Mitochondrial association of myocilin, product of a glaucoma gene, in human trabecular meshwork...

ORIGINAL ARTICLE 775J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology
Mitochondrial Association of
Myocilin, Product of a GlaucomaGene, in Human TrabecularMeshwork Cells
HIROSHI SAKAI, XIANG SHEN, TAKAHISA KOGA, BUM-CHAN PARK, YELINA NOSKINA,
MARTIN TIBUDAN, AND BEATRICE Y.J.T. YUE*

Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois

The trabecular meshwork (TM), an ocular tissue next to the cornea, is a major site for regulation of the aqueous humor outflow.Malfunctioning of this tissue is believed to be responsible for development of glaucoma, amajor blinding disease. Myocilin is a gene directlylinked to themost common formof glaucoma. Its protein product has been localized to both intra- and extra-cellular sites in TM cells. Thisstudy was to investigate the association of myocilin with mitochondria in TM cells. In vitro mitochondrial import assays showed thatmyocilin was imported to the TM mitochondria, targeting to mitochondrial membranes and/or the intermembrane space. The targetingwas mediated mostly via the amino-terminal region of myocilin. When myocilin expression was induced either by treatment withdexamethasone or transfection with amyocilin construct, themitochondrial membrane potential in TM cells, as assessed by JC-1 staining,was lowered. Subcellular fractionation andWestern blot analyses confirmed that a portion ofmyocilin sedimentedwith themitochondrialfractions. Upon anti-Fas treatment to provoke apoptosis, an increase of myocilin distribution in cytosolic fraction was observed,suggesting that myocilin was partially released from mitochondrial compartments. These results confirmed the association of myocilinwith TM cell mitochondria and indicated that myocilin may have a proapoptotic role in TM cells.J. Cell. Physiol. 213: 775–784, 2007. � 2007 Wiley-Liss, Inc.

Hiroshi Sakai and Xiang Shen contributed equally to this work.Contract grant sponsor: National Eye Institute;Contract grant numbers: EY 05628, EY 03890, EY 01792.

*Correspondence to: Beatrice Y.J.T. Yue, Department ofOphthalmology and Visual Sciences, University of Illinois atChicago, 1855 W. Taylor Street, Chicago, IL 60612.E-mail: [email protected]

Received 9 March 2007; Accepted 13 April 2007

DOI: 10.1002/jcp.21147

The trabecular meshwork (TM) is a major site for regulation ofthe aqueous humor outflow (Bill, 1975). This tissue iscomposed of sheets of trabecular beams made up ofextracellular matrix (ECM) components including fibronectinand collagens (Yue, 1996). Lining the beams areTMcells that arebelieved to be essential for maintenance of the normal outflowpathway and control of the intraocular pressure (IOP).Disturbance of either the vitality or functional status of TM cellsmay lead to glaucoma, a heterogeneous blinding diseasegenerally characterized by IOP elevation, loss of retinal ganglioncells and visual impairment.

Myocilin is the product of a gene that has been linked directlyto both juvenile- and adult-onset open angle glaucoma, themostcommon form of glaucomatous diseases (Stone et al., 1997).Multiple mutations were identified in a number of families(Gong et al., 2004). Myocilin was initially identified as a55/57-kilodalton (kDa) protein secreted into the media of TMcultures after induction with glucocorticoids such asdexamethasone (DEX) (Polansky et al., 1989, 1997). The genehas been cloned from human and other species (Tamm, 2002;Shepard et al., 2003; Fautsch et al., 2006a) and characterized.Containing 3 exons, the human gene encodes a protein of504 amino acids. There is a non-muscle myosin-like domainnear the amino (N)-terminus and an olfactomedin-like domainat the carboxyl (C)-terminus. TheN-terminus has two initiationsites, and within the myosin-like domain, a leucine zipper motifand a coiled-coil region (Nguyen et al., 1998). The leucine zipperand the olfactomedin domain are well conserved. At thesecondary level, theN-terminal region is primarilya-helical andthe C-terminal consists mostly of b-strands (Nagy et al., 2003;Kanagavalli et al., 2004).

Myocilin is a secreted protein in TMcultures (Caballero et al.,2000; Shepard et al., 2003; Hardy et al., 2005). It interacts withitself as well as a number of other proteins. The interaction siteis mainly at the N-terminus in the leucine zipper/coiled coilregion (Wentz-Hunter et al., 2002b; Gobeil et al., 2006;Fautsch et al., 2006b).

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Myocilin protein has been localized to both intra- andextra-cellular sites in TM cells. Immunofluorescence has shownthat the intracellular form of myocilin is distributed in thecytoplasm including perinuclear regions (Tamm, 2002).Subcellular fractionation indicated that intracellular myocilin inTMcells is associated not onlywith endoplasmic reticulum (ER),Golgi apparatus, vesicles, but also with mitochondria (Wentz-Hunter et al., 2002a). The mitochondrial association wasvisualized by immunoelectron microscopy (Ueda et al., 2000).Immunogold labeling also illustrated the extracellularlocalization of myocilin (Ueda et al., 2002; Ueda and Yue, 2003).

The myocilin transcript or protein has been detected notonly in the TM, cornea, and a number of other ocular andnonocular tissues (Tamm, 2002). One distinctive feature is thatthe myocilin expression is highly upregulated by DEX in TMcells but not in other cell types including corneal fibroblasts(Polansky et al., 1989, 1997; Tamm, 2002;Wentz-Hunter et al.,2002a, 2003). There was also little or no association of myocilinwith mitochondria in corneal fibroblasts (Wentz-Hunter et al.,2003). The TM cell-specific findings may explain why onlyglaucoma is manifested in patients with mutations of myocilin,

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despite its ubiquitous distribution throughout ocular andnonocular tissues.

In the current study, the association of myocilin withmitochondria was further examined. Myocilin was incubatedwith isolated mitochondria. The assays showed that myocilinwas robustly imported into the mitochondria isolated from TMcells, but only minimally into those from corneal fibroblasts andmouse liver. Mitochondria consist of four compartments, theouter membrane, intermembrane space, inner membraneand matrix (Truscott et al., 2003). Our study in additiondelineated that the myocilin imported into TM mitochondriawas targeted mostly to the membranes. When upregulatedor overexpressed, myocilin had an adverse effect on themitochondrial membrane potential. Furthermore, upontreatment of apoptotic stimuli, myocilin was found releasedfrom mitochondrial compartments.

Materials and MethodsPlasmid construction

Full-length myocilin (amino acid residues 1–504) was amplified byPCR using 50-CCACCATGGCTATGAGGTTCTTCTGTGCACGTTGC-30

sense and 50-TATCACATCTTGGAGAGCTTGATGTCATA-30

antisense primers. The 1.5-kilobase myocilin PCR product wassubcloned using TA-cloning into pTarget (Roche, Indianapolis, IN),a mammalian expression vector that utilizes CMV promoterand contains neomycin gene. For the N-terminal (amino acidresidues 1–270) and C-terminal (amino acid residues 271–504)myocilins, myocilin 1–270 and 271–504, DNA fragments wereamplified by PCRusing pTarget-myocilin as template. Primerswere50-CCACCATGGCTATGAGGTTCTTCTGTGCACGTTGC-30

(sense) and 50-TATCACCACACACCATACTTGCCAG-30

(antisense) for myocilin 1–270, and 50-CCACCATGCGAGACCCCAAGCCCAC-30 (sense) and50-TATCACATCTTGGAGAGCTTGATGTCATA-30 (antisense)for myocilin 271–504. The resulting PCR products wereinserted into pTarget vector, yielding pTarget-myocilin 1–270 andpTarget-myocilin 271–504. Optineurin is not associated with themitochondria (Park et al., 2006), thus pTarget-optineurin, made aspreviously described (Park et al., 2006), was used as a negativecontrol for mitochondrial import assay. The orientation andsequences of the constructs were confirmed by sequence analyses.The pGEM4Z plasmid containing yeast porin was a kind gift fromDr. Nikolaus Pfanner (University of Amsterdam, Amsterdam, TheNetherlands). Porin is an outer membrane protein (Dekker et al.,1997) and the porin plasmid was used as a positive control for themitochondrial import assay.

