Factors Controlling the Uptake of Yeast Copper/Zinc Superoxide Dismutase into Mitochondria*

Lori Sturtz Field {ddagger} §, Yoshiaki Furukawa ¶ ||, Thomas V. O'Halloran ¶ ** and Valeria Cizewski Culotta {ddagger} {ddagger}{ddagger}

From the {ddagger}Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205 and the Departments of Chemistry and **Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208

Received for publication, April 24, 2003 , and in revised form, May 9, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously shown that a fraction of yeast copper/zinc-superoxide dismutase (SOD1) and its copper chaperone CCS localize to the intermembrane space of mitochondria. In the present study, we have focused on the mechanism by which SOD1 is partitioned between cytosolic and mitochondrial pools. Using in vitro mitochondrial import assays, we show that only a very immature form of the SOD1 polypeptide that is apo for both copper and zinc can efficiently enter the mitochondria. Moreover, a conserved disulfide in SOD1 that is essential for activity must be reduced to facilitate mitochondrial uptake of SOD1. Once inside the mitochondria, SOD1 is converted to an active holo enzyme through the same post-translational modifications seen with cytosolic SOD1. The presence of high levels of CCS in the mitochondrial intermembrane space results in enhanced mitochondrial accumulation of SOD1, and this apparently involves CCS-mediated retention of SOD1 within mitochondria. This retention of SOD1 is not dependent on copper loading of the enzyme but does require protein-protein interactions at the heterodimerization interface of SOD1 and CCS as well as conserved cysteine residues in both molecules. A model for how CCS-mediated post-translational modification of SOD1 controls its partitioning between the mitochondria and cytosol will be presented.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotes have evolved with two intracellular superoxide dismutase (SOD)1 enzymes to combat toxicity from superoxide anion. One, a manganese containing enzyme (SOD2), is located in the mitochondrial matrix (1) in close proximity to a primary endogenous source of superoxide, the mitochondrial respiratory chain (25). The other, a copper- and zinc-containing SOD (SOD1), is an abundant cytosolic enzyme (6) that has also been found in the nucleus, peroxisomes, and lysosomes (79). More recently, we discovered that a small percentage of the total SOD1 protein in Saccharomyces cerevisiae is located in the intermembrane space (IMS) of the mitochondria (10). This has also been shown for mammalian SOD1 with the wild type enzyme (1, 11) as well as with mutant forms of SOD1 associated with familial amyotrophic lateral sclerosis (1215). A mitochondrial location for SOD1 presumably protects against superoxide radicals released into the IMS by the respiratory chain (5, 16).

Structural studies reveal that all eukaryotic SOD1s are homodimeric proteins that contain one copper, one zinc, and one intrasubunit disulfide bond per monomer (1719). Copper insertion into SOD1 is well understood and is mediated in vivo by CCS, the copper chaperone for SOD1 (20). CCS consists of three domains. N-terminal domain I is important for binding copper under conditions in which copper is limiting (21), as normally exists within the cell (22). Domain II possesses high homology with SOD1 and is important for target recognition of SOD1 by CCS, thus facilitating heterodimerization of the two proteins (23, 24). Finally, the 30-amino acid C-terminal domain III of CCS contains two conserved cysteine residues (CXC) that bind copper and facilitate its insertion into the active site of SOD1 (21). One of these cysteines is capable of forming an intermolecular disulfide bond with a conserved cysteine in SOD1 as part of the heterodimeric CCS-SOD1 complex (24).

Our previous work showed that a fraction of yeast CCS co-localizes with SOD1 in the IMS of the mitochondria. Moreover, CCS appears to greatly influence the subcellular distribution of yeast SOD1 (10). When the fraction of CCS that localizes to the mitochondria was reduced, mitochondrial SOD1 levels were also lowered. Likewise, when the bulk of CCS was targeted to the mitochondrial IMS, SOD1 protein levels in the IMS were greatly increased (10).

The mechanism by which CCS influences mitochondrial accumulation of SOD1 is not understood. Does CCS facilitate the transport of SOD1 across the outer mitochondrial membrane, or does the copper chaperone act at a later step to help retain the enzyme within the IMS? Furthermore, what is the physical state of SOD1 that enters the mitochondria? With the various post-translational modifications of the enzyme, any number of SOD1 "microstates" are possible substrates for mitochondrial uptake.

In the present study, we have examined how the mitochondrial localization of SOD1 is affected by its physical state and by its copper chaperone, CCS. We demonstrate that only an immature form of SOD1, lacking both copper and zinc and possessing a reduced disulfide, is eligible for mitochondrial entry. Moreover, CCS modulates the mitochondrial accumulation of SOD1 by apparently increasing retention of the enzyme within the IMS, without affecting the mitochondrial uptake of SOD1. Once inside the mitochondria, SOD1 is subject to the same post-translational maturation steps as cytosolic SOD1.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Culture Conditions—Strain LS101 (MAT{alpha} leu 2-3, 112 his3{Delta}1 trp1-289 ura3-52 sod1{Delta}::TRP1 lys7{Delta}::URA3) (10) lacking both endogenous SOD1 and CCS (encoded by the S. cerevisiae LYS7 gene) was used throughout this study. Transformation of LS101 followed the lithium acetate procedure (25). All of the strains and transformants were propagated at 30 °C using enriched yeast extract, peptone-based medium supplemented with 2% glucose (YPD) or minimal synthetic dextrose medium (26).

Plasmids—Plasmids pLS101 (LEU2 CEN) expressing SOD1, pLS113 (HIS3 CEN) expressing native yeast CCS, and pLS117 (HIS3 CEN) expressing yeast CCS fused to the IMS-targeting sequence of cytochrome b2 (10) and the empty vectors pRS313 (HIS3 CEN) and pRS315 (LEU2 CEN) (27) were described previously. The mutant yeast SOD1 expressing plasmids pLS119 (H46A,H48A), pLS130 (F50E,G51E), pLS131 (C57S), pLS132 (C146S), and pLS133 (C57S,C146S) were generated by site-directed mutagenesis of the SOD1 expression plasmid pLS101. pLS117 expressing the cytochrome b2-CCS fusion was also mutagenized to produce plasmids pLS026 (C229S with respect to the natural CCS start codon), pLS027 (C231S), and pLS009 (C229S,C231S). All of the mutagenesis reactions were performed using the QuikChange site-directed mutagenesis kit (Stratagene). Sequence integrity was confirmed by DNA sequencing (Johns Hopkins University Core Facility).

