Instituto Cajal, Consejo Superior de Investigaciones Científicas, 28002 Madrid, Spain
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Abstract |
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The characterization of scully, an essential
gene of Drosophila with phenocritical phases at embryonic and pupal stages, shows its extensive homology
with vertebrate type II L-3-hydroxyacyl-CoA dehydrogenase/ERAB. Genomic rescue demonstrates that four
different lethal mutations are scu alleles, the molecular
nature of which has been established. One of them,
scu3127, generates a nonfunctional truncated product.
scu4058 also produces a truncated protein, but it contains
most of the known functional domains of the enzyme.
The other two mutations, scu174 and scuS152, correspond
to single amino acid changes. The expression of scully mRNA is general to many tissues including the CNS;
however, it is highest in both embryonic gonadal primordia and mature ovaries and testes. Consistent with
this pattern, the phenotypic analysis suggests a role for
scully in germ line formation: mutant testis are reduced
in size and devoid of maturing sperm, and mutant ovarioles are not able to produce viable eggs. Ultrastructural analysis of mutant spermatocytes reveals the presence of cytoplasmic lipid inclusions and scarce
mitochondria. In addition, mutant photoreceptors contain morphologically aberrant mitochondria and large
multilayered accumulations of membranous material.
Some of these phenotypes are very similar to those
present in human pathologies caused by -oxidation
disorders.
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Introduction |
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ALTHOUGH energy storage and metabolism have been
well-studied in Drosophila (Clark, 1989), the enzymes implicated in fatty acid oxidation have not
been characterized, and the phenotypes associated with
genetic alterations in this metabolic pathway have not
been described.
-oxidation is a major metabolic process by which fatty acids are oxidized to provide a significant
source of energy, while also generating acetyl-CoA, a metabolite that is located at the crossroads of many metabolic
routes. In mammals, hepatic
-oxidation provides circulating ketone bodies. These ketone bodies are a very important fuel for other organs
especially the brain
when
blood glucose levels are low, for example, during long-lasting
exercise or starvation. By contrast, in muscles,
-oxidation
is almost exclusively used to obtain energy from complete oxidation of acetyl-CoA. In animal cells, both mitochondria and peroxisomes are the subcellular organelles where
-oxidation takes place (reviewed by Mannaerts and Van
Veldhoven, 1996; Eaton et al., 1996
), but the mitochondrion is the main site of energy production. As a secondary product of mitochondrial aerobic respiration, reactive oxygen species are generated (Boveris et al., 1973). Also,
mitochondria are important storage sites for intracellular
calcium, and are necessary for intracellular calcium buffering (Gunter et al., 1994
). Currently, mitochondria are considered a triggering factor in the onset of many neurodegenerative diseases (Beal et al., 1993
; Sims, 1996
).
During one passage through the -oxidation pathway,
saturated fatty acids with an even number of carbon atoms
release a pair of carbon residues. This release is achieved
by four consecutive reactions successively catalyzed by
acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (HADH),1 and 3-ketoacyl-CoA
thiolase. Over the last years, it has become clear that
-oxidation pathway enzymes consist of specificity groups of isoenzymes that catalyze the same reaction, but differ in their
affinity for carbon chain length of the various substrates. Complexity of this metabolic pathway is further increased
by tissue-specific isoenzymes. In mitochondria, the third
step of the pathway was known to be catalyzed by two
HADHs with overlapping substrate chain-length specificities. Long-chain HADH is a trifunctional protein that catalyzes the last three steps of
oxidation. It is tightly associated with the inner mitochondrial membrane, and is
active with medium and long chain-length substrates (El-Fakhri and Middleton, 1982
). In contrast, short-chain
HADH is a monofunctional soluble enzyme located in the
mitochondrial matrix that preferentially metabolizes short
chain-length substrates (He et al., 1989
). However, a new
type of HADH has been recently characterized (Kobayashi et al., 1996
) and cloned (Furuta et al., 1997
) from bovine liver. Termed type II short chain HADH, it differs
from the classical isozyme (type I) in its primary structure,
and also in its molecular and catalytic properties. It is clear
now that the
-oxidation pathway conceals a more elaborate specificity than previously thought.
Primary defects in mitochondrial function are implicated in a growing number of human diseases (Luft, 1994;
Roe and Coates, 1995
). Manifestation of these diseases are
thought to result from oxidative stress derived from energy imbalance. Oxidative stress, perhaps partly glutamate-mediated, has also been implicated in some age-
related neurodegenerative diseases such as Parkinson, Alzheimer, and Huntington diseases, and amyotrophic lateral sclerosis (Beal et al., 1993
; Coyle and Puttfarcken,
1993
). In several inherited enzymopathies of the mitochondrial fatty acid
-oxidation pathway (reviewed in Roe
and Coates, 1995
), the affected enzymatic activity remains
unknown, partly as consequence of the emerging complexity of the enzymatic repertoire. Studies of patients with
these genetic disorders suggest that mitochondrial
-oxidation may be essential only during periods of high energy
demand such as fasting, febrile illness, or muscular exertion. In addition, the levels of some of the
-oxidation mitochondrial enzymes have been shown to increase only after birth (Lopaschuk et al., 1992
; Hainline et al., 1993
). During prenatal development,
-oxidation seems to represent a minor energy source, and thus the role of the
-oxidation enzymes in this period is not well understood.