Cell cultures

Normal human eyes were obtained from the Illinois Eye Bank(Chicago, IL) or the National Disease Research Interchange(Philadelphia, PA). TM cells were derived from 4-, 15-, 21-, 22-, 24-,29-, 35-, 38-, 48-, 49-, and 54-year-old donors without any knownocular disease. Corneal fibroblasts were cultured from the stromaof donors aged 6, 15, 26, and 52 years. The procurement of tissueswas approved by the Institutional Review Board committee at theUniversity of Illinois at Chicago in accordance with the Declarationof Helsinki. The TM and corneal tissues were dissected andcultured as previously described (Zhou et al., 1996) in Dulbecco’smodified Eagle’s minimum essential medium (DMEM, Sigma, St.Louis, MO) supplemented with 4 mM glutamine, 10% fetal bovineserum, 5% calf serum, 1% nonessential and essential amino acids,and antibiotics. Second- or third-passaged cells were used for thestudies. To inducemyocilin expression, TM cells were treated with100 nM of DEX for 10 days (Polansky et al., 1997; Wentz-Hunteret al., 2002a). To provoke apoptosis, TM cells were treated

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

with anti-Fas (400 ng/ml, Upstate Biotechnology, Lake Placid, NY)for 18 h. pTarget-myocilin was transfected into human TM cells(Wentz-Hunter et al., 2004) using FuGene 6 reagent (Roche;1:3 DNA to FuGene 6 ratio). Cells were plated at 40–60%confluence 18 h prior to transfection. The transfected cells wereselected with G418 (100 mg/ml, Sigma).

Mitochondrial isolation

Mitochondria were isolated from cultured TM cells, cornealfibroblasts, or mouse liver using techniques adapted frompreviously reported, well-establishedmethods (Krause et al., 1997;Cavadini et al., 2002; Kaufmann et al., 2003). Cells or tissues werehomogenized in homogenization buffer (HB) containing 220 mMmannitol, 70 mM sucrose, 10 mM HEPES, pH 7.4, 1 mM EDTA,1 mM EGTA, 0.5% bovine serum albumin, and protease inhibitors.Cell debris and nuclei were removed by centrifugation at 1,000g.The mitochondria were pelleted at 12,000g for 15 min. Afterwashing in HB, the mitochondria were resuspended at 2 mg/ml inmitochondrial storage buffer and stored at �808C (Priest andHajduk, 1996).

Mitochondrial import assay

[35S]-labeledmyocilin, porin or optineurin was prepared by in vitrotranslation using pTarget-myocilin, pGEM4Z-porin, or pTarget-optineurin plasmid, T7 or SP6 RNA polymerase, and 20 mCi of[35S]-methionine per 50 ml of mixture, following instructions forthe TnT1 Coupled Reticulocyte Lysate System (Promega,Madison, WI). The enzyme susceptibility of [35S]-labeled myocilinin the translation mixture was tested by treatment of increasingconcentrations (1, 2, 5, 10, and 25mg/ml) of proteinaseK for 15min(Zhuang et al., 1992). Stored mitochondria were resuspended in9 volumes of HB and centrifuged at 12,000g for 15 min. The importof [35S]-labeled myocilin, porin, or optineurin was performed aspreviously described (Addya et al., 1997; Kurz et al., 1999; Cavadiniet al., 2002; Johnet al., 2002;Anandatheerthavarada et al., 2003) in a50 ml import reaction consisting 50 mg of mitochondria,import buffer (125 mM sucrose, 5 mM HEPES, pH 7.4, 1 mM ATP,1 mM DTT, 40 mM KCl, 5 mM MgCl2, 0.5 mM EDTA), and 10 mlof [35S]-labeled myocilin, porin, or optineurin-containing in vitrotranslationmixture. After 60min at room temperature, the importwas stopped on ice. The mitochondria were reisolated andprotease inhibitor cocktail was added to one of the importreactions. For proteinase K treatment, the import reactionmixture was incubated with 25 mg/ml of proteinase K on ice for15 min followed by addition of 50 mM of phenylmethylsulfonylfluoride. For alkaline treatment, the mitochondria were pelleted,solubilized in alkali buffer (100 mM Na2CO3, pH 11.1) andincubated at 308C for 30 min. To swell the mitochondria, theimport reaction mixture was solubilized in hypotonic buffer(10 mM HEPES, pH 7.2, 1 mM EDTA) and incubated on ice for10 min, then pelleted and treated as described above withproteinase K. At the end of all treatments, the mitochondriawere pelleted, washed, solubilized in protein sample buffer, andsubjected to 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). The gelwas dried and the radioactivebands were visualized using the Cyclone Storage Phosphor System(Packard Bioscience/Perkin Elmer, Boston, MA). Band intensitieswere analyzed by densitometry using Kodak Digital Science ImageStation (440CF, Kodak, Rochester, NY).

To determine whether the imported myocilin had assembledinto a complex, the re-isolated mitochondria were solubilized inextraction buffer (1.5% n-dodecyl-D-maltopyranoside, 40 mMBis-Tris–HCl, 600mM 6-aminocaproic acid), centrifuged (22,000g)at 48C for 30 min, and the supernatant was subjected to 8% bluenative gel electrophoresis (BN-PAGE) (Schagger and Von Jagow,1991; Johnston et al., 2002; Tatsuta et al., 2005; Tamura et al.,2006).

Fig. 1. A: In vitro translated [35S]-labeled porin (lane 1), optineurin(lane 2) and myocilin (lane 3). A major band for each was visualized byPhosphorImager analysis. The expected molecular sizes wererespectively, 29, 74, and 57 kDa. B: Mitochondrial import assays.[35S]-labeled porin (lane 1), optineurin (lane 2) and myocilin (lane 3)were incubated for 60 min with mitochondrial isolated from humanTM cells in the presence of ATP. The mitochondria were re-isolated,dissolved, and subjected to SDS-PAGE and PhosphorImager analyses.[35S]-labeled porin and myocilin, but not optineurin, were associatedwith the re-isolated mitochondria.

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Mitochondrial membrane potential

Control,DEX-treated, andmyocilin-transfectedTMcells in 35mmglass bottom culture dishes (MatTek, Ashland,MA)were incubatedat 378C with DMEM containing 5% fetal calf serum and 1 mM JC-1(5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocyanine iodide, Molecular Probes, Eugene, OR) for 15 minand washed with phosphate buffered saline. This dye exists eitheras a red fluorescent J-aggregate at hyperpolarized membranepotential or as a green fluorescent monomer at depolarizedmembrane potentials. With increasingly lower potentials of themitochondrial membrane, less J-aggregates form and JC-1undergoes a shift in emission peak from 590 to 527 nm.

Dual emission images were obtained using the Leica TC-SP2laser scanning confocal microscope (Heidelberg, Germany). Withthe Leica 63� 1.2NA objective, JC-1 was excited sequentially withthe 488 nm argon laser and the 543 nm green HeNe laser andvisualized at a range of 501–581 and 582–651 nm, respectively.Images were acquired from randomly selected fields and analyzedusing the Leica analysis software to measure fluorescence intensityin each cell. The ratio of the orange/red J-aggregate to the greenmonomer fluorescence intensity in a minimum of 100 cells wascalculated. To account for background images were taken ofunlabeled cells and their intensity was subtracted from that of thesamples.

