAs) in yeast. Tsc13p is a polytopic ER membrane protein that accumulates at nucleus-vacuole (NV) junctions, which are formed through Velcro-like interactions between Nvj1p in the perinuclear ER and Vac8p on the vacuole membrane. NV junctions mediate piecemeal microautophagy of the nucleus (PMN), during which bleb-like portions of the nucleus are extruded into invaginations of the vacuole membrane and degraded in the vacuole lumen. We report that Tsc13p is sequestered into NV junctions from the peripheral ER through Vac8p-independent interactions with Nvj1p. During nutrient limitation, Tsc13p is incorporated into PMN vesicles in an Nvj1p-dependent manner. The lumenal diameters of PMN blebs and vesicles are significantly reduced in tsc13-1 and tsc13-1 elo3-∆ mutant cells. PMN structures are also smaller in cells treated with cerulenin, an inhibitor of de novo fatty acid synthesis and elongation. The targeting of Tsc13p-GFP into NV junctions is perturbed by cerulenin, suggesting that its binding to Nvj1p depends on the availability of fatty acid substrates. These results indicate that Nvj1p retains and compartmentalizes Tsc13p at NV junctions, and that VLCFAs contribute to the normal biogenesis of trilaminar PMN structures in yeast. Introduction The endoplasmic reticulum (ER) is a mosaic of dynamic subdomains, including those involved in interorganelle “contact sites” or zones of close apposition between the ER membrane and other subcellular compartments (reviewed in Levine, 2004; Voeltz et al., 2002). Such contact sites have been observed between the ER and Golgi, mitochondrial, vacuolar (lysosomal), peroxisomal, endosomal, lipid droplet, or plasma membranes (Voeltz et al., 2002; Staehelin, 1997). ER contact sites have been proposed to mediate the non-vesicular trafficking of small molecules and lipids (reviewed in Levine, 2004). For instance, mitochondria-associated ER membrane domains are implicated in the transport of phospholipids and sterols to-and-from mitochondria (Achleitner et al., 1999; Rusinol et al., 1994). Plasma membrane-associated ER subdomains may likewise facilitate the transfer of specific lipids to the plasma membrane (Pichler et al., 2001). Accordingly, mitochondrial and plasma membrane-associated ER domains are enriched in enzymes involved in the biosynthesis of phospholipids and sterols (Pichler et al., 2001; Stone and Vance, 2000; Gaigg et al., 1995; Rusinol et al., 1994). The structural basis for the establishment and maintenance of ER contact sites in eukaryotes is largely unknown, except in the case of nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae (Pan et al., 2000; Levine, 2004; Voeltz et al., 2002). NV junctions occur where the vacuolar membrane protein, Vac8p, associates with Nvj1p in the outer nuclear membrane that is continuous with the perinuclear ER in yeast (Pan et al., 2000). The sizes of NV junctions increase proportionally to the expression level of Nvj1p, which is up-regulated though nutrient depletion following the diauxic shift or acute carbon or nitrogen starvation (Roberts et al., 2003; Gasch et al., 2000). The upstream promoter region of NVJ1 contains stress response elements that likely control expression in response to nutrient stress (Moskvina et al., 1998). NV junctions are sites of piecemeal microautophagy of the nucleus (PMN), during which nonessential portions of the nucleus are pinched-off into invaginations of the vacuole membrane and subsequently degraded by hydrolytic enzymes in the vacuole lumen (Roberts et al., 2003). PMN occurs at low frequencies in rapidly dividing cells, but is induced to higher levels upon carbon or nitrogen starvation through the TOR nutrient response pathway (Roberts et al., 2003). The granular nucleolus, which is composed largely of pre-ribosomes, is one non-essential nuclear constituent that have been observed to partition into PMN blebs (Roberts et al., 2003). Recently, two conserved proteins with roles in lipid metabolism, namely Tsc13p and Osh1p, were shown to localize at NV junctions (Kohlwein et al., 2001; Levine and Munro, 2001). Osh1p belongs to a family of proteins related to mammalian oxysterol binding