In this study, we report the molecular characterization
of a Drosophila gene, scully (scu), that encodes a protein
with high structural homology to type II HADH. This is
the first enzyme related to -oxidation that has been reported in Drosophila. Recently, the human homologue
ERAB has been shown to bind the amyloid-
peptide, and
has consequently been related with Alzheimer's disease neuronal dysfunction (Yan et al., 1997
). We also report the
phenotypes associated with different lethal alleles of this
gene. The mitochondrial phenotypes presented here suggest an identical localization for this new protein with that
of its vertebrate counterparts. Striking similarities between the cellular phenotypes of scu mutants and the human pathologies associated with alterations in this metabolic pathway are discussed.
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Materials and Methods |
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Fly Culture and Strains
Flies were grown and collected under standard conditions (Ashburner,
1989). We used Canton-S (CS) as the wild-type stock. Lethal mutations
scu174 and scu4058 were induced by ethyl methanesulphonate (EMS) and x
ray, respectively, on a f 5 os-marked chromosome (Ferrús et al., 1990
). scu3127
was induced by EMS on a y w hdp3-marked chromosome. scuS152 was generated by EMS mutagenesis, and was kindly provided by Dr. N. Perrimon
(Department of Genetics, Harvard Medical School, Boston, MA). All mutations were kept either balanced with chromosome FM6 or covered by duplication Dp(1,3)JC153 (stock was C[1]M3/scu*;Dp[1;3]JC153/TM3).
Biology of Mutants: Mosaics and Lethal Phase Analysis
To determine the lethality phase, scu*/+ females were crossed to CS males. We allowed fertilized females to lay eggs for a 24-h period at 25°C. Groups of 20-30 eggs were examined at 1-d intervals. For generating twin clones, progeny from the cross: y w scu*os/FM6 × f 5 os was x ray-treated 0-48 h after egg laying. The presence of somatic clones was monitored in the cuticle of adult y w scu*os/f 5 os F1-females. A twin clone is composed of two adjacent patches generated in the same event of recombination: one marked with forked and the other with yellow, where the yellow clone is homozygous for the lethal mutation scu*. Single forked clones were considered internal controls. Eye mosaics were generated in y w hdp3 scu3127/M(1)n flies that had been x ray-treated between 48 and 72 h of development.
Isolation and Molecular Cloning of cDNA and Genomic Clones
An adult MATCHMAKER cDNA library from Drosophila melanogaster
(CLONTECH Laboratories, Inc., Palo Alto, CA) was screened at high
stringency with the 422E1 genomic fragment (Fig. 1). Recombinant DNA
manipulations were performed using standard procedures (Sambrook et al.,
1989). All purified cDNA positives were sequenced by the dideoxy chain
termination method using a DNA sequencer (PE Applied Biosystems,
Foster City, CA). DNA sequence data were analyzed using DNAstar software (DNASTAR Inc., Madison, WI), and comparisons were done using
the BLASTX program (Gish et al., 1993; Altschul et al., 1990
). The complete genomic sequence of scully gene has been submitted to the DDBJ/
EMBL/GenBank databases under accession No. Y15102.
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Genomic Rescue
Genomic clones were previously obtained (described in Baumann et al.,
1987). EcoRI fragments were subcloned into pBluescript KS+ vector
(Stratagene, La Jolla, CA). For genomic rescue, EcoRI fragment 422E1
was inserted into the germ line transformation vector pCasper (Pirrotta,
1988
). P element-mediated germline transformation was performed according to described methods (Spradling and Rubin, 1982
). Df(1)w flies
were the parental strain for all germline transformations.
Northern Blot Analysis
Poly(A)+ mRNA was isolated from different developmental stages of
Drosophila melanogaster CS strain using a QuickPrep MicroTM mRNA
purification kit (Pharmacia Biotech, Inc., Piscataway, NJ). The same
amount of mRNA (5 µg) for each sample was loaded on 1.2% agarose
formaldehyde gels and transferred onto nylon membranes (Nycomed
Amersham Inc., Princeton, NJ). The hybridization was in 50% formamide, 6× SSC, 10× Denhardt's solution, and 100 µg/ml herring sperm
DNA using 2 × 106 cpm/ml of random priming labeled probe (Feinberg
and Vogelstein, 1983). Finally, the filter was washed in 0.2× SSC, 0.1%
SDS at 65°C. Exposure times ranged from several hours to 3 d. Loading and transfer efficiency were monitored by staining the ribosomal RNA on
the nylon membrane with 0.05% methylene blue in 0.5 M sodium salicylate (pH 5.2). Usually, filters were further hybridized with Dras-1 probe to
quantify mRNA content in each lane (Neuman-Silberberg et al., 1984
).
