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Human Molecular Genetics, 2003, Vol. 12, No. 22 2981-2991
DOI: 10.1093/hmg/ddg321
© 2003 Oxford University Press

NSDHL, an enzyme involved in cholesterol biosynthesis, traffics through the Golgi and accumulates on ER membranes and on the surface of lipid droplets

Hugo Caldas1 and Gail E. Herman1,2,*

1Center for Molecular and Human Genetics, Columbus Children's Research Institute, Columbus, OH 43205, USA and 2Department of Pediatrics, The Ohio State University, Columbus, OH 43205, USA

Received July 15, 2003; Revised August 29, 2003; Accepted September 11, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NSDHL, for NAD(P)H steroid dehydrogenase-like, encodes a sterol dehydrogenase or decarboxylase involved in the sequential removal of two C-4 methyl groups in post-squalene cholesterol biosynthesis. Mutations in this gene are associated with human CHILD syndrome (congenital hemidysplasia with ichthyosiform nevus and limb defects), an X-linked, male lethal disorder, as well as the mouse mutations bare patches and striated. In the present study, we have investigated the subcellular localization of tagged proteins encoded by wild-type and selected mutant murine Nsdhl alleles using confocal microscopy. In addition to an ER localization commonly found for enzymes of post-squalene cholesterol biosynthesis, we have identified a novel association of NSDHL with lipid droplets, which are endoplasmic reticulum (ER)-derived cytoplasmic structures that contain a neutral lipid core. We further demonstrate that trafficking through the Golgi is necessary for ER membrane localization of the protein and propose a model for the association of NSDHL with lipid droplets. The dual localization of NSDHL within ER membranes and on the surface of lipid droplets may provide another mechanism for regulation of the levels and sites of accumulation of intracellular cholesterol.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cholesterol is an essential component of the membranes of animal cells and a major determinant of membrane fluidity. In addition, cholesterol is the substrate for synthesis of steroid hormones and bile acids. Isoprenoid intermediates in early steps of the cholesterol biosynthetic pathway are precursors for the synthesis of isopentyl tRNAs for protein synthesis; dolichol for N-linked glycosylation of proteins; ubiquinone and heme A that are cofactors for respiratory chain enzymes in the mitochondria; and farnesyl and geranylgeranyl ligands that result in the prenylation of key cellular proteins and anchor them to cell membranes. Cholesterol and selected sterol intermediates can be converted to oxysterols that act as regulatory signaling molecules and can bind to orphan nuclear receptors including LXR{alpha} (reviewed in 1,2). Finally, hedgehog signaling proteins that are involved in many important developmental pathways are modified by the addition of cholesterol in autoprocessing reactions that are required for their proper function in vivo (reviewed in 3).

The 27-carbon cholesterol molecule is synthesized from acetate in a series of approximately 30 enzymatic reactions. The first sterol intermediate in the pathway, lanosterol, is formed by the condensation of squalene, a 30-carbon isoprenoid. This and subsequent reactions in ‘post-squalene’ cholesterol biosynthesis are known, or believed, to occur in the membranes of the endoplasmic reticulum (ER), while earlier steps in the biosynthetic pathway occur in the cytosol, peroxisome and/or ER membrane (4–6). The compartmentalization of enzymes involved in the early steps of the cholesterol biosynthetic pathway may provide a mechanism to help regulate its synthesis.

Nsdhl [NAD(P)H steroid dehydrogenase-like] encodes a 3ß-hydroxysterol dehydrogenase that was identified as part of a positional cloning effort; mutations in the murine gene are responsible for the X-linked dominant, male lethal mouse mutations bare patches (Bpa) and striated (Str) (7). Bpa females have a skeletal dysplasia and are dwarfed compared with normal littermates. They develop a hyperkeratotic skin eruption on postnatal day 5–7 that resolves producing a striping of the adult coat consistent with random X-inactivation (8, reviewed in 9). Milder Bpa alleles, originally thought to be a distinct locus called striated (Str) (10), are indistinguishable from normal littermates until day 12–14 when the striped coat is first apparent. Mutations in Nsdhl have been identified in seven Bpa and Str alleles, including a nonsense mutation (K103X) in the original Bpa1H mutant mouse that produces a truncated protein that lacks the putative catalytic site and is predicted to be a null allele (7,11). Heterozygous mutations in the human NSDHL gene were subsequently detected in several females with CHILD syndrome (congenital hemidysplasia with ichthyosiform nevus and limb defects, MIM 308050) (12–14), a rare X-linked, male-lethal malformation syndrome characterized by unilateral ichthyosiform skin lesions and limb reduction defects (reviewed in 9).