Purification of SOD1 and Determination of Metal and Disulfide Status—Expression of yeast SOD1 in Escherichia coli BL21 and cell lysis were previously described (28). The proteins were precipitated by 50 to 75% ammonium sulfate. The precipitate was collected, redissolved, and desalted using the HiPrepTM Desalting column (Amersham Biosciences). Yeast SOD1 protein was purified using an anion-exchange column (Uno-Q12; Bio-Rad) and a MonoQTM HR5/5 column (Amersham Biosciences). Protein concentration was determined with a calibrated Bradford (Bio-Rad) curve with immunoglobulin G as the standard and by monitoring the absorption at 280 nm using 1490 M1 cm1/monomer as the extinction coefficient (29).

Fully reduced and demetallated yeast SOD1 (E,ESH) was prepared by treating the as isolated yeast SOD1 protein with dithiothreitol to reduce the disulfide bond and adding EDTA, BCS, and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine and organic solvents (15% CH3CN, 10% CH3OH) to remove bound metals.2 Metal content of E,ESH was determined by inductively coupled plasma atomic emission spectroscopy, ICP-AES, using a Thermo Jarrell Ash Atomscan model 25 Sequential ICP Spectrometer.

E,ZnSH and Cu,ZnS-S were prepared by adding equimolar amounts of ZnSO4 and CuSO4 where appropriate in a glove box for 1 or 2 h, respectively, at 37 °C. Unbound metals were removed using a Centrifugal Filtration Device (Millipore). The metal contents were verified by ICP-AES.

E,ES-S and E,ZnS-S were prepared by the addition of 10-fold molar excess of ferricyanide to the solution of E,ESH and E,ZnSH, respectively. Excess oxidant was removed with a Centrifugal Filtration Device, and the metal content was determined by ICP-AES.

The thiol-disulfide status of the purified SOD1s was determined by chemical modification with the thiol-specific reagent 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (Molecular Probes, Inc.). 4-Acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid conjugation results in an ~1-kDa increase in the molecular mass of SOD1SH as visualized by nonreducing SDS-PAGE and Coomassie Blue staining. The increase in molecular mass was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

In Vitro Mitochondrial Import Assays—Yeast cells lacking endogenous SOD1 were grown with aeration to mid-late log phase in YPD at 30 °C. The cells were harvested, and the mitochondria were isolated as described (30). The mitochondria were resuspended in import buffer (0.6 M sorbitol, 50 mM HEPES, 50 mM KCl, 10 mM MgCl2, 2.5 mM EDTA, 2 mM KH2PO4, and 1 mg/ml fatty acid-free bovine serum albumin, pH 7.0) (31). 100-µl in vitro import reactions generally included 100 µg of crude mitochondria, 200 or 400 ng of purified SOD1 protein, and 2 mM NADH. Although NADH is generally prescribed for in vitro import reactions, we found that it is not needed for mitochondrial uptake of SOD1. Because treatment of mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone did not perturb SOD1 import (not shown), mitochondrial uptake of SOD1 appears to be independent of a membrane potential, as has been shown with other IMS proteins that lack cleavable presequences (3234). The in vitro import reaction proceeded for 1 h at 30 °C, and mitochondria were then pelleted by centrifugation at 13,000 rpm for 10 min. The SOD1 that did not enter the mitochondria was effectively removed by washing the mitochondria three times with 250 mM sucrose, 10 mM MOPS buffer containing 1 mg/ml fatty acid-free bovine serum albumin (to prevent nonspecific binding of SOD1 to the mitochondrial outer membrane). The washed pellets were resuspended in 20 µl of this same buffer. SDS loading dye and dithiothreitol were added to all of the samples. The samples were vortexed for 1 min, boiled for ~5 min, and filtered through Spin-x centrifuge tube filters (0.22-µm membrane; Corning Inc.) prior to analysis of mitochondrial uptake of SOD1 by SDS-PAGE on 14% Tris-glycine gels (Invitrogen) and Western blotting.

Mitochondrial Retention of SOD1—The mitochondria were isolated from cells containing SOD1 but expressing different levels of CCS. The samples containing 100 µg of mitochondria in 1 ml of import buffer supplemented with 1 mg/ml bovine serum albumin and 2 mM NADH were incubated at 30 °C for 0 or 60 min to allow for any possible exit/loss of SOD1 from the mitochondria. Following incubation, 40-µl aliquots of all samples were taken to compare the total SOD1 in the 60-min samples with that in the 0-min samples (as in Fig. 3D). The mitochondria remaining in both samples were then reisolated by centrifugation at 13,000 rpm for 10 min and washed once with 250 mM sucrose, 10 mM MOPS buffer containing 1 mg/ml bovine serum albumin to identify the portion of SOD1 retained within mitochondria (as in Fig. 3C). All of the samples were processed as detailed above for gel electrophoresis and immunoblotting for SOD1.



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FIG. 3.
Evidence for mitochondrial retention of SOD1 by mitochondrial CCS. A, strain LS101 was transformed with the SOD1 expression plasmid pLS101 and either the empty vector pRS313 (CCS, –), pLS113 containing wild type CCS (WT), or pLS117 expressing the Cyt b2-CCS (Cyt b2). The cells were fractionated into PMS and mitochondria and analyzed according to cell equivalents, where 100- or 25-fold more cell equivalents of mitochondrial fractions were loaded for detection of SOD1 and CCS, respectively (10). These fractions were immunostained for SOD1 (top panel) and CCS (bottom panel) protein levels. B, in vitro mitochondrial import assays were conducted where indicated (+ Mitochondria) by incubating apo and reduced SOD1 (E,ESH) with mitochondria isolated from strain LS101 that lacked SOD1 but expressed various levels of CCS as indicated in A. 2 ng of the total SOD1 added to the reactions is represented in the first lane. The samples were analyzed by immunoblotting with antibodies directed against SOD1 (top panel) and CCS (bottom panel). C and D, mitochondria were isolated from the cells described in A. Duplicate samples for each mitochondrial type were prepared. The mitochondria were either incubated under in vitro import conditions for 1 h at 30 °C (lanes 2, 4, and 6) or were not incubated (lanes 1, 3, and 5). The mitochondria were either reisolated and washed prior to immunoblot analysis (C, mitochondrial SOD1) or analyzed without further separation (D, Total SOD1). Right panels, a lighter exposure of lanes 5 and 6.