As a control for membrane depolarization, TM cells weretreated overnight with 2.5 mM of staurosporine (Sigma, followingthe protocol of Samraj et al. (2007)). The treatment was mild andthe integrity of the cells was intact. Parallel experiments were alsoperformed on human corneal fibroblasts without or with DEXtreatment for 10 days. In some experiments, DEX- andstaurosporine-treated and untreated cells were subjected toanalyses by flow cytometer (EPICS Elite ESP, Beckman Coulter,Hialeah, FL) after 15-min incubation with JC-1.

Subcellular fractionation and Western blotting

Control and DEX-treated TM cells harvested in 0.25 M sucrose,10 mM HEPES, pH 7.4, and 1 mM EDTA were broken with aDounce glass homogenizer. Cell debris and nuclei were removed.The post-nuclear supernatant was overlaid onto a discontinuousgradient of 10–30%, made with Optiprep (Accurate Chemical andScientific Company, Westbury, NY) in Optiprep buffer (0.25 Msucrose, 6mM EDTA, 120mMHEPES, pH 7.4), and centrifuged in afixed angle rotor for 3 h at 100,000g. Equal fractions (250 ml)removed from the top of the gradient were subjected to 10% SDS-PAGE. The densities in gradient fractions were determined bymeasuring absorbance at 340 nm according to the manufacturer’sprotocol.

The gels were electroblotted to Protran nitrocellulose(Midwest Scientific, St. Louis, MO) for 45 min. After blocking with5% non-fat milk, the membranes were probed with polyclonal anti-myocilin (1:5,000; a generous gift of Dr. W. Daniel Stamer,University of Arizona; Stamer et al., 1998), made against aglutathione-S-transferase fusion protein corresponding to the N-terminal functional domain (amino acid residues 3–230), polyclonalanti-human succinate dehydrogenase B (1:250; Santa CruzBiotechnology, Santa Cruz, CA), monoclonal anti-cytochrome c(1:1,000, Imgegex, Sorrento Valley, CA), or monoclonal anti-golgin97 (Invitrogen/Molecular Probes, Carlsbad, CA). The secondaryantibody was either horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:10,000; ICN/Cappel Biomedical,Westchester, PA). Protein bands were visualized using SuperSignalSubstrate (Pierce, Rockford, IL). For repeated probing, the blotwasstripped for 1 h at room temperature with ImmunoPure IgGElution buffer (Pierce).

DEX-treated TM cell without orwith exposure to anti-Fas wereharvested. The mitochondrial and cytosolic fractions wereseparated using a mitochondria isolation kit from Pierce followingthe manufacturer’s instructions. Equal aliquots of the separated


fractions were subjected to electrophoresis andWestern blottingusing anti-myocilin (Stamer et al., 1998), anti-cytochrome c, anti-golgin 97 (Invitrogen), or monoclonal anti-heat shock cognate 70(Hsc 70, Santa Cruz). Band intensity in each fraction was analyzedby densitometry. Experiments were repeated 5 times.

ResultsTargeting of myocilin to mitochondria

[35S]-labeled porin, optineurin and myocilin were prepared byin vitro translation using pGEM-porin, pTarget-optineurin andpTarget-myocilin plasmid (Fig. 1A). The [35S]-labeled proteinswere incubated with mitochondria isolated from culturedTM cells for import assays (Priest and Hajduk, 1996;

Fig. 2. A: Susceptibility of in vitro translated [35S]-labeled myocilinto proteinase K. The [35S]-labeled myocilin product was digested for15 min on ice with 0, 1, 2, 5, 10 or 25 mg/ml (lanes 1–6) prior toelectrophoresis on 10% SDS-PAGE. The 57-kDa protein band wasvisualized by PhosphorImager analysis. B: Mitochondrial importassays. [35S]-labeled myocilin was incubated for 60 min withmitochondrial isolated from human TM cells in the presence of ATP.The import reaction mixtures were divided into four fractions. To onefraction, protease inhibitors were added (lane 1). Other fractionswere treated either for 15 min with 25mg/ml proteinase K (lane 2), for30 min at 30-C with 0.1 M Na2CO3 (lane 3), or with hypotonic buffer(10 mM HEPES, 1mM EDTA) followed by incubation with 25 mg/mlproteinase K (lane 4). The mitochondria were re-isolated andsubjected to SDS-PAGE and PhosphorImager analyses. The majorband was the [35S]-labeled myocilin. The molecular size of theradioactive myocilin was unaffected by any of the treatments.C: Mitochondrial import was performed with [35S]-labeled myocilin.Either without treatment (lane 1), or after treatment of proteinaseK (lane 2), the mitochondria reisolated were solubilized in n-dodecyl-D-maltopyranoside-containing buffer. Proteins were then subjectedto 8% blue native gel electrophoresis (BN-PAGE). Arrow denotes thesample loading front.

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Cavadini et al., 2002). As anticipated, positive control[35S]-labeled porin was successfully imported into themitochondria (Fig. 1B, lane 1), pelleted together with there-isolated mitochondria and appeared as a 29 kDa-bandupon electrophoresis under reducing conditions. The[35S]-labeled myocilin band, like porin, also appeared,indicating that myocilin was imported into the mitochondria(Fig. 1B, lane 3). Parallel experiments performed with pTargetconstruct of optineurin, another glaucoma gene not known toassociate with the mitochondria (Rezaie et al., 2002; Park et al.,2006), showed no evidence of import into the TM cellmitochondria (Fig. 1B, lane 2).

The radioactive myocilin in vitro translated product washighly susceptible to proteinase K digestion. The band intensityor radioactivity was much reduced with an enzymeconcentration as low as 1 mg/ml.With 2 mg/ml of proteinase K,the radioactive band nearly disappeared after a 15-minincubation (Fig. 2A).

The [35S]-labeled myocilin was incubated with mitochondriaisolated from cultured TM cells. Aliquots of the reactionmixture were subsequently mixed with protease inhibitors, orsubjected to proteinase K, alkali (100 mM Na2CO3), orhypotonic swelling treatment. As can be seen in Figure 2B, arobust [35S]-labeled myocilin band, after incubation with TMcell mitochondria, was observed in the mitochondrial pellet(lane 1) upon electrophoresis. Again, myocilin was associatedand imported into themitochondria. The amount of radioactivemyocilin was substantially decreased after treatment of25 mg/ml of proteinase K (lane 2), although a distinctiveradioactive band did remain. A majority of the myocilinimported therefore appeared to be associated with the outermembrane liable to proteinase K digestion and the remainingenzyme-resistant portion was likely present inside themitochondria. Compared to Figure 2A, the [35S]-labeledmyocilin, after incubation with the TMmitochondria, was muchmore resistant to proteinase K, supporting the notion thatmyocilin was incorporated into, and protected by itsassociation with, this organelle.

The intensity ofmyocilin band recovered frommitochondrialpellet was also much reduced when the mitochondrial (outerand/or inner) membranes were disrupted (Fujiki et al., 1982a,b;Mihara and Omura, 1995) by alkali treatment (Fig. 2B, lane 3).The susceptibility indicated that a portion of the myocilinimported was targeted to the intermembrane space and/ormatrix and the remaining was integrated into mitochondrialmembranes. After sequential treatments of hypotonic bufferand proteinase K, the myocilin band completely disappeared(lane 4). Hypotonic treatment is known to cause the outermembrane to rupture. The inner membrane remains intact(Glick, 1995), while the inner membrane proteins becomeprotease sensitive. Proteins present in the matrix space, on theother hand, should survive the proteinase K digestion. Thecomplete disappearance of myocilin suggested that it was notpresent in thematrix space. Altogether, these findings led to theconclusion that myocilin was imported into the TMmitochondria, integrating into the outer and inner membranes,and/or present in the intermembrane space, but not thematrix.

The molecular size of radioactive myocilin remained at 57kDa, unaltered by any of the treatments. Myocilin did notappear to undergo additional processing subsequent to themitochondrial transport. When electrophoresed under non-reducing and blue native gel conditions, a radioactive band withhigher molecular weight (>250 kDa) resistant to proteinase Kwas observed (Fig. 2C).