The function of the enzyme as a sterol dehydrogenase involved in the removal of C-4 methyl groups from the cholesterol precursor lanosterol was initially suggested by the accumulation of 4-methyl and 4,4-dimethyl sterol intermediates in tissue samples and cultured skin fibroblasts from Bpa/Str mutant females and affected male embryos (7) (G. Herman and R. Kelley, unpublished data). Further support for this role of the enzyme is our recent demonstration that the murine NSDHL protein can rescue the lethality of erg26 deficient S. cerevisiae that lack the yeast ortholog (11). Finally, human X-linked dominant chondrodysplasia punctata (CDPX2; MIM 302960) and the X-linked, male-lethal tattered (Td) mouse, whose phenotypes are very similar to that of Bpa mice, result from mutations in the human and murine {Delta}8–{Delta}7 sterol isomerase genes, respectively, encoding the protein for the next step in the cholesterol biosynthetic pathway (15,16).

Based on homology with yeast (17,18), it is predicted that NSDHL would be part of a multisubunit ER membrane complex that also contains proteins for a sterol C-4 methyl oxidase, a 3-keto sterol reductase, and possibly one or more regulatory scaffold proteins. We describe here the subcellular localization of the NSDHL protein, confirming its ER localization. Surprisingly, the protein also appears to accumulate on the surface of lipid droplets (LDs) that function as intracellular storage compartments for neutral lipids and cholesterol esters. We further demonstrate that association of NSDHL with ER membranes and LDs requires prior trafficking through the Golgi, suggesting an additional possible mechanism for the regulation of cellular cholesterol content.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Subcellular localization of the NSDHL protein
The predicted amino acid sequence of murine NSDHL (7) includes 362 amino acids and contains an N-terminal NADH cofactor binding site and a central Tyr–X3–Lys motif that is present at the active site of all known 3ß-hydroxysteroid dehydrogenases (3ß-HSDs) (19). The SOSUI system, hydrophobic cluster analysis (HCA), and DNASTAR Protean 4.05 each predict that the enzyme contains a single 22-amino acid hydrophobic transmembrane domain between residues 285 and 307 organized in an {alpha}-helical conformation.

To examine the subcellular localization of the protein, GFP-tagged NSDHL was co-expressed transiently in COS7 cells with an ER marker, DsRed2-ER (Fig. 1). NSDHL was detected in the ER as evidenced by the complete overlap of the green (NSDHL) and red (DsRed2-ER) signals (Fig. 1F), while the GFP vector alone produced diffuse nuclear and cytoplasmic staining (Fig. 1A). Similar results were obtained using N-terminal FLAG tag and C-terminal myc tag NSDHL fusion proteins, demonstrating that the N-terminal GFP tag did not perturb the three-dimensional structure or the localization of the NSDHL protein. Further, the presence of the C-terminal myc tag did not prevent proper ER membrane targeting (data not shown). The localization was distinct from that of the peroxisomal integral membrane protein PMP70 (20) (data not shown). Most of the experiments described below were performed using the GFP-Nsdhl construct, which produced the best signals for immunofluorescence.



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  		Figure 1. Subcellular localization of murine wild type and mutant GFP-NSDHL fusion proteins. GFP–Nsdhl fusion constructs were transiently transfected into COS7 cells together with an ER-localization marker, DsRed2-ER. Cells were visualized by direct fluorescence using confocal microscopy. GFP fluorescence results in green pixels (A, D, G and J), DsRed2 produces red pixels (B, E, H and K) while co-localization of GFP and DsRed2 results in yellow pixels (C, F, I and L). (A, B, C) Empty GFP vector; (D, E, F) wild-type GFP-Nsdhl; (G, H, I) fusion construct containing the Bpa1H nonsense mutation; (J, K, L) fusion construct containing the Bpa8H missense mutation. Fusion proteins containing the wild type and Bpa8H mutation correctly localize to the ER as evidenced by the overlap of the two fluorescent channels. Protein containing the Bpa1H mutation presents a pattern of predominantly nuclear staining, similar to that observed with GFP vector alone.

 
In most cells, NSDHL also accumulated on the surface of spherical cytoplasmic structures that were often clustered together. To determine whether the NSDHL-positive structures were LDs, cells were stained with Oil Red O, which stains their neutral lipid core. As shown in Figure 2, the green fluorescence from GFP-NSDHL coated the red core of the LDs stained with Oil Red O, demonstrating the presence of the protein on their surface. In most successfully transfected cells, all Oil Red O-positive LDs were also positive for NSDHL.