 

For in vivo studies (Figs. 3A and 4, 5, 6), SOD1 and CCS protein levels in the postmitochondrial supernatant (PMS) and mitochondria were analyzed according to cell equivalents and monitored by immunoblot as previously described (10). SOD enzymatic activity in PMS and mitochondrial fractions was assayed by nondenaturing gel electrophoresis and nitro blue tetrazolium staining (10, 35).



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FIG. 4.
Copper insertion into SOD1 by CCS is not required for mitochondrial retention of SOD1. Strain LS101 transformed with the wild type (WT) or Cyt b2-CCS expression plasmid was also transformed with the wild type SOD1 expression vector, pLS101 (A and B), or the H46A, H48A SOD1 expression plasmid, pLS119 (B). A, cells were grown in YPD in the absence or presence of 500 µM BCS. PMS and mitochondrial fractions were prepared and monitored for SOD activity by native gel electrophoresis and nitro blue tetrazolium staining (top panel); positions of SOD1 and SOD2 are indicated. These same fractions were also analyzed for SOD1 (middle panel) and CCS (bottom panel) protein levels by immunostaining as in Fig. 3A. B, PMS and mitochondria were isolated and analyzed for SOD activity (top panel) and SOD1 (middle panel) and CCS (bottom panel) protein levels as in A.

 


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FIG. 5.
Mutations at the dimer interface of SOD1 prevent CCS-control of mitochondrial SOD1 accumulation. PMS and mitochondria were isolated from cells expressing either wild type (WT) or Cyt b2-CCS as in Fig. 4 in combination with wild type or F50E,G51E (pLS130) SOD1. The fractions were analyzed for SOD activity (top panel) or SOD1 (middle panel) and CCS (bottom panel) protein levels by immunoblot as in Fig. 4.

 


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FIG. 6.
CCS-mediated mitochondrial SOD1 accumulation is inhibited by cysteine mutations in both SOD1 and CCS. A, a computer model illustrating the crystal structure of a segment of the SOD1-CCS heterodimer (adapted from Ref. 24). SOD1 is in cyan, and CCS is in blue. Cysteine residues are red, and sulfhydryls and disulfide bridges are yellow. The structures depict either the SOD1 intramolecular disulfide bond between cysteines 57 and 146 (left panel) or the SOD1-CCS intermolecular disulfide bond between cysteine 57 of SOD1 and cysteine 229 of CCS (right panel). B and C, PMS and mitochondria were isolated from strain LS101 transformed with wild type or various mutant forms of SOD1 or Cyt b2-CCS. The fractions were analyzed for SOD activity (top panel) and SOD1 (middle panel) and CCS (bottom panel) protein levels. B, cells containing the Cyt b2-CCS expression plasmid, pLS117, and either wild type SOD1 (pLS101), C57S SOD1 (pLS131), C146S SOD1 (pLS132), or C57S,C146S SOD1 (pLS133) were analyzed. C, cells were monitored that expressed wild type SOD1 (pLS101) and one of the following Cyt b2-CCS expression plasmids: pLS117 (no mutations), pLS026 (C229S), pLS027 (C231S), or pLS009 (C229S,C231S).

 


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Effects of Metallation and the Conserved Disulfide on Mitochondrial Uptake of SOD1—To study the requirements for SOD1 import into mitochondria, we developed an in vitro assay for mitochondrial uptake of yeast SOD1. Mitochondria isolated from sod1{Delta} strains of yeast were incubated with various forms of purified yeast SOD1, and following import, the SOD1 that failed to enter the mitochondria was removed by extensive washing. (SOD1 is highly resistant to the protease treatment generally used to remove protein not taken up by mitochondria during in vitro import (31).) The SOD1 that was internalized by the mitochondria was monitored by immunoblotting.

Using the aforementioned in vitro import assay, we tested the effects of SOD1 metallation on transport into mitochondria. The experiment of Fig. 1 utilized purified SOD1 that was stripped of both copper and zinc (E,E) and, where indicated, reconstituted with both metals. As seen in Fig. 1A (top panel), only the completely apo form of the enzyme lacking both metals was taken up by isolated mitochondria in the in vitro import assay, even though equal amounts of apo and metallated protein were added to the reaction (Fig. 1A, bottom panel).



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FIG. 1.
Copper/zinc SOD1 cannot be taken up by the mitochondria in vitro. Mitochondria were isolated from cells expressing wild type CCS but lacking SOD1 (LS101 transformed with pLS113 and pRS315). A, mitochondria were incubated with purified apo SOD1 lacking both copper and zinc (E,E) or with SOD1 reconstituted with copper and zinc (Cu,Zn) as described under "Experimental Procedures." SOD1 present in the mitochondria (top panel) or 2 ng of the total SOD1 added to the reaction (bottom panel) were analyzed by immunoblotting with an anti-yeast SOD1 antibody. B, following an in vitro import reaction with purified apo SOD1, the mitochondria were washed and resuspended in MOPS/sucrose buffer (10 mM MOPS, 250 mM sucrose) (lanes 2 and 3) or MOPS buffer lacking sucrose (lanes 4 and 5) and incubated for 40 min on ice. The samples were centrifuged to pellet the mitochondria/mitoplasts. The supernatants (S) and pellets (P) were analyzed for CCS (top panel, a marker of the IMS) and SOD1 (bottom panel) by immunoblotting with anti-yeast CCS and SOD1 antibodies, respectively. Lane 1 represents mitochondria (Mito) not incubated.