The N-terminal fragment of myocilin (myocilin 1–270) wasimported into the mitochondrial membrane to a similarextent as the full-lengthwild type. By contrast, the import of theC-terminus (myocilin 271–504) was only at a 30% level(Fig. 3A and B).


For comparison purpose, import assays using mitochondriaisolated form corneal fibroblasts and mouse liver wereperformed. The import of full-length myocilin into thesemitochondria was markedly (10–20-fold) lower than that intothe TM cell mitochondria (Fig. 3C and D).

Fig. 3. A: [35S]-labeled full-length myocilin (lane 1 and 2), or truncated N-terminal (lanes 3 and 4) and C-terminal (lanes 5 and 6) fragments in thereticulocyte lysates prior to mitochondrial incubation (lanes 1, 3, and 5) or those targeted to mitochondrial membranes following import into TMcellmitochondriaandalkali treatment(lanes2,4,and6).Thealiquotsofradioactiveproteinor fragmentsusedformitochondrial import for lanes2,4,and6werethesameasthoseshownin lanes1,3,and5,respectively.ThemolecularsizesfortheN-andC-terminal fragmentswereapproximately30 and 27 kDa as expected. B: The import of myocilin protein or fragments in (A) was calculated as the ratio of band intensity after and beforemitochondrial import (lane 2/lane 1; lane 4/lane 3; and lane 6/lane 5). Percent import of the truncated N- and C-terminus was then normalized tothat of the myocilin full-length (100%). Data presented was from a representative experiment. Experiments were repeated 3 times with similarresults.C: Identical aliquotsof [35S]-labeled myocilin were incubatedwith50mgofmitochondria isolatedeither fromnormalhumanTMcells (TM,lane1),cornealfibroblasts (cornea, lane2),ormouse liver(liver, lane3) for60minandtreatedwith0.1MNa2CO3.Mitochondrialmembraneswereisolated and subjected to SDS-PAGE prior to PhosphorImager analysis. D: Densitometric analyses of protein bands in (C). As identical aliquots of[35S]-labeled myocilin was used for lanes 1, 2 and 3, a direct comparison of band intensities was made. The import to corneal fibroblast (lane 2) andmouse liver (lane 3) mitochondria was normalized to that of TM cell mitochondria (lane 1, 100%). Data presented was from a representativeexperiment. Experiments were repeated 3 times with similar results.


Myocilin effect on mitochondrial membrane potential

The effect of myocilin upregulation or overexpression onmitochondria was determined in DEX-treated ormyocilin-transfected human TM cells utilizing the fluorescentprobe JC-1. TM cells incubated with JC-1 were examined underthe Leica confocal microscope or analyzed by flow cytometer.This dye accumulates in the inner mitochondrial membranewhere it formsmonomers at depolarized potential, producing agreen fluorescence emission at 527 nm. At higher membranepotentials, indicative of mitochondrial activity, JC-1 formsmultimers, producing a red fluorescence emission at 590 nm(Reers et al., 1991). The amount of probe fluorescing at theincreased (red) membrane potential and that fluorescing at thelower (green)membrane potential is an indication of the activityof the mitochondria, and the lower the red fluorescence theless active are the mitochondria within the cells (Mathur et al.,2000; Busciglio et al., 2002).

Figure 4A shows the distribution of JC-1-stainedmitochondria in control TM cells. As was noted previously(Diaz et al., 1999), there were high and low energymitochondrial subpopulations even within the same cell. Themitochondria found in the cell soma closer to the nucleus werea combination of both low and high membrane potential


organelles, as was evident by the green and the orange(overlapping green and red) fluorescence, while themitochondria at the cell periphery were mainly red fluorescingwith a high membrane potential. In controls, the mitochondriawere predominantly in the red or high energy form. After DEXtreatment, red-stained mitochondria diminished and anincrease in green mitochondria was seen, suggesting a generaldecrease of the mitochondrial membrane potential (Fig. 4A).Fluorescence histograms obtained from flow cytometricanalyses of TM cells stained with JC-1 showed that thetreatment ofDEX caused a shift toward a higher intensity (or anincrease) in the green fluorescence emission. A mild treatmentof staurosporine (to serve as a control) also induced apopulation of cells to shift in the green channel (Fig. 4B).

The intensity of red and green fluorescence in control andDEX-treated cells was measured and the red to greenratios were determined using the Leica analysis software.At least 100 cells were examined for each treatment in eachexperiment and the values were compiled from nineexperiments. The mean red to green ratio was significantlyreduced (P< 0.024) in DEX-treated cells (0.70� 0.08,mean� SEM) compared to controls (0.99� 0.10).

A fluorescence shift from red to green or a drop ofmembrane potential was also observed after myocilin

Fig. 4. A: Mitochondrial membrane potential in control anddexamethasone (DEX)-treated TM cells as assessed with JC-1and analyzed by confocal microscopy. In controls, most of themitochondrial staining is red fluorescent. In DEX-treated cells, ashift from red to green fluorescence is observed, indicatingreduced mitochondrial membrane potential. Bar, 20 mm.B: Fluorescence histograms of untreated control (black line), andDEX-treated (blue line) and staurosporine-treated (green line)TM cells stained with JC-1. After JC-1 incubation for 15 min, thecells were detached and analyzed by flow cytometry. A shift to ahigher intensity (or an increase) of the green fluorescenceemission, signaling mitochondrial depolarization, was observed withDEX treatment. A mild treatment of staurosporine (2.5 mM,overnight), served as a control, also induced a shift of thegreen fluorescence emission. C: JC-1 staining in control andmyocilin-transfected TM cells. A shift from red to green fluorescencewas observed in a population of cells upon myocilin transfection. Bar,20 mm. D: JC-1 staining in control and DEX-treated humancorneal fibroblasts. DEX treatment did not affect the JC-1 staining.Bar, 20 mm.


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transfection in a populationof cells (Fig. 4C). It is of note that thetransfected cells were mixed with the non-transfected cells inthe culture. They were indistinguishable particularly at the livecell stage required for JC-1 staining. The commonly usedGFPorDsRed strategywas also not applicable in this scenario since theGFP or DsRed fluorescence would interfere with the JC-1fluorescence staining. Because of the difficulty in distinguishingthe two populations, the red to green fluorescence ratios werenot determined. However, we did use G418 to select andenrich the transfected cells and noted that a substantial numberof cells in culture exhibited a red to green fluorescence shiftafter JC-1 incubation (Fig. 4C). These cells represented in alllikelihood the myocilin-overexpressing cells and thefluorescence shift strongly suggested the mitochondrialdepolarization occurred upon myocilin transfection. Asanother control, the JC-1 staining was not changed in cornealfibroblasts by DEX treatment (Fig. 4D).

Subcellular fractionation

Subcellular fractionation was performed to examine thedistribution of myocilin in human TM cells without orwith DEXtreatment. TheOptiprep fractionation result produced with anantibody against the N-terminal (amino acid residues 30–230)region of myocilin (Stamer et al., 1998) was nearly identical tothat reported previously (Wentz-Hunter et al., 2002a) using apeptide (amino acid residues 33–43) antibody. Myocilin had abroad distribution. In fractions 6 and 7, its presence overlappedwith that of Golgi marker golgin 97. In fractions 9 and 10,myocilin did co-sedimentwithmitochondrialmarkers succinatedehydrogenase B (Fig. 5A) and cytochrome c (data not shown).

