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Targeted Mutation of the MLN64 START Domain Causes Only Modest Alterations in Cellular Sterol Metabolism

2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês

10.1074/jbc.m400717200

1083-351X

Tatsuro Kishida, Igor Kostetskii, Zhibing Zhang, Federico Martı́nez, Pei Liu, Steven U. Walkley, Nancy K. Dwyer, E. Joan Blanchette‐Mackie, Glenn L. Radice, Jerome F. Strauss,

Cholesterol and Lipid Metabolism

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice. The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice. The mechanisms by which hydrophobic lipids like sterols are moved within cells and targeted to specific membranes is not well understood. Recent findings indicate that a family of proteins related to the steroidogenic acute regulatory protein (StAR) 1The abbreviations used are: StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid; PBS, Dulbecco's phosphate-buffered saline; 8-Br-cAMP, 8-bromo-cyclic AMP; HMG, hydroxymethylglutaryl; GM3, NeuAcα 2,3Galβ1,4Glc-ceramide. perform critical functions in moving lipids within cells (1Christenson L.K. Strauss III, J.F. Biochim. Biophys. Acta. 2000; 1529: 175-187Google Scholar, 2Soccio R.E. Breslow J.L. J. Biol. Chem. 2003; 278: 22183-22186Google Scholar, 3Hanada K. Kumagai K. Yasuda S. Miura Y. Kawano M. Fukasawa M. Nishijima M. Nature. 2003; 426: 803-809Google Scholar, 4Soccio R.E. Adams R.M. Romanowski M.J. Sehayek E. Burley S.K. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6943-6948Google Scholar). StAR, the prototype of the family, promotes the translocation of cholesterol from the outer to the inner mitochondrial membrane and cholesterol side-chain cleavage enzyme in steroidogenic cells (1Christenson L.K. Strauss III, J.F. Biochim. Biophys. Acta. 2000; 1529: 175-187Google Scholar, 5Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss III, J.F. J. Biol. Chem. 1998; 273: 16339-16345Google Scholar, 6Kallen C.B. Billheimer J.T. Summers S.A. Stayrook S.E. Lewis M. Strauss III, J.F. J. Biol. Chem. 1998; 273: 26285-26288Google Scholar). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia, a cholesterol storage disorder in which synthesis of all gonadal and adrenal cortical steroid hormones is severely impaired; the cholesterol that is not efficiently moved into the mitochondria accumulates in cytoplasmic lipid droplets (7Lin D. Sugawara T. Strauss III, J.F. Clark B.J. Stocco D.M. Sanger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Google Scholar, 8Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Google Scholar). The C terminus of StAR possesses sterol transfer activity, and it has been named the StAR-related lipid transfer (START) domain (2Soccio R.E. Breslow J.L. J. Biol. Chem. 2003; 278: 22183-22186Google Scholar, 6Kallen C.B. Billheimer J.T. Summers S.A. Stayrook S.E. Lewis M. Strauss III, J.F. J. Biol. Chem. 1998; 273: 26285-26288Google Scholar, 9Ponting C.P. Aravind L. Trends Biochem. Sci. 1999; 24: 130-132Google Scholar). It consists of a 210-amino acid residue sequence that forms a compact α/β structure, a helix-grip fold, with a hydrophobic tunnel that can accommodate a sterol molecule (2Soccio R.E. Breslow J.L. J. Biol. Chem. 2003; 278: 22183-22186Google Scholar, 10Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Google Scholar, 11Romanowski M.J. Soccio R.E. Breslow J.L. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6949-6954Google Scholar, 12Roderick S.L. Chan W.W. Agate D.S. Olsen L.R. Vetting M.W. Rajashankar K.R. Cohen D.E. Nat. Struct. Biol. 2002; 9: 507-511Google Scholar). START domains can bind sterol, facilitate the transfer of cholesterol from sterol-rich unilammelar liposomes to acceptor membranes, and stimulate steroidogenesis when expressed in cells co-expressing the cholesterol side-chain cleavage system or when added to isolated steroidogenic mitochondria (5Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss III, J.F. J. Biol. Chem. 1998; 273: 16339-16345Google Scholar, 6Kallen C.B. Billheimer J.T. Summers S.A. Stayrook S.E. Lewis M. Strauss III, J.F. J. Biol. Chem. 1998; 