 

To verify that SOD1 actually entered the mitochondria, we monitored the mitochondrial IMS by hypotonic rupture of the mitochondrial outer membrane (36). The endogenous CCS of the mitochondrial IMS was used as a marker, and as seen in Fig. 1B (top panel), a good fraction of this IMS protein was released to the supernatant when mitochondria from the in vitro import reaction were incubated under hypotonic conditions (lanes 4 and 5).3 By comparison, there was no release of CCS when these mitochondria were incubated and washed in the same buffer supplemented with sucrose (lanes 2 and 3). As seen in Fig. 1B (bottom panel), the exogenously added SOD1 followed precisely the same fractionation pattern as the endogenous CCS of the IMS, only being released from the mitochondria under hypotonic conditions, indicating that the SOD1 and CCS are in the same IMS compartment. As with other IMS proteins that lack a presequence for mitochondrial import (e.g. cytochrome c), (3234) the in vitro uptake of SOD1 into mitochondria appeared to occur independently of a mitochondrial membrane potential (data not shown; see "Experimental Procedures").

To further delineate the physical state of SOD1 that enters mitochondria, we tested in the in vitro import assay forms of SOD1 that contained zinc but lacked copper. With three different preparations of the zinc-only enzyme (E,Zn), zinc binding to the protein hindered the ability of SOD1 to enter the mitochondria in the in vitro assay (Fig. 2A, top panel, compare lanes 2 and 4, lanes 3 and 5, and lanes 8 and 9). Although the degree of inhibition by zinc differed with the three protein preparations, this seemed to correlate well with the percentage of zinc occupancy in the enzyme (Fig. 2B). Therefore, zinc binding alone appears to inhibit the mitochondrial uptake of SOD1. Moreover, the additional binding of copper further prevents SOD1 uptake (Fig. 2A, lanes 6 and 10). These effects of metallation on mitochondrial uptake are seen regardless of whether the mitochondria contain CCS (Fig. 2A, top left panel) or not (top right panel).



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FIG. 2.
Completely apo and reduced SOD1 is optimal for entry into the mitochondria. SOD1 proteins were purified that either lacked both copper and zinc (E,E), contained only zinc (E,Zn), or contained both copper and zinc (Cu,Zn). The proteins were also subject to reduction (SH) or oxidation (S-S) of the disulfide bond between cysteine residues 57 and 146 of SOD1 as described under "Experimental Procedures." A, Top panel, the purified proteins were incubated with isolated mitochondria containing wild type CCS but lacking SOD1 (cells used in Fig. 1) (left panel) or with mitochondria null for both CCS and SOD1 (LS101 transformed with empty vectors pRS313 and pRS315) (right panel). Uptake of SOD1 proteins (top panel) and total SOD1 added to the import reaction (bottom panel) were analyzed by immunostaining as described in Fig. 1. B, the copper and zinc occupancy as well as the disulfide content of each SOD1 sample used in A was determined by ICP-AES and chemical modification with 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid, respectively, as described under "Experimental Procedures." The numbers on the left-hand side correspond to the lane in A in which that particular protein sample was used. Lanes 2–6 and 8–10 represent two independent sets of SOD1 preparations.

 

SOD1 contains a well conserved intramolecular disulfide bond between cysteines 57 and 146 (17). To assess the impact of this disulfide on mitochondrial import, we prepared forms of the apo and zinc-only enzyme that contained either reduced or oxidized disulfides and tested these for mitochondrial uptake. It was not possible to analyze the reduced form of the copper and zinc enzyme because copper ion itself is generally an efficient oxidant for disulfide formation (37). As seen in Fig. 2A (top panel), oxidation of these cysteines to form the disulfide greatly reduced the uptake of SOD1 by the mitochondria (compare lanes 2 and 3 and lanes 4 and 5) even in the completely apo form of the protein. The observed differences in protein uptake by the mitochondria are not due to differences in the amount of the various forms of SOD1 added to the import reactions (Fig. 2A, bottom panel). Overall, these in vitro import assays indicate that mitochondrial entry of SOD1 is optimized when SOD1 lacks both copper and zinc and has reduced cysteines at positions 57 and 146.

The Role of CCS in Mitochondrial Accumulation of SOD1—We examined how CCS may influence the mitochondrial accumulation of SOD1. As seen in the in vivo studies of Fig. 3A, SOD1 still localizes to the mitochondria of yeast lys7{Delta} cells lacking CCS. This is consistent with our in vitro import assays (Fig. 2A, right panels; Fig. 3B) demonstrating that CCS is not needed for mitochondrial uptake of SOD1. However, the yield of in vivo mitochondrial SOD1 is often reduced in cells lacking CCS (Fig. 3A) and in certain instances, even difficult to detect (10), which may reflect the loss of SOD1 from the mitochondria when CCS is absent (see below). Although CCS is not essential for the mitochondrial uptake of SOD1, the levels of mitochondrial SOD1 are greatly increased when cells express high levels of mitochondrial CCS. As we have previously shown, the targeting of CCS to the mitochondrial IMS through the use of the cytochrome b2 presequence (Cyt b2-CCS) resulted in a dramatic increase in mitochondrial SOD1 accumulation in vivo (Fig. 3A, top panel, compare lanes 4 and 6).

There are two potential explanations for how high mitochondrial CCS could increase mitochondrial levels of SOD1. The first possibility is a "dragging-in" effect, in which CCS on the outside of mitochondria facilitates SOD1 entry, perhaps through a co-transport mechanism. However, in our preliminary in vitro import assays for SOD1, we failed to detect any increase in SOD1 import when purified CCS was added to the outside of mitochondria (data not shown). The second possibility is a "retention" effect, in which CCS inside the mitochondria helps retain the SOD1 that enters the IMS. To investigate this possibility, we conducted in vitro import assays for apo and reduced SOD1 using mitochondria that contained varying levels of CCS. As shown in Fig. 3B, with increasing levels of CCS inside the mitochondria, there was also a moderate increase in the amount of SOD1 accumulated by mitochondria in vitro, suggesting that CCS may help retain SOD1 inside the mitochondria. Although the presence of mitochondrial CCS moderately affected mitochondrial accumulation of SOD1 in vitro (Fig. 3B), the effect was not nearly as strong as that seen with mitochondrial CCS in vivo (Fig. 3A). This may reflect the fact that CCS does not efficiently activate SOD1 following in vitro mitochondrial import, because we cannot detect enzymatic activity under these conditions (data not shown). One or more factors necessary for SOD1 activation (e.g. proper mitochondrial sources for zinc and copper and conditions for disulfide oxidation, see below) appear to be lost from the isolated mitochondria used for in vitro import.