protein (OSBP), which participates in the regulation of sterol homeostasis (Levine and Munro, 2001). Osh1p and related OSBP family members are hypothesized to function in the non-vesicular transport of sterol lipids between membrane compartments (reviewed in Levine, 2004). In support of this model, mammalian CERT, which shares a similar domain structure to Osh1p, has been shown to extract and transfer ceramide from the ER to nearby Golgi membranes (Kumagai et al., 2005; Hanada et al., 2003). We recently showed that Osh1p is sequestered into NV junctions through direct, Vac8pindependent interactions with Nvj1p, and that a redundant function of the OSH1-7 family of proteins is required for efficient PMN (Kvam and Goldfarb, 2004). Tsc13p is an essential protein required for the elongation of long-chain fatty acids (C16 and C18) to very-long-chain fatty acids (VLCFAs), which possess acyl-chain lengths of 20-carbons or more (Kohlwein et al., 2001). VLCFAs are constituents of several complex lipid species in yeast including ceramides, sphingolipids, inositolglycerophospholipids, and the phosphatidylinositol moiety of GPI anchors (Dickson, 1998), each of which are important components of lipid rafts and other detergent-insoluble lipid microdomains (Boukh-Viner et al., 2005; Dupre and Haguenauer-Tsapis, 2003; Eisenkolb et al., 2002). Accordingly, various studies indicate that VLCFAs strongly influence the structure, function, and fluidity of biological membranes (Millar et al., 1998; Schneiter et al., 1996; Ho et al., 1995). VLCFA biosynthesis in yeast occurs through a microsomal fatty acid elongation cycle that partially overlaps with the activity of the soluble fatty acid synthase (FAS) complex (Rossler et al., 2003). During VLCFA elongation, 2-carbon units from malonyl coenzyme A are sequentially condensed onto C16/C18 long-chain fatty acyl-CoA precursors, thereby increasing the acyl-chain length of the fatty acid substrate (Kohlwein et al., 2001; Schneiter and Kohlwein, 1997). C26 is the most abundant VLCFA produced in this manner in yeast (Welch and Burlingame, 1973). One round of elongation requires four consecutive biochemical reactions that are catalyzed by Elo2p, Elo3p, Tsc13p, and Ybr159p (Rossler et al., 2003). Elo2p and Elo3p are related elongases with partially overlapping fatty acyl-CoA substrate specificities, and thus form two separate elongase systems (Rossler et al., 2003; Oh et al., 1997). Co-purification studies indicate that Tsc13p and Ybr159p may associate in distinct elongase complexes with either Elo2p or Elo3p (Han et al., 2002; Kohlwein et al., 2001). Based on mutant phenotypes in yeast, and sequence similarity to reductases in higher eukaryotes, Tsc13p is thought to be the enoylCoA reductase that catalyzes the final step of VLCFA formation (Gable et al., 2004; Kohlwein et al., 2001). Here we show that the activity of Tsc13p in the VLCFA elongation cycle contributes to the biogenesis of PMN blebs and vesicles in yeast. This role is directly facilitated by the selective targeting of Tsc13p to NV junctions through a physical association with Nvj1p. The Nvj1p-mediated targeting of Tsc13p to NV junctions, and the incorporation of Tsc13p into PMN structures, provides a unique opportunity to investigate the interplay between the structure and function of a model ER membrane contact site. Materials and Methods Yeast strains, plasmids, and growth conditions Yeast strains used in this study are listed in Table I. Plasmids for the expression of NVJ1, NVJ1-EYFP, and NVJ1-Fc under CUP1 promoter control (PCUP1-NVJ1, PCUP1-NVJ1EYFP, and PCUP1-NVJ1-Fc) or NVJ1-myc under GAL1 promoter control (PGAL1-NVJ1-myc) were described previously (Kvam and Goldfarb, 2004; Roberts et al., 2003; Pan et al., 2000). PCUP1-TSC13-EYFP was created by ligating a SmaI-TSC13-HindIII PCR fragment and a HindIII-EYFP-XholI PCR fragment into the SmaI and SalI sites of pJN40 (Pan et al., 2000; Macreadie et al., 1989). PCR fragments were amplified from genomic yeast DNA or pEYFP (Clontech, CA) using Taq DNA polymerase (Invitrogen, CA). Construction of pRS425-TSC13, encoding TSC13 under the control of its native promoter, was