Subsequently, TM cells without or with DEX treatmentwere subjected to anti-Fas challenge as previously described(Wentz-Hunter et al., 2002a). The cellswere harvested, and themitochondrial and cytosolic fractions were separated andanalyzed. The purity of the mitochondrial fraction wasconfirmed by the absence of glogin 97, a Golgi marker, and Hsc70, a cytosolic marker (Ma and Lindquist, 2001) (Fig. 5B). It wasfound that after anti-Fas induction and under apoptoticconditions, the percentage of myocilin present in themitochondrial fraction was decreased from 33.4� 2.4 to19.6� 3.6 (mean� SEM, n¼ 5, P< 0.013) and that in thecytosolic fraction was increased from 66.6� 2.4 to 80.4� 3.6(P< 0.016). The shift suggested that myocilin was redistributedor released into cytosol from mitochondrial compartments(Fig. 5B). Concurrently, cytochrome c was also released fromthemitochondria in anti-Fas-treated cells to the cytoplasm (Fig.5B). The percentage of cytochrome c in the mitochondrialfraction was decreased from 75.2� 6.4 to 43.2� 4.3(P< 0.0035) and that distributed in the cytosol was increasedfrom 24.8� 6.2 to 56.8� 4.3 (P< 0.0032) by the anti-Fastreatment.

Discussion

The present study demonstrates that myocilin is imported intothe mitochondria of TM cells, corroborating previous resultsfrom immunogold, fluorescence labeling and subcellularfractionation experiments that myocilin is associated withmitochondria (Ueda et al., 2000;Wentz-Hunter et al., 2002a) inTM cells. The level of myocilin imported into the TM cellmitochondria is dramatically higher than that into themitochondria from corneal fibroblasts and mouse liver,consistent with the notion that myocilin processing andlocalization may be distinct in TM cells (Wentz-Hunter et al.,2003).

The alkali, osmotic shock and/or protease treatment ofmitochondrial import mixtures (Fig. 2B) established that themyocilin imported may target the mitochondrial outer

Fig. 5. A:SubcellularfractionationofTMcellextract.Proteins inthefractionatedsampleswereimmunoblottedwithanti-myocilin,anti-glogin97or anti-succinate dehydrogenaseB (SDHB). The molecular weight for myocilin, golgin 97andsuccinatedehydrogenase B is respectively, 55/57, 97,and 27 kDa. The densities in gradient fractions are stated at the bottom. B: Distribution of myocilin in cytosolic (cyto) and mitochondrial (mito)fractions of lysates from DEX-treated TM cells without or with exposure to anti-Fas to induce apoptosis. Equal aliquots of cytosolic andmitochondrial fractions were subjected to Western blotting for myocilin, golgin 97, Hsc 70, and cytochrome c. The expected molecular weight formyocilin, golgin 97, Hsc 70, and cytochrome c is respectively, 55/57, 97, 70, and 15 kDa. The purity of the mitochondrial isolated was indicated byabsence of glogin 97 and Hsc 70 in the mitochondrial fraction. The percent of myocilin or cytochrome c in each fraction was determined bydensitometric analyses. Experiments were repeated 5 times.


membrane, innermembrane, intermembrane space, but not thematrix. Notably, the molecular size of myocilin is not changedafter the mitochondrial import. Most of the mitochondrialproteins are known to have a signal peptide at their N-terminusthat is rapidly removed during mitochondrial import steps(Wiedemann et al., 2004). In the case of outer membraneproteins, theirmolecular sizes remain the same before and afterincorporation into the membrane (Mihara and Omura, 1995).From this point of view, myocilin seems to be largely one ofmitochondrial outer membrane proteins. On the other hand,we cannot exclude, based on the current data, the possibilitythat myocilin, albeit to a minor extent, also resides in the innermembrane. An association of myocilin with inner membraneswas also indicated in an earlier study by immunogold labeling(Ueda et al., 2000). Myocilin hence may have a dual membranetopology, localizing on both the outer and inner membranes ofmitochondria. Although uncommon, this topology has beenobserved and described in the literature with mitochondrialproteins such as Mgm1p and Taz1p (Claypool et al., 2006).

As has been discussed previously (Wentz-Hunter et al.,2002a), the association of myocilin with ER and Golgi complex(Stamer et al., 1998; O’Brien et al., 1999) is not surprising sincemyocilin is presumably processed through these organelles


(Nguyen et al., 1998; Shepard et al., 2003). The mitochondrialassociation, by contrast, is somewhat unexpected.Nevertheless, a mitochondrial localization of myocilin was alsonoted in astrocytes (Noda et al., 2000). An increasing number ofproteins have also been shown to have both mitochondrial andextramitochondrial localization, and a variety of mitochondrialproteins are found under physiological conditions in differentsubcellular compartments (Soltys and Gupta, 2000). Examplesinclude Bcl-2 (Borgese et al., 2003), presenilin-1 (Ankarcronaand Hultenby, 2002), and TNF-associated protein 1 (Cechettoand Gupta, 2000). Myocilin now may also be added to the list.

The presence of myocilin in TM cell mitochondria raises thequestion how the protein is targeted to this site. Myocilin doesnot appear to contain a typical N-terminal mitochondrial-targeting presequence and myocilin is not further processedonce imported. Nevertheless, myocilin is predicted to contain apotential mitochondrial transit peptide at its N-terminus(amino acid residues 1–47) using the PCGene program andfollowing the charge transition and R(�2) weak rule (Gavel andVon Heijne, 1990). Both the N- and C-terminus also contain alysine and arginine-rich mitochondrial tethering domain (aminoacid residues 33–46 and 460–504; Li et al., 2004). It is unclear atpresent whether or which targeting sequence can account for

782 S A K A I E T A L .

themitochondrial translocation of myocilin. However, our datadoes show that the mitochondrial incorporation of myocilininto the membranes is mainly due to the a-helicaltransmembrane-containing N-terminal fragment (Fig. 3A) andthat theb-sheet segment at theC-terminus is a relatively minorcontributor, similar to those predicted for other outermembrane proteins (Sogo and Yaffe, 1994; Wiedemann et al.,2004; Youngman et al., 2004). Myocilin may, at least in part, beimported into mitochondria with the aid of translocatorcomplexes in the outer and inner mitochondrial membranes, asis well established with other mitochondrial proteins (Truscottet al., 2003; Green and Kroemer, 2004; Rehling et al., 2004;Wiedemann et al., 2004; Mokranjac and Neupert, 2005).Consistent with this possibility, the imported myocilin appearsto have assembled into a high molecular weight complex(Fig. 3C). Identification of components is this complex howeverawaits further experimentation.

The association between myocilin and TM cell mitochondriasuggests that myocilin might also alter the function of thisorganelle. We investigated the effect of myocilin upregulationon mitochondrial function by examining the mitochondrialmembrane potential using a positively charged carbocyaninedye JC-1 (Cossarizza et al., 1993). Our data (Fig. 4)demonstrates that both DEX treatment and myocilintransfection resulted in a decrease of mitochondrial membranepotential in TM cells as compared with untreated or non-transfected controls. In direct contrast, DEX treatment causedlittle or no change in JC-1 staining or mitochondrial membranepotential in corneal fibroblasts (Fig. 4D), in which myocilin isdocumented not to be associated with the mitochondria andthemyocilin expression is not inducible byDEX (Polansky et al.,1989; Wentz-Hunter et al., 2003).

Mitochondrial membrane depolarization has been suggestedto be an early event of apoptosis (Zamzami et al., 1995; Susinet al., 1999; Zamzami and Kroemer, 2001). The depolarizationobserved with upregulated myocilin may, in analogy to thatdescribed for apoptosis-regulating Bcl-2 family proteins(Tsujimoto, 2003; Green and Kroemer, 2004), lead to anincreased mitochondrial membrane permeability, facilitatingthe release of pro-apoptotic factors including cytochromec into the cytoplasm (Ravagnan et al., 2002; Shih et al., 2003) toactivate downstream death programs. On the other hand,recent studies have indicated that the loss of mitochondrialpotential may be uncoupled with apoptotic events such asrelease of cytochrome c (Kluck et al., 1997; Diaz et al., 1999;Samraj et al., 2007). In line with this finding, previous studiesfrom our laboratory have shown that despite mitochondrialdepolarization, neither DEX treatment nor pTarget-myocilintransfection invoked apoptosis in TM cells. However, anadditional induction by anti-Fas resulted in a significantly higherlevel of apoptosis in these cells as compared to untreated ornon-transfected controls (Wentz-Hunter et al., 2002a, 2004).This suggests that myocilin targeting to the mitochondria maynot directly cause the cells to undergo apoptosis. Rather it mayinfluence the mitochondrial membranes to sensitize cells toapoptotic challenges.