273: 26285-26288Google Scholar, 10Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Google Scholar). Sixteen mammalian START domain proteins have been identified to date (2Soccio R.E. Breslow J.L. J. Biol. Chem. 2003; 278: 22183-22186Google Scholar). Of these, StAR (StarD1) and MLN64 (StarD3) consist of one subfamily. MLN64 was originally discovered as a gene amplified in breast and ovarian cancers. It contains a C-terminal domain with 37% amino acid identity and 60% amino acid similarity to the C-terminal domain of StAR (13Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Google Scholar, 14Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Google Scholar). The N terminus of MLN64 contains a leader sequence and four putative transmembrane domains, distinguishing it from StAR and suggesting that MLN64 functions in a different subcellular compartment. The MLN64 START domain was subsequently found to have StAR-like activity in that it could promote steroidogenesis in a model cell system (14Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Google Scholar). Like the StAR START domain, the MLN64 START domain binds cholesterol, stimulates the movement of free cholesterol from sterol-rich donor vesicles to acceptor membranes, and augments steroid synthesis when added to isolated mitochondria (10Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Google Scholar, 15Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss III, J.F. J. Biol. Chem. 2002; 277: 33300-33310Google Scholar). MLN64 was localized to late endosomes, a cellular compartment involved in the trafficking of cholesterol (15Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss III, J.F. J. Biol. Chem. 2002; 277: 33300-33310Google Scholar, 16Alpy F. Stoeckel M.E. Dierich A. Escola J.M. Wendling C. Chenard M.P. Vanier M.T. Gruenberg J. Tomasetto C. Rio M.C. J. Biol. Chem. 2001; 276: 4261-4269Google Scholar). The predicted topology of MLN64 in this compartment has the START domain facing the cytoplasm, being anchored to the vesicle wall by the four transmembrane domains, which positions the START domain to interact with apposing organelles or possibly other START domain proteins in the cytoplasm (16Alpy F. Stoeckel M.E. Dierich A. Escola J.M. Wendling C. Chenard M.P. Vanier M.T. Gruenberg J. Tomasetto C. Rio M.C. J. Biol. Chem. 2001; 276: 4261-4269Google Scholar). MLN64 is incorporated into this dynamic tabulating vesicular system that also contains NPC1, a protein with a sterol-sensing domain (17Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.Y. Liscum L. et al.Science. 1997; 277: 228-231Google Scholar, 18Loftus S.K. Morris J.A. Carstea E.D. Gu J.Z. Cummings C. Brown A. Ellison J. Ohno K. Rosenfeld M.A. Tagle D.A. Pentchev P.G. Pavan W.J. Science. 1997; 277: 232-235Google Scholar), and NPC2 (also known as HE1), a sterol-binding protein (19Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Science. 2000; 290: 2298-2301Google Scholar). Niemann-Pick type C disease, a lysosomal cholesterol storage disorder, is caused by deficiency of either NPC1 or NPC2. The intracellular cholesterol trafficking abnormalities in these Neimann-Pick type C mutant cells or drug-induced phenocopies of the disorder have been well studied (20Frolov A. Srivastava K. Daphna-Iken D. Traub L.M. Schaffer J.E. Ory D.S. J. Biol. Chem. 2001; 276: 46414-46421Google Scholar, 21Millard E.E. Srivastava K. Traub L.M. Schaffer J.E. Ory D.S. J. Biol. Chem. 2000; 275: 38445-38451Google Scholar, 22Cruz J.C. Chang T.Y. J. Biol. Chem. 2000; 275: 41309-41316Google Scholar, 23Cruz J.C. Sugii S. Yu C. Chang T.Y. J. Biol. Chem. 2000; 275: 4013-4021Google Scholar, 24Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Neurochem. 2002; 83: 1154-1163Google Scholar, 25Karten B. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 2003; 278: 4168-4175Google Scholar). The presence of a START domain protein in a subcellular compartment known to be involved in vesicular sterol trafficking by virtue of the presence of NPC1 and NPC2, raised the possibility that MLN64 participates in the intracellular distribution of sterols. This notion was supported by the finding that expression of a truncated MLN64 protein lacking the START domain coupled to green fluorescent protein (GFP) in COS-1 and Chinese hamster ovary cells caused accumulation of free cholesterol in lysosomes, mimicking the cellular sterol trafficking defect of Niemann-Pick type C disease (15Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss III, J.F. J. Biol. Chem. 2002; 277: 33300-33310Google Scholar). It was proposed that the sterol trafficking abnormality was the result of a dominant negative action of the truncated MLN64-GFP fusion protein. The present studies were conducted to probe the function of the MLN64 START domain using a gene targeting strategy with the goal of identifying the physiological role of Mln64 in sterol dynamics in the mouse. Based on observations summarized above, it was anticipated that disruption of the Mln64 START domain would result in a phenotype similar to that of spontaneous mutations in the Npc1 gene in mice, including a progressive neurodegenerative disorder (ataxia, progressive motor deficits, and later death) and, at a cellular level, accumulation of free cholesterol in lysosomes (26Xie C. Turley S.D. Pentchev P.G. Dietschy J.M. Am. J. Physiol. 1999; 276: E336-E344Google Scholar, 27Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A.M. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Google Scholar). Humans lacking functional NPC1 or NPC2 have a similar phenotype. Here we show that, unexpectedly, mice lacking the Mln64 START domain are healthy and display only minimal disturbances in sterol dynamics, indicating that the Mln64 START domain is largely dispensable for intracellular cholesterol trafficking. We determined that the murine Mln64 protein is encoded by a gene containing 14 exons and spanning ∼9 kb (Fig. 1). We disrupted the START domain in the Mln64 gene in murine embryonic stem cells by replacing exons 10-13 (GenBank™ accession number AL591390) with the fusion gene βgeo, which consists of an internal ribosome entry site-lacZ-Neor fusion gene (28Mountford P. Zevnik B. Duwel A. Nichols J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Google Scholar). This targeting construct removes the majority of the Mln64 START domain (from amino acid residues 267-380). Embryonic stem cells derived from 129/Sv mice were transfected with the linearized βgeo targeting vector, selected in medium supplemented with G-418, and analyzed by Southern blotting to identify correctly targeted clones. For Southern blotting, genomic DNA was digested with EcoRV, and the blots were probed with a 0.87-kb cDNA containing genomic sequence downstream from the targeted genomic sequence. Three correctly targeted embryonic stem cell clones were used to generate chimeric mice, which were crossed with C57BL/6J females to obtain heterozygous mutants. Mice used in these studies were the offspring of crosses between the F1 and/or F2 generations (129/SvJ/C57BL/6J genetic background). Mice were genotyped by PCR. Three set of primers were used in the PCR. One set of primers corresponded to a sequence in the Neo gene (5′-CACATCTGTAGAGGTTTTACTTGC-3′) and a sequence in exon 14 of the Mln64 gene (5′-CTACCTTGATTGAACCCCAGAAC-3′). Another set of primers corresponded to a sequence in the deleted region of exon 13 of the Mln64 gene (5′-GGGACTTTGTGAATGTCCGACG-3′) and the above noted primer for exon 14 of the Mln64 gene. The mice used in these experiments were maintained in a University Laboratory Animal Resource facility and all animal protocols were approved by the University of Pennsylvania's Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. To assess fertility and fecundity, littermate males (>6 weeks old) were placed in cages with two mature wild-type females for 1 month or more. Littermate females were caged with a wild-type male for a similar period. The number of mice achieving a pregnancy and the number of offspring from each mating set or pregnancy were recorded. Mice were fed ad libitum Purina Mouse Chow 5001 or one of three experimental diets (Teklad Research Diets) containing control diet (TD7001; Teklad), or 0.5% cholesterol-enriched diet (TD88137; Teklad) or 15.75% fat and 1.25% cholesterol (high fat) diet (TD90221; Teklad). The high fat diet (TD90221; Teklad) contained (by weight) 75% Purina Mouse Chow, 15.75% fat, 1.25% cholesterol, and 0.5% sodium cholate. Mice over 6 weeks of age were housed individually and fed the specified diet for 1 week before harvesting tissue and blood. Mice were weighed and anesthetized with isoflurane (Forane; Baxter), and a syringe was used to draw blood from the heart. The mice were then killed by cervical dislocation, and organs were harvested, weighed, and flash-frozen in dry ice. Serum was generated from the blood following coagulation. To assess ovarian steroidogenic function, six immature female mice (38-45 days old) were injected intraperitoneal with 5 IU of pregnant mare's serum gonadotropin (Calbiochem). After 44-48 h, all mice were injected with 5 IU of human chorionic gonadotropin (Sigma). Mice were anesthetized, and blood was collected by cardiac puncture. The mice were killed by cervical dislocation, and the ovaries and uterus were removed, cleaned, and weighed. Cholesterol and triglyceride were quantified in serum and bile using cholesterol reagent and triglyceride reagent (2350 and TR22321, respectively (Thermo DMA). Liver tissue (1 g) was homogenized in 10 ml of ice-cold buffer (phosphate-buffered saline, pH 7.4, 1 mm EDTA, 1% Nonidet P-40, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride). Aliquots of liver homogenate were extracted with hexane/isopropyl alcohol (3:2, v/v) at 4 °C, and the solvent was evaporated under nitrogen gas. Hepatic total and free cholesterol and triglyceride in the dried extract were quantified using cholesterol reagent, triglyceride reagent, and free cholesterol reagent (401-100P and TR22321, Thermo DMA; and 274-47109; Wako Chemicals, respectively). The level of cholesterol ester was calculated by subtracting the free cholesterol content from the total cholesterol content. Hepatic and bile phospholipids levels were quantified by a modification of the Bartlett procedure (29Bartlett E.M. Lewis D.H. Anal. Biochem. 1970; 36: 159-167Google Scholar). Serum nonesterified free fatty acid and glucose were quantified using enzymatic colorimetric assays (Wako Chemicals and Stanbio Laboratories, respectively). Testosterone, corticosterone, and progesterone were quantified using radioimmunoassay kits (TKTT1, TKRC1, and TKPG2, respectively; Diagnostic Products Corp.). Total RNA was isolated using TRIzol reagent (Invitrogen). Real time PCR analysis was performed on an ABI PRISM 7900HT sequence detection system with target-specific probes and primers designed with Primer Express (PerkinElmer Life Sciences). Total RNA (5 μg) was treated with DNase I (Promega) followed by cDNA synthesis using the Moloney murine leukemia virus reverse transcriptase (Promega) and oligo(dT) primer (Promega). The resulting cDNAs were diluted 1:10 in sterile water, and 1-μl aliquots were used in the quantitative real time PCR. The primers used to quantitate mRNA are listed in Table I. In order to account for differences in starting material, glyceraldehyde-3-phosphate dehydrogenase primers and probe reagents from Applied Biosystems were used as described by the manufacturer. In order to quantify differences, the samples were compared with standard curves for each gene and glyceraldehyde-3-phosphate dehydrogenase, and the average value for the triplicate determinations was used in all subsequent calculations.Table IPrimers used to generate probes for Northern analysis and for quantitative real time PCRGeneForward primer sequenceReverse primer sequenceMln64 exons 1-95′-ACTGTCCCACAGCCAGAGCC-3′5′-CTCCTCTCGAACTTCCAGTTC-3′Mln64 exons 10-135′-GAGTATGGGGACACTGTGTAC-3′5′-CTGACATATTTGTGCGTGGG-3′Npc15′-CAATCCTGTGTTTGGTATGGAGAG-3′5′-ATCTACAGACTCATTGCAGCCTTTG-3′Npc25′-ATGCGTTTTCTGGCCGCCACGATC-3′5′-CTAGCTTGTGATCTGAACTGGGATC-3′StarD45′-ATGGCTGACCCTGAGAGCCCG-3′5′-TCATGCCTTGCGTAGACCTTTTCG-3′StarD55′-ATGGACCCGTCCTGGGCCACGCAAG-3′5′-TCAGTGATGGAACTTCCTCACTGCCTTC-3′Mentho5′-ATGAACCATCTTCCAGAACAC-3′5′-CCTCAGCTTCTTCTTCAGATC-3′Actin5′-GTGGGCCGCTCTAGGCACCAA-3′5′-CTCTTTGATGTCACGCACGATTTC-3′Cyp7a15′-ACTCTCTGAAGCCATGATGCAA-3′5′-GACAGCGCTCTTTGATTTAGGAA-3′HMG-CoA