Transcript sizes were estimated using mRNA markers (Boehringer Mannheim Corp., Indianapolis, IN).
In Situ Hybridization
For in situ hybridization, we used a probe corresponding to the longest
cDNA. cRNA was labeled with digoxigenin and hybridized to 12-µm paraffin sections according to Schaeren-Wiemers and Gerfin-Moser (1993)
with the following modification. Tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.5) for 24 h at 4°C. After embedding
and cutting, paraffin sections were deparaffinized in xylene, rehydrated
through a series of ethanol/PBS solutions, permeabelized in 1% Tween
for 30 min, and postfixed. Sections were prehybridized in 50% formamide,
1.3× SSC (pH 4.5), 0.5% CHAPS, 0.2% Tween, 100 µg/ml heparine, 50 µg/ml t-RNA, and 5 mM EDTA for 2 h at 65°C. Overnight hybridization was
performed at the same temperature and in the same solution with the probe at a concentration of 500 ng/ml. Sections were then extensively washed in
0.2× SSC at 65°C, passed to 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and
blocked with the addition of 10% normal goat serum and 0.1% Tween. After
incubating with anti-DIG-alkaline phosphatase (1:1,000; Boehringer Mannheim) and washing several times in 100 mM maleic acid, 150 mM NaCl, 2 mM levamisole (Sigma Chemical Co., St. Louis, MO) at pH 7.5, alkaline
phosphatase activity was developed with NBT/BCIP in 0.1 M Tris-HCl pH
9.5, 0.1 M NaCl, 50 mM MgCl2. Reaction was stopped in 1 mM EDTA.
Histological Procedures
Testes of white-to-light brown pupae (0-12 h after puparium formation)
of CS or scully mutants and adult mosaic eyes were dissected and prefixed
for 1 h in an ice-cold solution of 4% paraformaldehyde plus 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. After rinsing the specimens in
phosphate buffer, postfixation was performed in 2% OsO4 for 90 min in
the dark. Samples were dehydrated in an ascending alcohol series and embedded in SPURR resin. Semithin sections (2 mm) were stained with
Toluidine blue. Ultrathin sections (60-70 nm) were contrasted with uranyl
acetate and lead citrate, and were examined using an electron microscope
(JEOL USA, Inc., Peabody, MA). For lipid staining, testes were dissected
in 5 mM Hepes pH 7.4 containing 115 mM sucrose, 100 mM NaCl, 5 mM
KCl, 20 mM MgCl2, and 0.15 mM CaCl2, fixed in 4% formaldehyde for
1 h, cryoprotected with 30% sucrose in 0.1 M phosphate buffer, pH 7.4, and embedded in Tissue TekTM (Salavra, The Netherlands). 10-µm cryostat sections were stained with red oil O solution following a described
protocol (Humason, 1972).
Enzymatic Assay
Testes were dissected as above and homogenized in 50 mM Tris-HCl, pH
7.4. Crude extracts were assayed for the HADH reverse reaction as described in Osumi and Hashimoto (1979) using acetoacetyl-CoA as substrate. One enzyme unit corresponds to the amount of enzyme protein
that catalyzes the oxidation of 1 µM NADH/min. Reaction specificity was
monitored, omitting the substrate in a control assay. Protein content in extracts was quantified using the Protein AssayTM reagent (Bio-Rad Laboratories, Hercules, CA), and equal amounts of protein was used for comparison of HADH activity in different extracts.
PCR Analysis of the Mutants
To find the molecular alterations of scu mutants, both larval genomic
DNA and cDNA were PCR-amplified under standard conditions (Innis
and Gelfand, 1990). The oligonucleotides used to amplify genomic DNA
were: 5'-ccggatcctcactgtttgcctgctc-3' (sense) and 5'-ccgaattccccgccagctgccctac-3' (antisense). To amplify cDNA, we used the primers: 5'-caggatcctttcgcacccacaaca-3' (sense) and 5'-ccgaattcgagccacttccacagg-3' (antisense).
The products of two independent amplifications were cloned in pBluescript and sequenced in each case to confirm any possible alteration found.