Subcellular fractionation of TM cells followed by Westernblotting analysis confirms that after the anti-Fas apoptoticinsult, a portion of the mitochondrial myocilin was shifted orre-distributed to a more cytosolic presence. This fractionapparently is released from the mitochondrial compartmentupon apoptotic challenge. An accompanied release ofcytochrome c from mitochondria to the cytosol is alsoobserved. These results imply a role of myocilin in thepro-apoptotic pathway.

In summary, we provided evidence to document thatmyocilin is imported into the mitochondria of TM cells and thatupregulation or overexpression of myocilin results in a declineof the mitochondrial membrane potential. Under challenges


such as oxidative stress or in less than optimal metabolicconditions, cells with upregulated myocilin may be primed formitochondrial depolarization and demise. TM cell loss has beenidentified as an important feature in POAG (Alvarado et al.,1984). We hypothesize that the cell vulnerability induced byupregulated myocilin, along with additional stress of factors,may be the key basis in the development of conditions such ascorticosteroid glaucoma. This notion is also consistent withclinical observations that certain glaucoma cases develop slowly(Quigley and Vitale, 1997) and that only a subset of thepopulation displays glaucoma when treated with steroids(Becker, 1965). The mitochondrial association in additionappears to be a common theme for neurodegenerative diseases(Green and Kroemer, 2004). For example, mutations ofpresenilin-1, a gene responsible for early onset Alzheimer’sdisease that has mitochondrial localization, also are known tosensitize cells to apoptotic stimuli in vitro (Ankarcrona andHultenby, 2002).

In glaucoma patients, more than 70 mutations of myocilinhave been detected (Gong et al., 2004). Most of the mutationsare mapped to the third exon of the myocilin gene. Thesemutants have been found to be misfolded to form aggregates(Yam et al., 2006). They also suppress the secretion of theendogenous myocilin (Jacobson et al., 2001) and are cytotoxic(Joe et al., 2003; Yam et al., 2006) in cultured cells. Recenttransgenic mouse studies (Senatorov et al., 2006; Shepard et al.,2007) have also provided evidence that mutations in myocilinmay affect the intracellular secretory pathway and lead tointraocular pressure (IOP) elevation and glaucoma. Forexample, transgenic mice expressing Tyr423His point mutationof myocilin (Senatorov et al., 2006) demonstrate moderateelevation of IOP, minor loss of retinal ganglion cells and axonaldegeneration in the optic nerve, possibly by blocking of thesecretion and accumulation of mutant myocilin in the cellcytoplasm. Significant IOP elevation was also achieved bytargeting and mislocating mutant myocilins to peroxisomes inmice (Shepard et al., 2007).

We herein suggest an alternative mechanism for myocilinglaucoma (Fingert et al., 2002) that via the mitochondrialassociation, myocilin may sensitize and help trigger apoptosis ofTM cells, with subsequent elevation of IOP. This may be thescenario in human steroid induced glaucoma when expressionof myocilin (without mutations) is induced by steroidtreatment. In support of this hypothesis, loss of TM cells hasbeen documented in human tissues with glaucoma (Alvaradoet al., 1984). In addition, dexamethasone administration hasbeen shown to result in increased IOP in rabbits (Knepper et al.,1985; Hester et al., 1987; Pang et al., 2001), cats (Zhan et al.,1992; Bhattacherjee et al., 1999), dogs (Gelatt and Mackay,1998), cattle (Gerometta et al., 2004) and primates (Fingertet al., 2001).

Acknowledgments

The authors thankDr.W. Daniel Stamer, University of Arizonafor generous gift of myocilin antibody, Dr. Nikolaus Pfanner,University of Amsterdam, Amsterdam, TheNetherlands for giftof pGEM4Z-porin plasmid, Ms. Ruth Zelkha for expert confocalmicroscopy assistance, and Dr. Paul Knepper for helpfuldiscussions. This work was supported by grants EY 05628(BYJTY), EY 03890 (BYJTY), and core grant EY 01792 from theNational Institutes of Health, Bethesda, MD.

Literature Cited

Addya S, Anandatheerthavarada HK, Biswas G, Bhagwat SV, Mullick J, Avadhani NG. 1997.Targeting of NH2-terminal-processed microsomal protein to mitochondria: A novelpathway for the biogenesis of hepatic mitochondrial P450 MT2. J Cell Biol 139:589–599.

Alvarado J, Murphy CG, Juster R. 1984. Trabecular meshwork cellularity in primary openangle glaucoma and non-glaucomatous normals. Ophthalmology 91: 564–579.


Anandatheerthavarada HK, Biswas G, Robin M, Avadhani NG. 2003. Mitochondrial targetingand a novel transmembrane arrest of Alzheimer’s amyloid Precursor protein impairsmitochondrial function in neuronal cells. J Cell Biol 161:41–54.

Ankarcrona M, Hultenby K. 2002. Presenilin-1 is located in rat mitochondria. BiochemBiophys Res Commun 295:766–770.

Becker B. 1965. Intraocular pressure response to topical corticosteroids. Invest OphthalmolVis Sci 4:198–205.

Bhattacherjee P, Paterson CA, Spellman JM, Graff G, Yanni JM. 1999. Pharmacologicalvalidation of a feline model of steroid-induced ocular hypertension. Arch Ophthalmol117:361–364.

Bill A. 1975. The drainage of aqueous humor. Invest Ophthalmol Vis Sci 14:1–3.Borgese N, Colombo S, Pedrazzini E. 2003. The tale of tail-anchored proteins: Coming fromthe cytosol and looking for a membrane. J Cell Biol 161:1013–1019.

Busciglio J, Pelsman A, Wong C, Pigino G, Yuan M, Mori H, Yankner BA. 2002. Alteredmetabolism of the amyloid b precursor protein is associated with mitochondrialdysfunction in Down’s syndrome. Neuron 33:677–688.

Caballero M, Rowlette LL, Borras T. 2000. Altered secretion of a TIGR/MYOC mutantlacking the olfactomedin domain. Biochim Biophys Acta 1502:447–460.

Cavadini P, GakhO, IsayaG. 2002. Protein import and processing reconstitutedwith isolatedrat liver mitochondria and recombinant mitochondrial processing peptidase. Methods26:298–306.

Cechetto JD, Gupta RS. 2000. Immunoelectron microscopy provides evidence that tumornecrosis factor receptor-associated protein 1 (TRAP-1) is a mitochondrial protein whichalso localizes at specific extramitochondrial sites. Exp Cell Res 260:30–39.

Claypool SM, McCaffery JM, Koehler CM. 2006. Mitochondrial mislocalization and alteredassembly of a cluster of Barth syndrome mutant tafazzins. J Cell Biol 174:379–390.

Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franeceschi C. 1993. A new method forthe cytofluorometric analysis of mitochondrial membrane potential using the J-aggregateforming lipophilic cation JC-1. Biochem Biophys Res Commun 197:40–45.

Dekker PJT, Martin F, Maarse AC, Bomer U, Muller H, Fuiard B, Meijer M, Rassow J, PfannerN. 1997. The Tim core complex defines the number of mitochondrial translocationcontact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.EMBO J 16:5408–5419.

Diaz G, Setzu MD, Zucca A, Isola R, Diana A, Murru R, Sogos V, Gremo F. 1999. Subcellularheterogeneity of mitochondrial membrane potential: Relationship with organelledistribution and intercellular contacts in normal, hypoxic and apoptotic cells. J Cell Sci112:1077–1084.