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  		Figure 2. Localization of GFP–NSDHL in LDs and requirement of C-terminal RKDK sequence for ER and LD targeting. The transfected constructs were the GFP vector (A–C), wild-type GFP-Nsdhl (D–F, J), GFP–Nsdhl containing the Bpa8H mutation (G–I), or GFP–Nsdhl {Delta}RKDK containing a stop codon at amino acid 359 (K, L). NSDHL protein localized to the ER is not well seen in (A–I) since images were optimized for visualization of LD targeted protein. Cells in (J–L) were counterstained with DAPI to visualize the cell nuclei.

 
Subcellular localization of selected mutant NSDHL proteins
Recently, we have described two ENU-induced alleles, Bpa7H and Bpa8H, that result from missense mutations in the conserved amino acids V53D and A94T, respectively, yet demonstrate the more severe ‘bare patches’ phenotype in surviving females (11). Two previous missense mutations, P98L and V109M, as well as a 3 bp in-frame insertion of a tyrosine residue within the predicted membrane spanning domain, had all been associated with the milder ‘striated’ phenotype (7). Thus, we were interested to examine the in vivo effects of these new mutations, including possible perturbations in proper protein folding. We compared the subcellular localization of mutant proteins for the Bpa7H and Bpa8H alleles with those of wild-type NSDHL and the Bpa1H nonsense allele.

Each mutation was generated from the wild type GFP-Nsdhl expression construct by site-directed mutagenesis. The protein representing the Bpa1H allele was found throughout the cytoplasm and to some extent in the nucleus (Fig. 1G–I), similar to the GFP vector itself (Fig. 1A–C). It did not co-localize with the ER, as evidenced by a lack of substantial overlap with the DsRed2-ER marker. There was also some accumulation in unidentified cytoplasmic inclusions that did not stain with Oil Red O (not shown) and, hence, were not LDs. The Bpa7H (V53D) protein demonstrated a diffuse cytoplasmic and nuclear subcellular localization very similar to that of the Bpa1H protein (not shown). The protein produced from the Bpa8H allele (A94T) correctly localized to the ER (Fig. 1J–L) and also accumulated on the surface of LDs (Fig. 2G–I), similar to the wild type NSDHL protein.

To assess whether the mutant Bpa1H and Bpa7H proteins were degraded and the resulting immunofluorescence was derived solely from GFP and whether any of the mutant proteins were membrane-bound, we performed western blotting on membrane and soluble cytosolic fractions isolated from cells transiently transfected with wild-type and mutant GFP-Nsdhl constructs (Fig. 3). As expected, both the wild-type and Bpa8H GFP-NSDHL fusion proteins were found exclusively in the membrane fraction and were detected by both anti-GFP antibodies and polyclonal antibodies directed against a C-terminal epitope of the murine NSDHL protein (Fig. 3 top and middle). The truncated GFP-NSDHL protein corresponding to the Bpa1H allele, with a predicted size of 39 kDa, produced an appropriately sized protein that was found primarily in the cytosolic fraction, consistent with the lack of a transmembrane domain (Fig. 3 bottom). This fusion was detected solely by the anti-GFP antibody since the anti-NSDHL C-terminal epitope is not present in the Bpa1H mutant protein. The GFP-NSDHL protein encoded by the Bpa7H allele was not detected with either antibody, suggesting that this fusion protein is not stable, at least under the conditions studied here.



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  		Figure 3. Subcellular fractionation and western blotting of COS7 cells transfected with GFP–Nsdhl fusion protein constructs. Cells were harvested for subcellular fractionation 48 h post-transfection. Equal amounts (50 µg) of membrane and cytosolic protein fractions were subjected to 12% SDS–PAGE, electroblotted and immunoprobed with polyclonal anti-NSDHL antibody (top), or polyclonal anti-GFP antibody (middle and bottom). Molecular size markers (Kaleidoscope pre-stained markers, BioRad) were used to estimate molecular weights. The GFP protein is predicted to have a molecular weight of 28 kDa, while the N-terminal 102 amino acids of NSDHL have a predicted molecular weight of 11 kDa. The full length, unmodified NSDHL protein has a predicted molecular weight of 41 kDa. M, membrane fraction, C, cytosolic fraction. Adequate separation of soluble and membrane fractions as well as equal protein loading was assessed by stripping and reprobing membranes with an anti-calnexin antibody (data not shown), which detects an abundant 90 kDa ER-resident transmembrane protein (25).