described elsewhere (Kohlwein et al., 2001). Plasmids for the expression of Elo2p-EGFP (pUG35ELO2-EGFP) were generously obtained from S. Kohlwein. The plasmid-based VAC8 myristoylation and palmitoylation double mutant (vac8-4) was obtained from L. Weisman (Wang et al., 1998). Unless otherwise indicated, cells were cultured in standard YPD or synthetic complete media (SC) with 2% glucose (SCGlu) at 30°C (Sherman, 1991). Nitrogen starvation media (SD-N) contained 0.17% yeast nitrogen base and 2% glucose without amino acids or ammonium sulfate. The antibiotic cerulenin (Sigma) was dissolved in ethanol and used at a final concentration of 45 μM. Myriocin (ISP-1, Sigma) was suspended in methanol and used at 1μg/ml. Where indicated, myristic acid (Sigma) was supplemented to media at a final concentration of 1 mM from a 100 mM stock in ethanol. To disperse myristic acid, 0.2% Tergitol (Type NP-40, Sigma) was added to the media. In vivo Nvj1p-Fc co-purification Yeast harboring PCUP1-TSC13-EYFP and either PCUP1-NVJ1-Fc or empty vector, respectively, were grown to log phase in 100 mLs SCGlu and induced for 3 hrs with 0.1 mM CuSO4. Nvj1p-Fc was isolated from whole cell extract using protein-A conjugated agarose as previously described (Kvam and Goldfarb, 2004). Nvj1p-Fc complexes were denatured from the agarose matrix with 40 μl of 2x sample loading buffer (100 mM TrisHCl pH 6.8, 2% SDS, 20% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoethanol) and boiled for 5 min. 10-20 μl of eluate was analyzed by SDS-PAGE and the co-purification of Tsc13p-EYFP was assessed by Immuno blot. Vac8p Fractionation Approximately 25 OD600 units of log phase cells expressing cTsc13p-EGFP were shifted into 50 mLs of SD-N media supplemented with either cerulenin or an equal volume of ethanol (mock treatment). Cells were starved overnight (~16 hrs) at 30°C and then harvested. Whole cell extracts were prepared by bead-beating in extraction buffer (0.3M sorbitol, 10 mM Tris-Cl pH 7.5, 0.1 M NaCl, 1 mM MgCl2, 1 mM EDTA) containing Complete protease inhibitors (Roche, Mannheim, Germany) and 1 mM PMSF. Extracts were pre-cleared by centrifugation (2,000 rpm x 10 min), and the resulting lysate was fractionated by differential centrifugation as described by Wang et al., 1998. Briefly, precleared lysates were centrifuged at 13,000 g for 10 min and separated into P13 pellet and S13 supernatant fractions. S13 fractions were then separated into P100 pellet and S100 supernatant fractions by ultracentrifugation (100,000 g for 30 min). Pelleted membranes in P13 and P100 fractions were solubilized in extraction buffer containing 1% NP-40 detergent and protease inhibitors. The protein content of each P13, S100, and P100 fraction was determined using Bradford reagent. Equal amounts of protein were mixed with 2x sample loading buffer, boiled for 5 min, and separated by SDS-PAGE. Vac8p was probed in each fraction by Immuno blot and compared to the fractionation profile of vac84 cells that were starved and processed in a similar manner. Fatty acid analysis Cells expressing cTsc13p-EGFP were grown in YPD to approximately 1.0 OD600, at which time they were transferred into SD-N media containing cerulenin or an equal amount of ethanol (mock treatment) to an OD600 of 0.5 and then starved for 24 hrs in the presence or absence of myristic acid. Fatty acid methyl esters (FAMEs) were extracted from 2 OD600 units of cells by acid methanolysis as described previously (Kohlwein et al., 2001). Briefly, cells were treated with 1M methanolic HCl for 40 min at 78°C and then extracted into hexane:ether (1:1). FAMEs were dried, dissolved in hexane, and separated on an Agilent GC 6890 using an HP INNOWax column at constant flow (0.5 ml/min). The oven was ramped from 90°C to 250°C at 40°C/min and held at 250°C for 90 min. FAMEs were detected using an Agilent MS 5973. Internal C21 (heneicosanoic) and C23 (tricosanoic) fatty acid standards, added to the cells prior to extraction, were used to normalize the data. The percentage of each fatty acid species was calculated from the sum of each FAME peak value and averaged among three independent experiments. Immuno blotting