Fautsch MP, Vrabel AM, Johnson DH. 2006a. Characterization of the Felix domesticus (cat)glaucoma-associated protein myocilin. Exp Eye Res 82:1037–1045.

Fautsch MP, Vrabel AM, Johnson DH. 2006b. The identification of myocilin-associatedproteins in the human trabecular meshwork. Exp Eye Res 82:1046–1052.

Fingert JH, Clark AF, Craig JE, AlwardWL, Snibson GR, McLaughlin M, Tuttle L, Mackey DA,Sheffield VC, Stone EM. 2001. Evaluation of the myocilin (MYOC) glaucoma gene inmonkey and human steroid-induced ocular hypertension. Invest Ophthalmol Vis Sci42:145–152.

Fingert JH, Stone EM, Sheffield VC, Alward WL. 2002. Myocilin glaucoma. Surv Ophthalmol47:547–561.

Fujiki Y, Hubbard AL, Fowler S, Lazarow PB. 1982a. Isolation of intracellular membranes bymeans of sodium carbonate treatment: Application to endoplasmic reticulum. J Cell Biol93:97–102.

Fujiki Y, Fowler S, Shio H, Hubbard AL, Lazarow PB. 1982b. Polypeptide and phospholipidcomposition of the membrane of rat liver peroxisomes: Comparison with endoplasmicreticulum and mitochondrial membranes. J Cell Biol 93:103–110.

Gavel Y, Von Heijne G. 1990. Cleavage site motifs in mitochondrial targeting peptides. ProtEng 4:33–37.

Gelatt KN,Mackay EO. 1998. The ocular hypertensive effects of topical 0.1% dexamethasonein beagles with inherited glaucoma. J Ocular Pharmacol Therapeutics 14:57–66.

Gerometta R, Podos SM, Candia OA, Wu B, Malgor LA, Mittag T, Danias J. 2004. Steroid-induced ocular hypertension in normal cattle. Arch Ophthalmol 122:1492–1497.

Glick BS. 1995. Pathways and energetics of mitochondrial protein import in Saccharomycescerevisiae. Method Enzymol 260:224–227.

Gobeil S, Letartre L, Raymond V. 2006. Functional analysis of the glaucoma-causing TIGR/myocilin protein: Integrity of amino-terminal coiled-coil regions and olfactomedinhomology domain is essential for extracellular adhesion and secretion. Exp Eye Res82:1017–1029.

Gong G, Kosoko-Lasaki O, Haynatzki GR, Wilson MR. 2004. Genetic dissection of myocilinglaucoma. Hum Mol Genet 13:R91–R102.

Green DG, Kroemer G. 2004. The pathophysiology of mitochondrial cell death. Science305:626–629.

HardyKM,Hoffman EA,Gonzalez P,McKay BS, StamerWD. 2005. Extracellular trafficking ofmyocilin in human trabecular meshwork cells. J Biol Chem 280:28917–28926.

HesterDE, Trites PN, Peiffer RL, PetrowV. 1987. Steroid-inducedocular hypertension in therabbit: A model using subconjunctival injections. J Ocular Pharmacol 3:185–189.

JacobsonN,AndrewsM, ShepardAR,NishimuraD, SearbyC, Fingert JH,HagemanG,MullinsR, Davidson BL, Kwon YH, Alward WL, Stone EM, Clark AF, Sheffield VC. 2001. Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecularmeshwork cells and in aqueous humor. Hum Mol Genet 10:117–125.

Joe MK, Sohn S, HurW, Moon Y, Choi YR, Kee C. 2003. Accumulation of mutant myocilin inER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells and inaqueous humor. Biochem Biophys Res Commun 312:592–600.

John GB, Anjum R, Khar A, Nagaraj R. 2002. Subcellular localization and physiologicalconsequences of introducing a mitochondrial matrix targeting signal sequence in Bax andits mutants. Exp Cell Res 278:198–208.

Johnston AJ, Hoogenaad J, DouganDA, Truscott KN, YanoM,Mori M, HoogenraadNJ, RyanMT. 2002. Insertion and assembly of humanTom7 into the preprotein translocase complexof the outer mitochondrial membrane. J Biol Chem 277:42197–42204.

Kanagavalli J, Krishnadas SR, Pandaranayaka E, Krishnaswamy S, Sundaresan P. 2004.Evaluation and understandingofmyocilinmutations in Indian open angle glaucoma patients.Mol Vis 9:606–614.

Kaufmann T, Schlipf S, Sanz J, Neubert K, Stein R, Borner C. 2003. Characterization of thesignal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane. J Cell Biol160:53–64.

Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. 1997. The release of cytochrome cfrom mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 275:1132–1136.

Knepper PA, Collins JA, Frederick R. 1985. Effects of dexamethasone, progesterone, andtestosterone on IOP andGAGs in the rabbit eye. InvestOphthalmol Vis Sci 26:1093–1100.


Krause DR, Piva TJ, Brown SB, Ellem KAO. 1997. Characterization and localization ofmitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervicaladenocarcinoma cell line HeLa. J Cell Biochem 66:297–308.

Kurz M, Martin H, Rassow J, Pfanner N, Ryan MT. 1999. Biogenesis of Tim proteins of themitochondrial carrier import pathway: Differential targeting mechanisms and crossingover with the main import pathway. Mol Biol Cell 10:2461–2474.

Li W, Kedersha N, Chen S, Gilks N, Lee G, Anderson P. 2004. FAST is a BCL-XL-associatedmitochondrial protein. Biochem Biophys Res Commun 318:95–102.

Ma J, Lindquist S. 2001. Wild-type PrP and a mutant associated with prion disease aresubjected to retrograde transport and proteasome degradation. Proc Natl Acad Sci USA98:14955–14960.

Mathur A, Hong Y, KempK, Barrientos AA, Erusalimsky JD. 2000. Evaluation of fluorescencedyes for the detection of mitochondrial membrane potential changes in culturedcardiomyocytes. Cardiovas Res 46:126–138.

Mihara K, Omura T. 1995. Protein import into mammalian mitochondria. Methods Enzymol260:302–310.

Mokranjac D, Neupert W. 2005. Protein import into mitochondria. Biochem Soc Trans33:1019–1023.

Nagy I, TrexlerM, Pathy L. 2003. Expression and characterizationof the olfactomedin domainof human myocilin. Biochem Biophys Res Commun 302:554–561.

Nguyen T, Chen P, Huang W, Chen H, Johnson D, Polansky J. 1998. Gene structure andproperties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J Biol Chem 273:6341–6350.

Noda S,MashimaY,ObazawaM,KubotaR,OguchiY, Kudoh J, MinoshimaS, ShimizuN. 2000.Myocilin expression in the astrocytes of the optic nerve head. Biochem Biophys ResCommun 276:1129–1135.

O’Brien ET, Metheney CD, Polansky JR. 1999. Immunofluorescence method for quantifyingthe trabecularmeshwork glucocorticoid response (TIGR) protein in trabecularmeshworkand Schlemm’s canal cells. Curr Eye Res 19:517–524.

Pang IH,MollH,McLaughlinMA,Knepper PA,DeSantis L, EpsteinDL,ClarkAF. 2001.Ocularhypotensive and aqueous outflow-enhancing effects of AL-3037A (sodium ferriethylenediaminetetraacetate). Exp Eye Res 73:815–825.

Park BC, Shen X, Samaraweera M, Yue BYJT. 2006. Studies of optineurin, a glaucoma gene:Golgi fragmentation and cell death fromoverexpressionofwild type andmutantoptineurinin two ocular cell types. Am J Pathol 169:1976–1989.

Polansky J, Kurtz R, Alvarado J, Weinreb R, Mitchell M. 1989. Eicosanoid production andglucocorticoid regulatorymechanisms in cultured human trabecular meshwork cells. ProgClin Biol Res 312:113–138.