 
The C-terminal amino acids RKDK act as an ‘ER retrieval’ signal
The C-terminal amino acid sequence ‘KKXX’ has been demonstrated to act as a consensus signal sequence for retrograde transport of ER membrane proteins from the Golgi by the COPI complex (21–23). Murine NSDHL contains a modified version of this sequence, RKDK. To test the hypothesis that the C-terminal RKDK motif is responsible for the ER targeting of the wild-type NSDHL protein, we performed site-directed mutagenesis and converted the first amino acid in this motif to a stop codon (creating the mutant protein designated GFP–NSDHL–{Delta}RKDK). The GFP–NSDHL–{Delta}RKDK protein remained membrane-bound (Fig. 4); however it was localized exclusively in cytoplasmic perinuclear structures consistent with the Golgi apparatus (Fig. 2K and L). These results are consistent with the function of the C-terminal RKDK as a COPI-complex mediated ER retrieval signal.



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  		Figure 4. Subcellular fractionation and western blotting of NSDHL protein lacking the putative C-terminal ER targeting signal. COS7 cells were transfected with plasmids encoding either wild-type GFP–NSDHL or GFP–NSDHL–{Delta}RKDK. After 48 h, protein extracts were subjected to subcellular fractionation, and 25 µg of membrane or cytosolic proteins analyzed as in Figure 3 with an anti-GFP antibody (top) or anti-NSDHL antibody (bottom). NSDHL–{Delta}RKDK is not detected by the anti-NSDHL antibody due to loss of part of the C-terminal epitope.

 
There was also no accumulation of the GFP–NSDHL–{Delta}RKDK protein on the surface of cytoplasmic LDs. LDs are known to derive from the ER, where triacylglycerides and cholesteryl esters are synthesized, and recent data are consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the ER membrane (24). As discussed below, some proteins found on LDs are believed to bud from the ER membrane with the droplet as it forms. Thus, we believe that the lack of accumulation of GFP–NSDHL–{Delta}RKDK on LDs strongly suggests that a prior ER membrane localization is required for this association. However, we cannot completely exclude the possibility that NSDHL accumulates on LDs without prior retrieval to the ER, that LD localization precedes incorporation into the ER membrane, and/or that the C-terminal RKDK sequence is specifically required for LD association of NSDHL.

Some of the known proteins found on the surface of LDs demonstrate an ER membrane topology in which both the N- and C-termini are exposed to the cytoplasmic side of the membrane. Movement of such proteins onto the surface of LDs occurs as the droplet buds from the ER membrane (see Fig. 5). In this model, ER lumenal proteins and those spanning the ER membrane would be excluded from entry into the LD, although exceptions do occur (22).



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  		Figure 5. Model of trafficking of selected ER membrane proteins to the surface of LDs (modified from 32). Neutral lipids accumulate in the interior of the ER bilayer, making a bulge that eventually buds into the cytoplasm to form a droplet surrounded by an ER-derived phospholipid monolayer. This process would drive opposite ER membrane monolayers apart, thickening the hydrophobic portion of the membrane. ER membrane proteins, such as NSDHL (blue), with N- and C-terminal surfaces exposed to the cytoplasm can easily diffuse laterally between the ER membrane proper and the monolayer surrounding the nascent droplets. Other proteins on the surface of the LD are shown in green and orange.

 
Surface probability plots predict the N-terminal major portion of NSDHL flanking the hydrophobic transmembrane domain to be localized on the cytoplasmic side of the ER membrane, while predictions for the smaller C-terminal region were uncertain (data not shown). To determine whether NSDHL may be recruited to LDs as they bud from the ER, we determined whether the C-terminus of the protein is exposed to the cytoplasm or ER lumen by exploiting the differential sensitivity of the plasma and ER membranes to permeabilization with non-ionic detergents. As shown in Figure 6, when HeLa cells are permeabilized with saponin, the ER membrane protein calnexin with a C-terminal epitope exposed to the cytoplasm is readily detected (25), while a C-terminal epitope of protein disulfide isomerase (PDI), which resides in the ER lumen, is only exposed upon harsher treatment with Triton X-100. The carboxy-terminus of the NSDHL protein detected with an antibody to either a C-terminal myc tag or the C-terminal amino acids of the murine protein is readily accessible in saponin-treated cells, strongly favoring a topology of the protein in the ER membrane as shown in Figure 5 in which both the N- and C-termini are exposed to the cytoplasm.