To further test the possible role of CCS in mitochondrial retention of SOD1, we employed mitochondria that had accumulated active SOD1 enzyme in vivo. Mitochondria isolated from cells expressing SOD1 and various levels of mitochondrial CCS (as in Fig. 3A) were either placed on ice (Fig. 3C, lanes 1, 3, and 5) or incubated for 1 h to allow for any possible export of SOD1 (lanes 2, 4, and 6). Mitochondria were then washed and reisolated, and the level of SOD1 remaining in mitochondria was determined by immunoblot. As seen in Fig. 3C, the amount of SOD1 retained in the mitochondria seemed proportional to the amount of mitochondrial CCS. The greatest loss of SOD1 was seen with mitochondria lacking CCS (compare lanes 1 and 2), whereas there was no significant loss of SOD1 from mitochondria harboring high levels of CCS (compare lanes 5 and 6 in darker (left panel) and lighter (right panel) exposures). The loss of SOD1 in the absence of high mitochondrial CCS was not due to protein degradation, because the total amount of SOD1 remaining following incubation was unchanged (Fig. 3D, compare lanes 1 and 2); only the portion of retained mitochondrial SOD1 was altered (Fig. 3C). Therefore, it appears that the presence of CCS in the mitochondria helps to retain SOD1 in the mitochondria.

The Requirements for CCS Retention of SOD1 within the Mitochondria—How is CCS causing the apparent mitochondrial retention of SOD1? One plausible explanation is that the CCS-mediated conversion of SOD1 into the copper-bound form traps SOD1 in the intermembrane space of the mitochondria. Two approaches were used to test the hypothesis that copper insertion affects mitochondrial accumulation of SOD1. First, the cells were grown aerobically in the absence or presence of the copper chelator BCS. As seen in Fig. 4A (top panel), BCS treatment greatly reduced the activity of SOD1, indicative of a decrease in copper loading. However, this reduction in copper loading of SOD1 did not prevent its accumulation in the mitochondria by high mitochondrial CCS (Cyt b2-CCS) (Fig. 4A, middle panel, compare lanes 6 and 8).

Because there was residual SOD1 activity in the BCS-treated cells, we wanted to more rigorously test whether copper insertion was needed. This was accomplished by expressing a mutant SOD1 (H46A,H48A) that cannot appropriately bind copper in the active site and is therefore devoid of activity (Fig. 4B, top panel, lanes 3, 4, 7, and 8). However, this mutant SOD1, which has lost its copper site, still accumulates to elevated levels in the mitochondria of cells expressing high mitochondrial CCS (Fig. 4B, middle panel, lane 8). Thus, it appears that the CCS effect on mitochondrial accumulation of SOD1 is not due to copper insertion into SOD1 by CCS.

Because the lack of copper loading had no effect, we tested whether CCS could facilitate SOD1 accumulation through protein-protein interactions. Mutations F50E and G51E in SOD1 have been shown to disrupt formation of the CCS-SOD1 heterodimer (38, 39). This mutant is inactive because of the lack of copper loading by CCS (Ref. 38 and Fig. 5, top panel, lanes 3, 4, 7, and 8). Interestingly, F50E,G51E SOD1 is resistant to CCS control, i.e. mitochondrial levels of this mutant did not increase in cells expressing high levels of mitochondrial CCS (Fig. 5, middle panel, compare lanes 3 and 4 with lanes 7 and 8). Thus, interactions at the dimer interface of SOD1 and CCS appear to be critical for mitochondrial accumulation of SOD1.

An additional site of protein interaction between CCS and SOD1 involves an intermolecular disulfide bond. In the crystal structure of the SOD1-CCS heterodimer, Cys57 of SOD1 that normally forms an intramolecular disulfide bond with Cys146 of the enzyme is instead bridged to Cys229 of CCS (Ref. 24 and Fig. 6A). If this intermolecular disulfide is essential for the effect of CCS on mitochondrial SOD1, then mutating either Cys57 in SOD1 or Cys229 in CCS should abolish mitochondrial accumulation of SOD1. As shown in Fig. 6B, the C57S variant of SOD1 is no longer increased in cells expressing high mitochondrial CCS (compare lanes 2 and 4, middle panel), suggesting that the intermolecular disulfide may be important for CCS control of mitochondrial SOD1. However, the effect of CCS on mitochondrial SOD1 was also abolished by a mutation in the other conserved cysteine of SOD1, Cys146 (Fig. 6B, lanes 5 and 6 and lanes 7 and 8). Clearly, both cysteines of SOD1 are required for CCS control of mitochondrial SOD1 accumulation. It is noteworthy that these two cysteines that together make up the conserved disulfide bond in SOD1 are also essential for enzymatic activity (Fig. 6B, top panel, lanes 3–8).

In parallel, we tested whether Cys229 in CCS was needed for the CCS control of mitochondrial SOD1 accumulation. For comparison, Cys231 in CCS, which does not participate in the intermolecular disulfide bond with SOD1 (Ref. 24 and Fig. 6A), was also analyzed. As seen in Fig. 6C, mutating Cys229 somewhat reduced the ability of high mitochondrial CCS to increase mitochondrial levels of SOD1. However, this inhibitory effect was more profound with Cys231 (Fig. 6B, compare lanes 4 and 6), which is known to be more essential for CCS activity (21). Overall, the results of Fig. 6 demonstrate that both Cys229 of CCS and Cys57 of SOD1 can affect the CCS control of mitochondrial SOD1 accumulation. However, Cys231 in CCS and Cys146 in SOD1 are also required, suggesting that mitochondrial retention of SOD1 by CCS involves more than the formation of the structurally characterized intermolecular disulfide bond between the two proteins. All four cysteines play a critical role in the maturation of SOD1 to the fully metallated and oxidized state (21).2 A model for how SOD1 maturation affects mitochondrial accumulation of the enzyme will be presented in the discussion.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, we have shown in yeast that a fraction of the "cytosolic" proteins copper/zinc SOD and its copper chaperone, CCS, reside in the intermembrane space of the mitochondria. In the experiments presented herein, we have attempted to more fully understand how the mitochondrial uptake and accumulation of SOD1 can be influenced by the post-translational modification of the protein and by interactions with its copper chaperone, CCS.