Polansky JR, Fauss DJ, Chen P, Chen H, Lutjen-Drecoll E, Johnson D, Kurtz RM, Ma ZD,BloomE,NguyenTD. 1997.Cellular pharmacology andmolecular biology of the trabecularmeshwork inducible glucocorticoid response gene product. Ophthalmologica 211:126–139.

Priest JW, Hajduk SL. 1996. In vitro import of the Rieske iron-sulfur protein by trypanosomemitochondria. J Biol Chem 271:20060–20069.

Quigley HA, Vitale S. 1997. Models of open-angle glaucoma prevalence and incidence in theUnited States. Invest Ophthalmol Vis Sci 38:83–91.

Ravagnan L, Roumier T, Kroemer G. 2002. Mitochondria, the killer organelles and theirweapons. J Cell Physiol 192:131–137.

Reers M, Smith TW,Chen LB. 1991. J-aggregate formation of a carbocyanine as a quantitativefluorescent indicator of membrane potential. Biochemistry 7:4480–4486.

Rehling P, BrandnerK, PfannerN. 2004.Mitochondrial import and the twin-pore translocase.Nat Rev Mol Cell Biol 5:519–530.

Rezaie T, Child A, Hitchings R, Brice G, Miller L, Coca-Prados M, Heon E, Krupin T, Ritch R,KreutzerD, Crick RP, Sarfarazi M. 2002. Adult-onset primary open-angle glaucoma causedby mutations in optineurin. Science 295:1077–1079.

Samraj AK, SohnD, Schulze-Osthoff K, Schmitz I. 2007. Loss of caspase-9 reveals its essentialrole for caspase-2 activation and mitochondrial membrane depolarization. Mol Biol Cell18:84–93.

Schagger H, Von Jagow G. 1991. Blue native electrophoresis for isolation of membraneprotein complexes in enzymatically active form. Anal Biochem 199:223–231.

Senatorov V, Malyukova I, Fariss R, Warrousek EF, Swamunathan S, Sharan SK, Tomarev S.2006. Expression of mutated mouse myocilin induces open-angle glaucoma in transgenicmice. J Neurosci 26:11903–11914.

Shepard AR, Jacobson N, Sui R, Steely HT, Lotery AJ, Stone EM, Clark AF. 2003.Characterization of rabbit myocilin: Implications for human myocilin glycosylation andsignal peptide usage. BMC Genet 4:5.

Shepard AR, Jacobson N, Millar JC, Pang TH, Steely HT, Searby CC, Sheffield VC, Stone EM,Clark AF. 2007. Glaucoma-causing myocilin mutants require the Peroxisomal targetingsignal-1 receptor (PTS1R) to elevate intraocular pressure. Hum Mol Genet 16:609–617.

Shih CM, Wu JS, Ko WC, Wang LF, Wei YH, Liang HF, Chen YC, Chen CT. 2003.Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normalhuman lung cells. J Cell Biochem 89:335–347.

Sogo LF, Yaffe MP. 1994. Regulation of mitochondrial morphology and inheritance byMdm10p, a protein of the mitochondrial outer membrane. J Cell Biol 126:1361–1373.

Soltys BJ, Gupta RS. 2000. Mitochondrial proteins at unexpected cellular locations: Export ofproteins frommitochondria from an evolutionary perspective. Int RevCytol 194:133–196.

Stamer WD, Roberts BC, Howell DN, Epstein DL. 1998. Isolation, culture, andcharacterization of endothelial cells from Schlemm’s canal. Invest Ophthalmol Vis Sci39:9143–9153.

Stone EM, Fingert HL, AlwardWL, Nguyen TD, Polansky JR, Sunden SL, Nishimura D, ClarkAF, Nystuen A, Nichols BE, Mackey DA, Ritch R, Kalenak JW, Craven ER, Sheffield VC.1997. Identification of a gene that causes primary open angle glaucoma. Science 275:668–670.

Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E,Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP,Penninger JM, Kroemer G. 1999. Molecular characterization of mitochondrial apoptosisinducing factor. Nature 397:441–446.

Tamm ER. 2002. Myocilin and glaucoma: Facts and ideas. Prog Retin Eye Res 21:395–428.Tamura Y, Harada Y, Yamano K, Watanabe K, Ishikawa D, Ohshima C, Nishikawa S,Yamamoto H, Endo T. 2006. Identification of Tam41 maintaining integrity of the TIM23protein translocator complex in mitochondria. J Cell Biol 174:631–637.

Tatsuta T, Model K, Langer T. 2005. Formation of membrane-bound ring complexes byprohibitins in mitochondria. Mol Biol Cell 16:248–259.

Truscott KN, Brandner K, Pfanner N. 2003. Mechanisms of protein import intomitochondria. Curr Biol 13:R326–R337.

TsujimotoY. 2003.Cell death regulation by theBcl-2 protein family in themitochondria. J CellPhysiol 195:158–167.

784 S A K A I E T A L .

Ueda J, Yue BYJT. 2003. Distribution of myocilin and extracellular matrix components in thecorneoscleral meshwork of human eyes. Invest Ophthalmol Vis Sci 44:4772–4779.

Ueda J, Wentz-Hunter K, Cheng EL, Fukuchi T, Abe H, Yue BYJT. 2000. Ultrastructurallocalization of myocilin in human trabecular meshwork cells and tissues. J HistochemCytochem 48:1321–1329.

Ueda J, Wentz-Hunter K, Yue BYJT. 2002. Distribution of myocilin and extracellular matrixcomponents in the juxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci43:1068–1076.

Wentz-Hunter K, Ueda J, Shimizu N, Yue BYJT. 2002a. Myocilin is associated withmitochondria in human trabecular meshwork cells. J Cell Physiol 190:46–53.

Wentz-Hunter K, Ueda J, Yue BYJT. 2002b. Protein interactions with myocilin. InvestOphthalmol Vis Sci 43:176–182.

Wentz-Hunter K, Shen X, Yue BYJT. 2003. Distribution of myocilin, product of a glaucomagene, in human corneal fibroblasts. Mol Vis 9:308–314.

Wentz-Hunter K, Shen X, Okazaki K, Tanihara H, Yue BYJT. 2004.Overexpression of myocilin in cultured human trabecular meshwork cells. Exp Cell Res297:39–48.

WiedemannN, Frazier AE, Pfanner N. 2004. The protein import machinery of mitochondria.J Biol Chem 279:14473–14476.


YamGHF, Gaplovska-Kysela K, Roth J. 2006. Aggregatedmyocilin induces Russell bodies andcauses apoptosis. Am J Pathol 170:100–109.

Youngman MJ, Hobbs AEA, Burgess SM, Srinivasan M, Jensen RE. 2004. Mmm2p, amitochondrial outer membrane protein required for yeast mitochondrial shape andmaintenance of mtDNA nucleoids. J Cell Biol 164:77–688.

Yue BYJT. 1996. The extracellular matrix and its modulation in the trabecular meshwork.Surv Ophthalmol 40:379–390.

Zamzami N, Kroemer G. 2001. The mitochondrion in apoptosis: How Pandora’s box opens.Nat Rev Mol Cell Biol 2:67–71.

Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX,Mignotte B, Kroemer G. 1995. Sequential reduction of mitochondrial transmembranepotential and generation of reactive oxygen species in early programmed cell death. J ExpMed 182:367–377.

Zhan GL, Miranda OC, Bito LZ. 1992. Steroid glaucoma: Corticosteroid-induced ocularhypertension in cats. Exp Eye Res 54:211–218.

Zhou L, Zhang S, Yue BYJT. 1996. Adhesion of human trabecular meshwork cells toextracellular matrix proteins. Invest Ophthalmol Vis Res 37:104–113.

Zhuang Z, Marks B, McCauley RB. 1992. The insertion of monoamine oxidase A into theouter membrane of rat liver mitochondria. J Biol Chem 267:591–596.

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