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  		Figure 6. Analysis of the topology of NSDHL in the ER membrane by differential permeabilization of transiently transfected HeLa cells. Transfected constructs are shown to the left and the non-ionic detergent used for permeabilization is shown at the top. NSDHL protein was detected with antibody to the C-terminal myc tag (myc) or antibody to a C-terminal NSDHL epitope (NSDHL). The blue signal represents DAPI staining and nuclear fluorescence. PDI=protein disulfide isomerase, a protein found in the ER lumen.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The conversion of lanosterol to cholesterol involves the removal of three methyl groups, one at the C-14 position and two at the C-4 position; the reduction of double bonds at the C-24 and C-14 positions; and the ‘migration’ of the C-8(9) double bond to the C-5 position (reviewed in 5) (Fig. 7). Nine enzymes complete this series of reactions, including three enzymes involved in the sequential removal of the two C-4 methyl groups—a sterol-C4-methyl-oxidase (encoded by the SC4MOL gene in human) (26), the NSDHL 3ß-hydroxysterol dehydrogenase and a 3ß-ketosteroid reductase (encoded by the HSD17B7 gene) (27). It has been suggested (4,5) that all of these enzymatic reactions occur on the ER membrane, due in part to the poor solubility of their sterol substrates. An ER membrane localization has been suggested based on partial enzyme purification for some of the enzymes (summarized in 5); however, direct demonstration of a subcellular localization using tagged constructs from cloned genes has been reported for only four of the mammalian enzymes: 3ß-hydroxysterol-{Delta}14-reductase activity has recently been demonstrated in the Lamin B receptor (28,29), which is localized on the inner nuclear membrane (29). GFP-tagged 3ß-ketosterol reductase protein has been localized to the ER membrane (27). The {Delta}8–{Delta}7–sterol isomerase has been localized to ER membranes as well as the nuclear membrane (30). Finally, human 3ß-hydroxysteroid-{Delta}7-reductase (DHCR7) has been localized to the ER membrane by immunofluorescence of tagged protein in COS7 cells (29). Subcellular localizations for the remainder of the mammalian proteins have not been directly determined, although analysis of predicted protein sequences suggests an integral membrane location for all of them.



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  		Figure 7. Schematic representation of ‘post-squalene’ cholesterol biosynthesis. Individual enzymes, denoted by numbers in parentheses, are: (1) 3ß-hydroxysteroid-{Delta}24-reductase, (2) lanosterol 14{alpha}-demethylase, (3) 3ß-hydroxysteroid-{Delta}14-reductase, (4) 3ß-hydroxysteroid C-4 methyl oxidase, (5) NSDHL, 3ß-hydroxysteroid dehydrogenase, (6) 3ß-keto-steroid reductase, (7) 3ß-hydroxysteroid–{Delta}8–{Delta}7–sterol isomerase, (8) 3ß-hydroxysteroid-{Delta}5-desaturase (lathosterol dehydrogenase), and (9) 3ß-hydroxysteroid-{Delta}7-reductase (DHCR7). Reduction of the C-24 double bond can occur at several points along the pathway, but is shown only as the first step for simplification.

 
We have demonstrated here that exogenously expressed murine NSDHL is localized on ER membranes and also accumulates on the surface of LDs. This is the first known association of a cholesterol biosynthetic enzyme with this ubiquitous cytoplasmic organelle. LDs are involved in the storage of neutral lipids, specifically, triacylglycerols and cholesterol esters. They are found in selected prokaryotes and in a wide variety of cell types of virtually all eukaryotes, including fungi and plants (reviewed in 31–33). However, we are only beginning to understand their biogenesis, as well as their possible functions in intracellular cholesterol transport and signaling, in addition to lipid storage.