synthase5′-GGGCCAAACGCTCCTCTAAT-3′5′-AGTCATAGGCATGCTGCATGTG-3′Abcg55′-GTGGCGGACCAAATGATTG-3′5′-TTGGGCTGCGATGGAAAC-3′Abcg85′-GAGCTGCCCGGGATGATA-3′5′-CCGGAAGTCATTGGAAATCTG-3′Map3k75′-TGCCATGAGCTGGTGTTTACAG-3′5′-AGCGCTTTGGGCTGCAT-3′ Open table in a new tab For Northern blot studies, the total RNA samples (50 μg/lane) were separated in 1.0% agarose gels containing formaldehyde and transferred to nylon membrane (Hybond N; Amersham Biosciences) according to the manufacturer's recommendations. Blots were subsequently hybridized with [α-32P]dCTP-labeled probes prepared by reverse transcriptase PCR using primers listed in Table I and wild-type mouse liver template. Hybridization was performed with Quikhyb (Stratagene). Membranes were exposed to X-AR films (Eastman Kodak Co.). Target Preparation and Hybridization—All protocols were conducted as described in the Affymetrix GeneChip Expression Analysis technical manual. Briefly, 7 μg of total RNA was converted to first strand cDNA using Superscript II reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 promoter. Second strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP. The cRNA products were fragmented to 200 nucleotides or less, heated at 99 °C for 5 min, and hybridized for 16 h at 45 °C to mouse MOE 430A Affymetrix microarrays. The microarrays were then washed at low (6× SSPE) and high (100 mm MES, 0.1 m NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain. A confocal scanner was used to collect fluorescence signal at 3-μm resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature. Initial Data Analysis—Affymetrix Microarray Suite 5.0 was used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe pairs were scored positive or negative for detection of the targeted sequence by comparing signals from the perfect match and mismatch probe features. The number of probe pairs meeting the default discrimination threshold (τ = 0.015) was used to assign a call of absent, present, or marginal for each assayed gene, and a p value was calculated to reflect confidence in the detection call. A weighted mean of probe fluorescence (corrected for nonspecific signal by subtracting the mismatch probe value) was calculated using the one-step Tukey's biweight estimate. This signal value, a relative measure of the expression level, was computed for each assayed gene. Global scaling was applied to allow comparison of gene signals across multiple microarrays; after exclusion of the highest and lowest 2%, the average total chip signal was calculated and used to determine what scaling factor was required to adjust the chip average to an arbitrary target of 150. All signal values from one microarray were then multiplied by the appropriate scaling factor (Genespring version 6.1 (Silicon Genetics Software)). Equal amounts of liver protein (50 μg/lane) were loaded onto 10% SDS-polyacrylamide gels and then transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corp.). Western blot analysis was carried out using a rabbit anti-recMLN64 N-terminal (amino acid residues 1-52) and anti-C-terminal MLN64 antibodies recognizing either the START domain (amino acids 216-440) or a peptide in the START domain (amino acid residues 370-385) as previously described (14Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Google Scholar, 15Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss III, J.F. J. Biol. Chem. 2002; 277: 33300-33310Google Scholar) For the detection of antibody protein complexes, the SuperSignal West Pico or Femto Kit (Pierce) was used according to the manufacturer's instructions. Mouse embryonic fibroblasts were prepared from embryonic day 14.5 embryos from the mated Mln64-/+ littermates (30George E.L. Hynes R.O. Methods Enzymol. 