Unless otherwise indicated, protein extracts were prepared by TCA precipitation as previously described (Roberts et al., 2003). Tsc13p-EYFP and cTsc13p-EGFP were probed by immunoblot with polyclonal BD Living Colors A.v. peptide GFP antibodies (Clontech). Nvj1p-myc was probed using mouse anti-c-Myc monoclonal antibody (Zymed). Vac8p was probed with polyclonal antibodies raised against purified Vac8p (Pan et al., 2000). Nvj1p and Nvj1p-Fc were probed with polyclonal antibodies raised against a segment of Nvj1p (Kvam and Goldfarb, 2004). All immuno-probed proteins were detected with either horseradish peroxidase-coupled donkey anti-rabbit antibody (Santa Cruz, CA) or horseradish peroxidase-coupled goat anti-mouse antibody (Santa Cruz, CA) where appropriate, and developed by chemilluminescence using Luminol Reagent (Santa Cruz, CA). Quantitative degradation analysis of Nvj1p-myc and cTsc13p-EGFP Cells expressing cTsc13p-EGFP were grown in YPD to log phase (~1 OD600), washed, and shifted into starvation media to an OD600 of 0.5. Cells harboring PGAL1-NVJ1-myc were grown, induced, and shifted into SD-N starvation media in a manner analogous to previously published degradation analyses of Nvj1p (Roberts et al., 2003). Cerulenin was applied to SD-N media at a final concentration of 45 μM and control cells were treated with an equivalent volume of ethanol. Two OD units of culture were then collected immediately (zero point) and equivalent volumes of culture were harvested after 10 and 20 hrs of starvation, respectively. Protein extracts were prepared by TCA precipitation as previously described (Roberts et al., 2003). Samples were separated by SDS-PAGE and immuno-probed as indicated. Cell Imaging Cells were grown in YPD or SCGlu to the indicated OD600 with or without induction of specific reporters. Elo2p-EGFP expression was induced in cells harboring pUG35-ELO2EGFP by culturing in medium lacking methionine for 1.5 hrs. For PCUP1-TSC13-EYFP, PCUP1-NVJ1, and PCUP1-NVJ1-Fc, high levels of expression were obtained by fortifying media with 0.1 mM CuSO4 for the times indicated, while basal levels of expression were achieved by withholding CuSO4. For all experiments SD-N starvation media, CuSO4 was withheld due to the positive feedback of nitrogen starvation on the CUP1 promoter (Gasch et al., 2000). Vacuoles were stained with FM4-64 in rich medium as described previously (Pan and Goldfarb, 1998). In cells starved of nitrogen (SD-N), vacuoles were first stained with FM4-64 in rich media, and then the cells were washed three times with SD-N and starved for the times indicated. For overnight starvation experiments, the localization of cNvj1p-EYFP or cTsc13p-EGFP was analyzed from SCGlu overnight starter cultures after ~15 hrs of starvation (SD-N) in the presence of cerulenin, myriocin, or equivalent amounts of ethanol, with or without myristic acid. DNA was stained immediately prior to microscopic analysis with 5 M Hoechst reagent H-1398 (Molecular Probes). Confocal Microscopy Confocal microscopy was performed on a Leica TCS NT microscope equipped with a 100X Fluorotar lens and UV, Ar, Kr/Ar, and He/Ne lasers (Leica Microsystems, Chantilly VA). Laser power and PMT settings were kept constant for all experiments unless otherwise indicated. PMN morphology was digitally analyzed using the Profile function of the Leica Confocal Software package (Version 2.5, Build 1227), which reports the pixel profile and length of a line segment applied to an image. PMN structures were captured by scanning the Z-axis of the cell for the section that yielded the largest surface area of colocalization between Nvj1p-GFP and FM4-64 labeled vacuole membranes. Line segments were drawn across the lumen of the largest representative PMN structures, starting from the nucleus-vacuole interface and ending at the opposing face of the PMN structure, in a manner that approximately bisected the nuclear PMN bleb or vesicle. The boundaries of each line segment were determined in part by the pixel intensity of Nvj1p-EYFP and/or FM4-64 along the measured section as well as a visual fit. All images were processed using Adobe PhotoShop 5.0 (Adobe Systems, CA).