Using an in vitro assay for mitochondrial import, we find that the completely immature form of yeast SOD1 lacking copper, zinc, and the conserved disulfide bond is most efficiently taken up by mitochondria. The mitochondrial uptake of apo yeast SOD1 is in accordance with a previously published study on human SOD1 (15). However, unlike this previous study (15), we find that even zinc binding to the enzyme can prevent mitochondrial uptake of SOD1. This apparent discrepancy may reflect differences between yeast and human SOD1, although this is unlikely based on the extensive homology between the two proteins. Alternatively, the discrepancy may be due to variations in zinc content of the reconstituted SOD1s. We did observe limited mitochondrial uptake of preparations of SOD1 that were not fully occupied with zinc (Fig. 2). Incomplete zinc occupancy and/or adventitious reduction of the disulfide may explain the mitochondrial uptake reported for preparations of the human zinc enzyme (15).

Because only the most immature form of SOD1 may enter mitochondria and because the mitochondrial enzyme is fully active in vivo, all three post-translational modifications of the enzyme that occur in the cytosol (i.e. insertion of zinc and copper and formation of the conserved disulfide) must also take place in the mitochondria. Are identical mechanisms for SOD1 maturation employed in the cytosol and mitochondria? The mechanism for zinc insertion remains unknown for both compartments; however, it is clear that copper loading into mitochondrial SOD1, like cytosolic SOD1, requires CCS. Yeast mutants of the copper chaperone exhibit no SOD1 activity in either the cytosol or the mitochondria.

Another essential step in the maturation of SOD1 is the formation of the disulfide bond between cysteine residues 57 and 146.2 We show here that these residues are essential for activity of both cytosolic and mitochondrial SOD1. There are several candidates that could possibly facilitate the oxidation of these cysteines in SOD1 including copper (II) (37) and glutathione disulfide (40). Free copper (II) is unlikely because both the cytosol (22) and mitochondrial IMS (this study) show an apparent absence of freely available copper, based on the strict CCS dependence of SOD1 for obtaining copper. Although GSSG constitutes less than 6% of total glutathione in the yeast cell (41), we cannot exclude a role for GSSG in the intermembrane space (42) in promoting protein disulfide oxidation in this cellular compartment. As an alternative possibility, recent work in the O'Halloran lab has shown that purified copper CCS can directly catalyze air oxidation of the SOD1 disulfide in vitro2 and in vivo.4 Assuming that both cytosolic and mitochondrial CCS can perform this reaction, the mechanism of SOD1 maturation appears to be mirrored in the cytosol and mitochondrial IMS (see cartoon of Fig. 7).



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FIG. 7.
Model of SOD1 partitioning between the cytosol and the mitochondria. The vast majority of newly synthesized SOD1 in the cytosol undergoes several post-translational modifications (i.e. addition of zinc by an unknown mechanism and copper insertion and disulfide oxidation by CCS) that prevent its entry into the mitochondria. However, the small fraction of SOD1 molecules that remain apo and contain a reduced disulfide are eligible for mitochondrial uptake. Once inside the intermembrane space of the mitochondria, SOD1 is subject to the same maturation steps as in the cytosol. Through its direct involvement in the maturation of SOD1, CCS can greatly influence the partitioning of SOD1 between cytosolic and mitochondrial pools.

 

Mitochondrial accumulation of SOD1 can be greatly influenced by mitochondrial CCS, and our studies suggest that this control reflects mitochondrial retention of SOD1 rather than enhanced SOD1 import into mitochondria. In the absence of mitochondrial CCS, a fraction of SOD1 is lost from mitochondria without any apparent protein degradation, suggesting that apo SOD1 may exit mitochondria. Such a phenomenon would not be unique to SOD1 because apocytochrome c can also reversibly pass through the outer membrane of the mitochondria (4345). The ability of apocytochrome c to exit the mitochondria is inhibited by interactions with its "metallochaperone," cytochrome c heme lyase, in the IMS. In fact, mitochondrial accumulation of cytochrome c is even more pronounced when mitochondrial cytochrome c heme lyase is overexpressed (43), and this is precisely what we observed with CCS and SOD1. Therefore, it is possible that small metalloproteins in their apo and unfolded state have the potential to exit the mitochondrial IMS if they fail to interact with their respective metal maturation factors.

How does CCS evidently retain SOD1 in the mitochondrial IMS? Protein-protein interactions between SOD1 and CCS are required. Disruption of the dimerization interface of SOD1 that interacts with CCS blocks this accumulation. In addition, two conserved cysteines (Cys57 and Cys146) in SOD1 that form the critical disulfide in the enzyme are also required. Essential sequences in CCS include the conserved cysteines in domain III. Although these cysteines facilitate copper transfer to SOD1 (21), copper insertion is not responsible for CCS retention of mitochondrial SOD1. We find that limiting or blocking copper loading of SOD1 did not prevent its accumulation in the mitochondria. We instead propose that these cysteines in CCS help retain SOD1 in the mitochondria by participating in oxidation of the SOD1 disulfide, as has been suggested by O'Halloran and colleagues.2,4 This model would readily explain why both conserved cysteines in SOD1 as well as the two conserved cysteines in domain III of CCS are needed for mitochondrial accumulation of SOD1.

Our overall model for how CCS and post-translational modification of SOD1 controls localization of the enzyme is presented in Fig. 7. The vast majority of SOD1 is converted to a metallated and oxidized protein in the cytosol, largely from interactions with cytosolic CCS. This mature SOD1 enzyme cannot enter mitochondria. However, the low percentage of SOD1 that escapes the maturation process in the cytosol remains eligible for uptake into mitochondria. It is possible that mitochondrial uptake of SOD1 occurs immediately following translation of the protein.

Because of its important role in maturation of SOD1, CCS can greatly influence the partitioning of SOD1 between the mitochondria and cytosol. When the bulk of CCS is made mitochondrial, the equilibrium of SOD1 is shifted toward the mitochondria. This presumably reflects both an increase in the cytosolic pool of apo and reduced SOD1 that may enter the mitochondria, as well as an increase in CCS-mediated retention of mitochondrial SOD1.