In animal cells, LDs are most abundant in adipocytes, where they range from 2 to 100 µm in diameter, and in steroidogenic tissues such as the adrenal cortex and liver. The major proteins coating the surface of LDs in animal cells demonstrate regions of homology in their amino acid sequences and have been called PAT proteins [for perilipins, adipose-differentiation-related-protein (ADRP), and TIP47 (34)]. The proteins are tissue-specific with ADRP found in LDs in all tissues as well as in pre-adipocytes, while perilipins are found only in mature adipocytes and in steroidogenic cells (35). In addition to their structural role, altered gene expression during differentiation or after hormone-stimulated lipolysis suggests that the association of these proteins with LDs may have regulatory roles in cellular lipid metabolism. Recently, additional proteins that demonstrate homology to perilipins and ADRP have been noted to associate with LDs, including S3-12 (36) and TIP47 (34), initially identified as a protein involved in trafficking of the mannose-6-phosphate receptor. In addition, caveolins that are overexpressed or modified by truncations or the addition of ER retrieval sequences have been found on the surface of LDs, although the physiologic significance of these findings is not known (32,37). Caveolins are found primarily in invaginations of the plasma membrane called caveolae that are enriched in cholesterol and glycosphingolipids and represent a subset of lipid rafts (38). They are involved in a variety of cellular processes including endocytosis, cell signaling and intracellular trafficking, suggesting that their presence on LDs could have physiologic importance. LDs in some plant seeds contain a continuous coating of proteins called oleosins (39).

As shown in Figure 5, LDs are believed to bud from the ER membrane and contain a single phospholipid bilayer derived from the outer (cytoplasmic) ER membrane surrounding a central lipid core. Proteins are excluded from the core but can coat the surface of the droplet. The mechanism of association of the known lipid droplet proteins with the organelle is not completely understood. Perilipins and ADRP appear to be translated on free ribosomes and associate with preformed LDs (35). Oleosins and caveolins have N- and C-terminal hydrophilic domains that face the cytoplasm flanking a central hydrophobic one and could bud from the ER with the newly formed lipid droplet (32,35,40).

We have demonstrated that ER retrieval and trafficking is likely necessary for the accumulation of NSDHL protein on LDs. Protein lacking the ER retrieval signal that is sequestered in the Golgi complex does not coat LDs, despite the presence of the transmembrane domain and all but the last four amino acids of the protein (Fig. 2J–L). Further, like oleosins and caveolins, NSDHL demonstrates a topology in which both the N- and C-terminal domains extrude from the ER membrane into the cytoplasm and flank a single transmembrane region. We hypothesize that NSDHL in the ER membrane can diffuse laterally to nascent LDs. Despite having no transmembrane domain, it is known that the yeast ortholog of NSDHL (ERG26) is intimately associated with ER membranes (17,41,42), suggesting that other regions of the protein may facilitate lipid binding.

We have also examined the subcellular localization of selected mutant NSDHL proteins. NSDHL protein from the Bpa1H allele that is missing the C-terminal 260 amino acids, including the 22 amino acid hydrophobic domain, is not membrane associated and does not associate with LDs (Figs 1G–I and 3). These data suggest that this domain is necessary for ER membrane association and, hence, indirectly for LD association. Protein from the Bpa7H allele, which contains the missense mutation V53D, retains both the central hydrophobic domain and the ER retrieval signal, yet had a diffuse nuclear and cytoplasmic localization similar to Bpa1H and the empty GFP vector (not shown). The immunofluorescence likely derives from GFP plus variable portions of an unstable mutant protein since we could not detect it on western blots with antibodies to either GFP or NSDHL (Fig. 3). This instability could be related to improper protein folding since the mutation results in the substitution of a charged aspartate residue within a small cluster of hydrophobic residues, breaking the predicted secondary conformation of a ß-strand. In addition, the preceding amino acid, T52, is predicted to be phosphorylated; after introduction of the mutation, this phosphorylation site is lost. In contrast, protein for Bpa8H allele demonstrates proper targeting to the ER as well as to LDs (Figs 1J–L and 2G–I). The mechanism by which the A94T mutation produces a non-functional protein remains unknown and may require determination of the three-dimensional structure of the protein.

Finally, we have shown that the RKDK sequence found at the C-terminus of the NSDHL protein acts as an ER retrieval signal as evidenced by the retention in the Golgi complex of protein lacking these four amino acids (Fig. 3J–L). The traditional consensus signal sequence for retrograde transport of ER membrane proteins from the Golgi by the COPI complex is the C-terminal amino acids KKXX (21–23). Our data suggest that, despite divergence in sequence, the variant RKDK motif is still sufficient to promote retrieval of NSDHL to the ER via the retrograde pathway.

The functional significance of the association of NSDHL with LDs is not known. Unesterified cholesterol has been reported on the surface of LDs, but not within its core (43,44), raising the possibility that one or more steps of cholesterol biosynthesis could occur on the LD surface. In S. cerevisiae, squalene epoxidase encoded by the erg1 gene, lanosterol synthase or oxidosqualene cyclase encoded by the erg7, and erg6 encoded sterol-{Delta}24-methyltransferase are located primarily in LDs (33,45,46) suggesting a possible evolutionarily conserved role for the LD localization of enzymes involved in sterol biosynthesis. The yeast NSDHL ortholog (ERG26 protein) has not been found on lipid droplets; however, the erg27 encoded 3-ketosterol reductase has recently been demonstrated to interact with and facilitate association of the yeast oxidosqualene cyclase with LDs and is necessary for enzymatic activity of the cyclase in this organelle (46).