1994; 245: 386-420Google Scholar). DNA extracted from embryos was genotyped by PCR and Southern blot using standard techniques to identify wild-type and homozygous mutants. Mouse granulosa cells were collected from periovulatory follicles from mature female mice given a follicular stimulation protocol. Three wild type and three nullizygous female mice were injected intraperitoneal with 5 IU of pregnant mare's serum gonadotropin (Calbiochem). After 44-48 h, the mice were killed by cervical dislocation, and the ovaries were carefully dissected away and placed into 4 °C Dulbecco's phosphate-buffered saline (PBS; Invitrogen) containing 0.1% bovine serum albumin (Sigma). To release the granulosa cells from the follicles, the ovaries were repeatedly punctured with a 30-gauge needle. The expressed granulosa cells and minor contaminants (i.e. oocytes, red blood cells, and stromal cells) were collected and centrifuged at 770 × g to pellet the cells in a 1.5-ml Eppendorf tube. The cells were cultured in Dulbecco's minimal essential medium/F-12 supplemented with 10% fetal calf serum and 100 μg of penicillin-streptomycin/ml in 5% CO2, 95% air at 37 °C. Dishes were coated with fibronectin from human plasma (Sigma) for at least 30 min prior to plating. Cells were plated at 100,000 cells/well in 12-well plates on day 0. On day 1, after washing with PBS, medium was changed to Dulbecco's minimal essential medium/F-12 containing 10% fetal calf serum and 100 μg of penicillin-streptomycin/ml without or with 5 μg/ml 22(R)-hydroxycholesterol, 1 mm 8-bromo-cyclic AMP (8-Br-cAMP), or ethanol (added in the same volume as for 22(R)-hydroxycholesterol). Each treatment group consisted of triplicate wells. After 24 h, medium was collected and frozen at -20 °C until progesterone was assayed. Cells were scraped into ice-cold buffer (PBS, pH 7.4, 1 mm EDTA, 1% Nonidet P-40, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride). The protein concentrations were determined with the Bio-Rad protein assay reagents. These experiments were conducted on three separate occasions. Progesterone secretion in the basal and 8-Br-cAMP-stimulated conditions was normalized to steroidogenic potential of the cells using progesterone production in the presence of 22(R)-hydroxycholesterol. Mouse livers were perfusion fixed through the heart with ice-cold 4% paraformaldehyde in PBS from homozygous mutant Mln64 mice and wild-type mice. The livers were removed and immersed in the same fixative for an additional 2 days at 4 °C. Following perfusion fixation, 10-μm frozen sections were cut from the whole left, right, and medial lobes of each liver. The sections were stained with Nile Red (0.1 μg/ml PBS) for 15 min at room temperature, washed three times in PBS, and mounted in 90% glycerol in PBS containing 1 mg/ml n-propyl gallate as an antifade agent. Images were taken on a Zeiss LSM410 confocal microscope equipped with a Melles Griot Omnichrome krypton argon laser using a 488-nm excitation line and 515-540 band pass emission filter. Transmission electron microscopy was carried out on thin sections of liver as previously described (31Matsuo H. Strauss III, J.F. Endocrinology. 1994; 135: 1135-1145Google Scholar) Brain tissue was stained with filipin and an anti-GM3 ganglioside antibody as described by Zervas et al. (32Zervas M. Dobrenis K. Walkley S.U. J. Neuropathol. Exp. Neurol. 2001; 60: 49-64Google Scholar). Mouse embryonic fibroblasts were grown on Nunc LabTek two-chambered glass slides. They were fixed at room temperature for 30 min in 3% paraformaldehyde in PBS, pH 7.4, washed 3 × 5 min in PBS, and stained with Nile Red, mounted, and visualized as described above for liver sections. Cells were grown and fixed as above and stained overnight in 50 μg/ml filipin in PBS, washed three times for 5 min each in PBS, and mounted as for the liver sections. They were imaged on a Zeiss LSM410 confocal microscope equipped with a Coherent Enterprise argon laser using a 364-nm excitation line and 400-435 band pass emission filter. For assays of steroidogenic activity (15Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss III, J.F. J. Biol. Chem. 2002; 277: 33300-33310Google Scholar), embryonic fibroblasts were cultured in Dulbecco's minimal essential medium supplemented with 10% fetal calf serum and 50 μg of gentamycin/ml in 5% CO2, 95% air at