Finally, these studies may have important relevance to the etiology of familial amyotrophic lateral sclerosis (ALS). ALS is a fatal, adult onset, neurodegenerative disorder. Approximately 20% of all familial ALS cases are due to gain-of-function mutations in SOD1 (46, 47). It is noteworthy that a number of mitochondrial pathologies have been observed in both transgenic mouse models of the disease as well as in familial ALS patients (13, 14, 4853). Recently, familial ALS mutant SOD1s have been found in the intermembrane space of the mitochondria in brain and spinal cord tissues of diseased mice (1214). Thus, it seems plausible that the fraction of SOD1 residing in the intermembrane space may be responsible for the disease. Mechanisms for controlling mitochondrial SOD1 may provide a new tool for exploring the possible role of mitochondrial SOD1 in this disease.


    FOOTNOTES
 
* This work was supported by the Johns Hopkins University NIEHS, National Institutes of Health Center, by National Institutes of Health Grants GM 50016 (to V. C. C.) and GM 54111 (to T. V. O.), by the Robert Packard Center for ALS Research at Johns Hopkins (to V. C. C.), and by a grant from the ALS Association (to T. V. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by NIEHS, National Institutes of Health Training Grant ES 07141. Back

|| Supported by Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Johns Hopkins University, 615 N. Wolfe St., Rm. 7032, Baltimore, MD 21205. Tel.: 410-955-3029; Fax: 410-955-0116; E-mail: vculotta{at}jhsph.edu.

1 The abbreviations used are: SOD, superoxide dismutase; IMS, intermembrane space; BCS, bathocuproinedisulfonic acid; ICP-AES, inductively coupled plasma atomic emission spectroscopy; PMS, postmitochondrial supernatant; ALS, amyotrophic lateral sclerosis; MOPS, 4-morpholinepropanesulfonic acid. Back

2 Y. Furukawa, A. S. Torres, and T. V. O'Halloran, submitted for publication. Back

3 It is generally difficult to obtain complete recovery of IMS proteins from in vitro import mitochondria isolated from glucose grown cells (R. Jensen, personal communication). Back