Recent demonstrations of distinct subcellular locations for several of the mammalian enzymes of post-squalene cholesterol biosynthesis—the ER, the nuclear membrane and LDs—could provide another level of complexity in regulating the amount of cholesterol synthesized in the cell. Alternatively, since the pattern of staining of NSDHL on LDs suggests that the protein coats most, if not all, of the surface of droplets, similarly to PAT proteins, it could have a function unrelated to its role as a cholesterol biosynthetic enzyme. To help differentiate between these two possibilities, it will be important to determine whether the other proteins of the C-4 demethylase complex co-localize with NSDHL on LDs.

Finally, just as we completed these studies, Ohashi et al. (47) reported a similar localization of endogenous and C-terminal VSV-G-tagged human NSDHL protein on the surface of LDs of CHO cells. The protein demonstrated a co-localization with ADRP and TIP47. They found significant localization in the ER only in cells expressing high levels of the protein. Treatment with sterol-depleted medium reduced the development of LDs in cells, resulting in re-distribution of NSDHL to the ER. Their studies complement those reported here that demonstrate the likely mechanism of LD targeting of NSDHL. Together, these results stress the importance of and need for further studies to understand the biological processes in which LDs participate and to examine whether other cholesterol biosynthetic or regulatory proteins are found on these organelles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials and plasmid constructs
The pDsRed2-ER subcellular localization vector was obtained from Clontech. Oil Red O and saponin were purchased from Sigma Chemical Co. Custom synthetic oligonucleotides were purchased from Sigma-Genosys. GFP-tagged NSDHL was generated by subcloning the murine Nsdhl cDNA (7) into the BamHI site of pEGFP-C1 (Clontech) yielding an in-frame fusion 3' to GFP. Mutations to generate the Bpa1H, Bpa7H and Bpa8H alleles were introduced into the wild-type fusion construct using the Quikchange® XL site-directed mutagenesis kit (Stratagene) and specific complementary oligonucleotides designed to contain the desired mutations. NSDHL–{Delta}RKDK, lacking the C-terminal residues 359–RKDK–362, was similarly generated by introducing a stop codon at the position of R359. All mutations were confirmed by sequencing.

Antibodies
Polyclonal rabbit antisera were raised against a C-terminal peptide VERTVQSFHHLRKDK corresponding to amino acids 348–362 of the mouse NSDHL protein (Cocalico Biologicals) and affinity purified using a Sulfolink kit (Pierce). Rabbit anti-GFP(FL) (Santa Cruz Biotechnology Inc.), rabbit anti-calnexin (Stressgen), mouse anti-myc (Invitrogen), mouse anti-PDI (Affinity Bioreagents), goat anti-mouse FITC and goat anti-rabbit FITC (Sigma), goat anti-rabbit Texas red (Jackson Laboratories), and goat anti-rabbit IgG horseradish peroxidase conjugate (Cell Signalling) were used at the dilutions described below. The antibody to the peroxisomal membrane protein PMP70 was the generous gift of D. Valle and G. Jimenez-Sanchez, The Johns Hopkins University. Vectashield and Vectashield-DAPI mounting media were obtained from Vector Labs.

Cell culture and immunofluorescence
COS7 and HeLa cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's media (Mediatech) supplemented with 10% BCS (bovine calf serum, defined/supplemented; HyClone) containing 2 mM L-glutamine (Invitrogen) at 37°C and 5% CO2. Transient transfections with Effectene transfection reagent were performed as recommended by the supplier (Qiagen), and cells were studied 48 h after transfection.

