Human Molecular Genetics, 2003, Vol. 12, No. 22 2981–2991 DOI: 10.1093/hmg/ddg321 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,* Downloaded from https://academic.oup.com/hmg/article/12/22/2981/606598 by guest on 27 September 2021 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 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 The 27-carbon cholesterol molecule is synthesized from acetate in a series of approximately 30 enzymatic reactions. Cholesterol is an essential component of the membranes of The first sterol intermediate in the pathway, lanosterol, is animal cells and a major determinant of membrane fluidity. In formed by the condensation of squalene, a 30-carbon isopre- addition, cholesterol is the substrate for synthesis of steroid noid. This and subsequent reactions in ‘post-squalene’ hormones and bile acids. Isoprenoid intermediates in early cholesterol biosynthesis are known, or believed, to occur in steps of the cholesterol biosynthetic pathway are precursors for the membranes of the endoplasmic reticulum (ER), while the synthesis of isopentyl tRNAs for protein synthesis; dolichol earlier steps in the biosynthetic pathway occur in the cytosol, for N-linked glycosylation of proteins; ubiquinone and heme A peroxisome and/or ER membrane (4–6). The compartmentali- that are cofactors for respiratory chain enzymes in the zation of enzymes involved in the early steps of the cholesterol mitochondria; and farnesyl and geranylgeranyl ligands that biosynthetic pathway may provide a mechanism to help result in the prenylation of key cellular proteins and anchor regulate its synthesis. them to cell membranes. Cholesterol and selected sterol inter- Nsdhl [NAD(P)H steroid dehydrogenase-like] encodes a mediates can be converted to oxysterols that act as regulatory 3b-hydroxysterol dehydrogenase that was identified as part of a signaling molecules and can bind to orphan nuclear receptors positional cloning effort; mutations in the murine gene are including LXRa (reviewed in 1,2). Finally, hedgehog signaling responsible for the X-linked dominant, male lethal mouse proteins that are involved in many important developmental mutations bare patches (Bpa) and striated (Str) (7). Bpa females pathways are modified by the addition of cholesterol in auto- have a skeletal dysplasia and are dwarfed compared with processing reactions that are required for their proper function normal littermates. They develop a hyperkeratotic skin eruption in vivo (reviewed in 3). on postnatal day 5–7 that resolves producing a striping of the *To whom correspondence should be addressed at: Columbus Children’s Research Institute, 700 Children’s Drive, Room W403, Columbus, OH 43205, USA. Tel: þ1 6147222848/9; Fax: þ1 6147222817; Email: [email protected] Human Molecular Genetics, Vol. 12, No. 22 # Oxford University Press 2003; all rights reserved 2982 Human Molecular Genetics, 2003, Vol. 12, No. 22 adult coat consistent with random X-inactivation (8, reviewed To examine the subcellular localization of the protein, GFP- in 9). Milder Bpa alleles, originally thought to be a distinct tagged NSDHL was co-expressed transiently in COS7 cells locus called striated (Str) (10), are indistinguishable from with an ER marker, DsRed2-ER (Fig. 1). NSDHL was detected normal littermates until day 12–14 when the striped coat is first in the ER as evidenced by the complete overlap of the green apparent. Mutations in Nsdhl have been identified in seven Bpa (NSDHL) and red (DsRed2-ER) signals (Fig. 1F), while the and Str alleles, including a nonsense mutation (K103X) in the GFP vector alone produced diffuse nuclear and cytoplasmic original Bpa1H mutant mouse that produces a truncated protein staining (Fig. 1A). Similar results were obtained using that lacks the putative catalytic site and is predicted to be a null N-terminal FLAG tag and C-terminal myc tag NSDHL fusion allele (7,11). Heterozygous mutations in the human NSDHL proteins, demonstrating that the N-terminal GFP tag did not gene were subsequently detected in several females with perturb the three-dimensional structure or the localization of CHILD syndrome (congenital hemidysplasia with ichthyosi- the NSDHL protein. Further, the presence of the C-terminal form nevus and limb defects, MIM 308050) (12–14), a rare myc tag did not prevent proper ER membrane targeting (data Downloaded from https://academic.oup.com/hmg/article/12/22/2981/606598 by guest on 27 September 2021 X-linked, male-lethal malformation syndrome characterized by not shown). The localization was distinct from that of the unilateral ichthyosiform skin lesions and limb reduction defects peroxisomal integral membrane protein PMP70 (20) (data not (reviewed in 9). shown). Most of the experiments described below were The function of the enzyme as a sterol dehydrogenase involved performed using the GFP-Nsdhl construct, which produced in the removal of C-4 methyl groups from the cholesterol the best signals for immunofluorescence. precursor lanosterol was initially suggested by the accumulation In most cells, NSDHL also accumulated on the surface of of 4-methyl and 4,4-dimethyl sterol intermediates in tissue spherical cytoplasmic structures that were often clustered samples and cultured skin fibroblasts from Bpa/Str mutant together. To determine whether the NSDHL-positive structures females and affected male embryos (7) (G. Herman and were LDs, cells were stained with Oil Red O, which stains their R. Kelley, unpublished data). Further support for this role of the neutral lipid core. As shown in Figure 2, the green fluorescence enzyme is our recent demonstration that the murine NSDHL from GFP-NSDHL coated the red core of the LDs stained with protein can rescue the lethality of erg26 deficient S. cerevisiae Oil Red O, demonstrating the presence of the protein on their that lack the yeast ortholog (11). Finally, human X-linked surface. In most successfully transfected cells, all Oil Red dominant chondrodysplasia punctata (CDPX2; MIM 302960) O-positive LDs were also positive for NSDHL. and the X-linked, male-lethal tattered (Td) mouse, whose phenotypes are very similar to that of Bpa mice, result from 8 7 Subcellular localization of selected mutant mutations in the human and murine D –D sterol isomerase NSDHL proteins genes, respectively, encoding the protein for the next step in the cholesterol biosynthetic pathway (15,16). Recently, we have described two ENU-induced alleles, Bpa7H Based on homology with yeast (17,18), it is predicted that and Bpa8H, that result from missense mutations in the NSDHL would be part of a multisubunit ER membrane conserved amino acids V53D and A94T, respectively, yet complex that also contains proteins for a sterol C-4 methyl demonstrate the more severe ‘bare patches’ phenotype in oxidase, a 3-keto sterol reductase, and possibly one or more surviving females (11). Two previous missense mutations, regulatory scaffold proteins. We describe here the subcellu- P98L and V109M, as well as a 3 bp in-frame insertion of a lar localization of the NSDHL protein, confirming its ER tyrosine residue within the predicted membrane spanning localization. Surprisingly, the protein also appears to accu- domain, had all been associated with the milder ‘striated’ mulate on the surface of lipid droplets (LDs) that function as phenotype (7). Thus, we were interested to examine the in vivo intracellular storage compartments for neutral lipids and choles- effects of these new mutations, including possible perturbations terol esters. We further demonstrate that association of NSDHL in proper protein folding. We compared the subcellular with ER membranes and LDs requires prior trafficking through localization of mutant proteins for the Bpa7H and Bpa8H alleles the Golgi, suggesting an additional possible mechanism for with those of wild-type NSDHL and the Bpa1H nonsense allele. the regulation of cellular cholesterol content. Each mutation was generated from the wild type GFP-Nsdhl expression construct