4 N. M. Brown, A. S. Torres, P. Doan, and T. V. O'Halloran, submitted manuscript. Back


    ACKNOWLEDGMENTS
 
Yeast SOD1 antibody was a generous gift of D. Kosman. We also thank R. Jensen for helpful discussions and A. Torres for preparation of protein samples for preliminary studies.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Weisiger, R. A., and Fridovich, I. (1973) J. Biol. Chem. 248, 4793–4796[Abstract/Free Full Text]
  2. Boveris, A., and Cadenas, E. (1975) FEBS Lett. 54, 311–314[CrossRef][Medline] [Order article via Infotrieve]
  3. Turrens, J. F., and Boveris, A. (1980) Biochem. J. 191, 421–427[Medline] [Order article via Infotrieve]
  4. Turrens, J. F., Alexandre, A., and Lehninger, A. L. (1985) Arch. Biochem. Biophys. 237, 408–414[Medline] [Order article via Infotrieve]
  5. Zhang, L., Yu, L., and Yu, C. A. (1998) J. Biol. Chem. 273, 33972–33976[Abstract/Free Full Text]
  6. Crapo, J. D., Oury, T., Rabouille, C., Slot, J. W., and Chang, L. Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10405–10409[Abstract]
  7. Chang, L., Slot, J. W., Geuza, H. J., and Crapo, J. D. (1988) J. Cell Biol. 107, 2169–2179[Abstract]
  8. Keller, G. A., Warner, T. G., Steimer, K. S., and Hallewell, R. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7381–7385[Abstract]
  9. Geller, B. L., and Winge, D. R. (1982) J. Biol. Chem. 257, 8945–8952[Abstract/Free Full Text]
  10. Sturtz, L. A., Diekert, K., Jensen, L. T., Lill, R., and Culotta, V. C. (2001) J. Biol. Chem. 276, 38084–38089[Abstract/Free Full Text]
  11. Okado-Matsumoto, A., and Fridovich, I. (2001) J. Biol. Chem. 276, 38388–38393[Abstract/Free Full Text]
  12. Higgins, C. M., Jung, C., Ding, H., and Xu, Z. (2002) J. Neurosci. 22, RC215[Abstract/Free Full Text]
  13. Mattiazzi, M., D'Aurelio, M., Gajewski, C. D., Martushova, K., Kiaei, M., Beal, M. F., and Manfredi, G. (2002) J. Biol. Chem. 277, 29626–29633[Abstract/Free Full Text]
  14. Jaarsma, D., Rognoni, F., Duijn, W. V., Verspaget, H. W., Hassdijk, E. D., and Holstege, J. C. (2001) Acta Neuropathol. 102, 293–305[Medline] [Order article via Infotrieve]
  15. Okado-Matsumoto, A., and Fridovich, I. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 9010–9014[Abstract/Free Full Text]
  16. Han, D., Williams, E., and Cadenas, E. (2001) Biochem. J. 353, 411–416[CrossRef][Medline] [Order article via Infotrieve]
  17. Bordo, D., Djinovic, K., and Bolognesi, M. (1994) J. Mol. Biol. 238, 366–386[CrossRef][Medline] [Order article via Infotrieve]
  18. Bertini, I., Mangani, S., and Viezzoli, M. S. (1998) in Advanced Inorganic Chemistry (Sykes, A. G., ed) Vol. 45, pp. 127–250, Academic Press, San Diego, CA
  19. Abernethy, J. L., Steinman, H. M., and Hill, R. L. (1974) J. Biol. Chem. 249, 7339–7347[Abstract/Free Full Text]
  20. Culotta, V. C., Klomp, L., Strain, J., Casareno, R., Krems, B., and Gitlin, J. D. (1997) J. Biol. Chem. 272, 23469–23472[Abstract/Free Full Text]
  21. Schmidt, P., Rae, T. D., Pufahl, R. A., Hamma, T., Strain, J., O'Halloran, T. V., and Culotta, V. C. (1999) J. Biol. Chem. 274, 23719–23725[Abstract/Free Full Text]
  22. Rae, T. D., Schmidt, P. J., Pufhal, R. A., Culotta, V. C., and O'Halloran, T. V. (1999) Science 284, 805–808[Abstract/Free Full Text]
  23. Lamb, A. L., Torres, A. S., O'Halloran, T. V., and Rosenzweig, A. C. (2000) Biochemistry 39, 14720–14727[CrossRef][Medline] [Order article via Infotrieve]
  24. Lamb, A. L., Torres, A. S., O'Halloran, T. V., and Rosenzweig, A. C. (2001) Nat. Struct. Biol. 8, 751–755[CrossRef][Medline] [Order article via Infotrieve]
  25. Gietz, R. D., and Schiestl, R. H. (1991) Yeast 7, 253–263[Medline] [Order article via Infotrieve]
  26. Sherman, F., Fink, G. R., and Lawrence, C. W. (1978) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  27. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19–27[Abstract/Free Full Text]
  28. Lamb, A. L., Wernimont, A. K., Pufahl, R. A., O'Halloran, T. V., and Rosenzweig, A. C. (2000) Biochemistry 39, 1589–1595[CrossRef][Medline] [Order article via Infotrieve]
  29. Lyons, T. J., Nerissian, A., Goto, J. J., Zhu, H., Gralla, E. B., and Valentine, J. S. (1998) J. Biol. Inorg. Chem. 3, 650–662[CrossRef]
  30. Daum, G., Bohni, P. C., and Schatz, G. (1982) J. Biol. Chem. 257, 13028–13033[Abstract/Free Full Text]
  31. Glick, B. S. (1991) Methods Cell Biol. 34, 389–399[Medline] [Order article via Infotrieve]
  32. Diekert, K., Kroon, A. I. D., Ahting, U., Niggemeyer, B., Neupert, W., Kruijff, B. D., and Lill, R. (2001) EMBO J. 20, 5626–5635[Abstract/Free Full Text]
  33. Lill, R., Stuart, R. A., Drygas, M. E., Nargang, F. E., and Neupert, W. (1992) EMBO J. 11, 449–456[Abstract]
  34. Steiner, H., Zollner, A., Haid, A., Neupert, W., and Lill, R. (1995) J. Biol. Chem. 39, 22842–22849
  35. Flohe, L., and Otting, F. (1984) Methods Enzymol. 105, 93–104[Medline] [Order article via Infotrieve]
  36. Glick, B. S., and Pon, L. A. (1995) Methods Enzymol. 260, 213–223[Medline] [Order article via Infotrieve]
  37. Kachur, A. V., Koch, C. J., and Biaglow, J. E. (1999) Free Radical Res. 31, 23–34[Medline] [Order article via Infotrieve]
  38. Schmidt, P., Kunst, C., and Culotta, V. C. (2000) J. Biol. Chem. 275, 33771–33776[Abstract/Free Full Text]
  39. Bertini, I., Piccioli, M., Viezzoli, M. S., Chiu, C. Y., and Mullenbach, G. T. (1994) Eur. Biophys. J. 23, 167–176[Medline] [Order article via Infotrieve]
  40. Sevier, C. S., and Kaiser, C. A. (2002) Nat. Rev. Mol. Cell. Biol. 3, 836–847[CrossRef][Medline] [Order article via Infotrieve]
  41. Muller, E. (1996) Mol. Biol. Cell 7, 1805–1813[Abstract]
  42. Martensson, J., Lai, J. C. K., and Meister, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7185–7189[Abstract]
  43. Dumont, M. E., Cardillo, T. S., Hayes, M. K., and Sherman, F. (1991) Mol. Cell. Biol. 11, 5487–5496[Medline] [Order article via Infotrieve]
  44. Hakvoort, T. B. M., Sprinkle, J. R., and Margoliash, E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4996–5000[Abstract]
  45. Mayer, A., Neupert, W., and Lill, R. (1995) J. Biol. Chem. 21, 12390–12397
  46. Deng, H. X., Hentati, A., Tainer, J. A., Iqbal, Z., Cayabyabi, A., Hung, W. Y., Getzoff, E. D., Hu, P., Herzfeld, B., Roos, R. P., Warner, C., Deng, G., Soriano, E., Smyth, C., Parge, H., Ahmed, A., Roses, A. D., Hallewell, R. A., Pericak-Vance, M. A., and Siddique, T. (1993) Science 261, 1047–1051[Medline] [Order article via Infotrieve]
  47. Gurney, M. E., Pu, H., Chiu, A. U., Canto, M. C. D., Polchow, C. Y., Alexander, D. D., Caliendo, J., Hentati, A., Kwon, Y., Deng, H. S., Ehen, W., Zhai, P., Sufit, R. L., and Siddique, T. (1994) Science 264, 1772–1775[Medline] [Order article via Infotrieve]
  48. Dhaliwal, G. K., and Grewal, R. P. (2000) Neuroreport 11, 2507–2509[Medline] [Order article via Infotrieve]
  49. Menzies, F. M., Cookson, M. R., Taylor, R. W., Turnbull, D. M., Chrzanowska-Lightowlers, Z. M., Dong, L., Figlewicz, D. A., and Shaw, P. J. (2002) Brain 125, 1522–1533[Abstract/Free Full Text]
  50. Wong, P. C., Pardo, C. A., Borchelt, D. R., Lee, M. K., Copeland, N. G., Jenkins, N. J., Sisodia, S. S., Cleveland, D. W., and Price, D. L. (1995) Neuron 14, 1105–1116[Medline] [Order article via Infotrieve]
  51. Kong, J., and Xu, Z. (1998) J. Neurosci. 18, 3241–3250[Abstract/Free Full Text]
  52. Jaarsma, D., Haasdijk, E. D., Grashorn, J. A., Hawkins, R., Duijn, W. v., Verspaget, H. W., London, J., and Holstege, J. C. (2000) Neurobiol. Dis. 7, 623–643[CrossRef][Medline] [Order article via Infotrieve]
  53. Jung, C., Higgins, C. M., and Xu, Z. (2002) J. Neurochem. 83, 535–545[CrossRef][Medline] [Order article via Infotrieve]