Transiently transfected COS7 cells grown on 13 mm glass coverslips were fixed in a solution of 2% sucrose and 1.8% formaldehyde in PBS for 10 min at room temperature and mounted with Vectashield. For staining of LDs, fixed cells were briefly rinsed in 60% propan-2-ol followed by incubation with 0.6% Oil Red O in 60% propan-2-ol for 2 min at room temperature, rinsed in 60% propan-2-ol, PBS and water, and mounted as above. For selective permeabilization of the plasma membrane, HeLa cells grown on 13 mm glass coverslips were fixed in 2% paraformaldehyde for 30 min, permeabilized with either 20 µg/ml saponin (selectively permeabilized cells) or 0.2% Triton X-100 (permeabilized cells). The cells were washed with PBS, blocked with PBS/5% FBS and incubated with primary antibodies (anti-myc, 1 : 500; anti-NSDHL, 1 : 200; anti-PDI, 1 : 100; anti-calnexin, 1 : 200). The cells were washed and incubated with a secondary antibody (anti-mouse FITC, 1 : 32; anti-rabbit Texas Red, 1 : 200; anti-rabbit FITC, 1 : 80), washed and mounted as above. For selectively permeabilized cells, all incubations and washes were performed in the presence of 20 µg/ml saponin.

Confocal microscopy was performed on a Zeiss LSM510 Laser Scanning Microscope. Image analysis was performed using the standard system operating software provided with the microscope. GFP was detectable in the FITC channel (green), DsRed2 and Oil Red O in the Texas Red channel (red), and DAPI in the near infrared channel (blue). Co-localization of green (GFP/FITC) and red (DsRed2/Texas Red) signals in a single pixel produced yellow, while separated signals remained green or red.

Membrane and cytosol separation
Membrane and cytosolic protein separation was performed with modifications of a procedure described by Martinez-Botas et al. (48). Briefly, transiently transfected 100 mM dishes of cells from different experimental conditions were rinsed with cold PBS and collected in sonication buffer [2 mM EDTA, 2 mM PMSF, and complete protease inhibitor cocktail (Roche) in PBS]. Cell suspensions were subjected to three freeze–thaw cycles, followed by a 5 min sonication in an ice-bath (2 s pulse, 50% amplitude) using a 100 W high intensity ultrasonic processor (Sonics and Materials Inc.). Cell membranes were pelleted by centrifugation at 50 000g for 30 min at 4°C in a Beckman Optima TLX ultracentrifuge using a TLA100.2 rotor. The supernatant containing the cytosol fraction was removed and the membrane pellet dissolved in resuspension buffer (100 mM Tris, pH 7.5, 300 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 2 mM PMSF, complete protease inhibitor cocktail). The amount of protein in the membrane and cytosol extracts was quantified using the BCA method (Pierce). Both fractions were boiled in Laemmli sample buffer [100 mM Tris–HCl pH 6.8, 4% (w/v) SDS, 200 mM ß-mercaptoethanol, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue] and stored at -20°C.

Immunoblot analysis
Equal amounts of protein were electrophoretically resolved on 12% SDS-polyacrylamide gels at 150 V for 1 h 30 min using vertical slab units, and transferred onto Hybond-ECL membranes (Amersham) for 1 h at 180 mA. Non-specific sites on the membrane were blocked by incubation for 1 h at room temperature in 10% non-fat dry milk powder (Carnation) in TBS (20 mM Tris–HCl, 137 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBST). Membranes were probed with NSDHL antibody (1 : 500), GFP antibody (1 : 200) or calnexin antibody (1 : 5000) in TBST containing 5% non-fat dry milk. Detection of the antigen–antibody complexes was performed with horseradish peroxidase conjugated secondary antibody (1 : 1000) and the enhanced chemiluminescence kit (Amersham). Membranes were exposed to X-Omat film S (Kodak) for variable lengths of time (1 s to 1 min 30 s). Prior to reprobing the membranes, they were stripped by treatment with 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris pH 6.7 for 30 min at 55°C with agitation. The membranes were washed with large volumes of TBST 2x10 min. Blocking and immunodetection of stripped membranes was performed as above.

Protein motif and structure prediction
DNASTAR family of programs, Hydrophobic Cluster Analysis (HCA) (49), and SOSUI system (50) were used for secondary structure prediction, hydrophobicity and surface probability plots. NetPhos 2.0 was used for the prediction of phosphorylation sites (51).


   ACKNOWLEDGEMENTS
 
The authors thank R. Burry, The Ohio State University for helpful discussions concerning cell permeabilization. This work was supported by NIH R01 HD38572 to G.E.H. DNA sequences were determined with the help of the Sequencing Core Facility at Columbus Children's Research Institute and confocal microscopy was performed in a Core Facility of the Comprehensive Cancer Center of The Ohio State University.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Columbus Children's Research Institute, 700 Children's Drive, Room W403, Columbus, OH 43205, USA. Tel: +1 6147222848/9; Fax: +1 6147222817; Email: hermang{at}pediatrics.ohio-state.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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