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Results |
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Genetic Mapping of scully
The gene scully (scu) is defined as the complementation
group composed by the lethal mutations scu174, scu4058,
scu3127, and scuS152. These alleles have been mapped by recombination and segmental aneuploidies to the interval
between the Shaker (Sh; Baumann et al., 1987) and Troponin I (TnI; Barbas et al., 1991
) genes in the 16F region of
the X chromosome. The genomic DNA between the Sh
and TnI transcription units extends over 65 kb, and has
been previously cloned (Baumann et al., 1987
). Six independent transcripts have been identified in this interval by
Northern blot analysis using different genomic probes (not
shown). To identify the genomic region containing the scu
gene, we generated transgenic flies carrying different genomic fragments, and tested their ability to rescue scu lethal mutations (see Materials and Methods). The smallest
fragment used, 422E1, spans 2.4 kb (Fig. 1) and completely rescued the male lethality of the four alleles, but it did not rescue any other complementation group present in the
area (data not shown). Three independent insertions of
this fragment were tested in the rescue experiments. These
results demonstrate that the scu gene is fully contained
within these 2.4 kb.
Molecular Characterization of the scully Transcription Unit
Northern blot analysis of this transcription unit using the
genomic fragment 422E1 as a probe revealed a single 1.0-kb
transcript. Screening of an adult cDNA library with the
same fragment rendered four cDNA clones whose sequences were compared with that of the genomic counterpart, and provided the gene structure represented in Fig. 1.
The transcription unit is composed of two exons separated by a 551-bp intervening sequence. The intron/exon borders precisely match consensus splice sequences (Mount
et al., 1992). The four cDNAs start at different but nearby
sites, suggesting that initiation of transcription occurs close
to this region. A GCAGT sequence close to the first nucleotide of the longest cDNA and preceded by an AT-rich
zone is a good candidate for a functional arthropod transcription initiator (Cherbas and Cherbas, 1993
). Furthermore, 20-40 nucleotides downstream of position +1 there
are three sequences
TCGA, AACA and ACAA
that
have been reported as downstream elements important for
initiation of transcription (Arkhipova, 1995). There is also a TATA box-like sequence in the upstream genomic region. Finally, three of the cDNAs are polyadenylated at
the same site 18 nucleotides downstream of the polyadenylation signal AUUAAA (Wickens and Stephenson, 1984
). Thus, our analysis predicts a full-length mRNA of
1026 nucleotides in agreement with the size of the band
detected by Northern blot analysis.
scully Encodes a Short Chain L-3-hydroxyacyl-CoA Dehydrogenase
All four cDNAs comprise a complete and identical open
reading frame of 765 nucleotides. The start codon 118ATG
matches the consensus sequence described for Drosophila
(Cavener and Ray, 1991). Translation of the cDNA predicted a 255-amino acid protein with an estimated Mr of
27 kg/mol. A PROSITE pattern search of this protein sequence detected the consensus motif for short-chain alcohol dehydrogenase between amino acids 149 and 177. In
agreement, a basic local alignment search tool search revealed a high degree of homology with proteins of the
short-chain dehydrogenase/reductase (SDR) family, identifying the protein Scully as a new member. Highest homology scores revealed a bovine protein (AB002156), biochemically characterized as a new type of HADH (Furuta et al.,
1997
), and its human counterpart (U73514; Zhuchenko et
al., unpublished data). The last sequence is identical to human ERAB (U96132), a protein that binds amyloid-
peptide (ERAB) and mediates neurotoxicity in Alzheimer's
disease (Yan et al., 1997
). Fig. 2 shows the alignment of
Scully with the consensus sequence derived from its three
mammalian homologues. Based on the high degree of
identity between Scully and this consensus (61% along the
whole sequence), we propose that this new protein is the
Drosophila homologue of the enzyme L-3-hydroxyacyl-CoA dehydrogenase type II, a new type of monofunctional enzyme involved in the mitochondrial
-oxidation
of fatty acids. Scully and its homologues seem to define a
new subfamily in the SDR family of proteins.
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Molecular Characterization of scully Mutations
Three allelesscu174, scu3127, and scuS152
were induced
by EMS mutagenesis; scu4058 was generated by x rays.
Southern analysis detected an alteration only in scu3127. Sequencing of at least two independent PCR amplifications
of genomic DNA and cDNA from male third instar mutant larvae identified the molecular alteration in each of
the four scu alleles (Fig. 2). scu3127 is a 254-bp deletion
within exon 2. The putative altered transcript encodes a
truncated peptide that contains the first 86 amino acids followed by seven out-of-frame residues and a premature
stop codon. The other three mutations are point mutations. In scu174 the codon change 1136CTG
CAG results in
the amino acid substitution L33Q, affecting a residue that
is strictly conserved in all Scully homologues. In scuS152,
the mutation 1397TTC
ATC produces the amino acid
substitution F120I in another well-conserved residue.
scu4058 is a deletion of two nucleotides (1651GG) that causes
a frame shift at E205 and generates a stop codon 60 nucleotides farther downstream. Note that in scu4058 the truncated putative protein has both the catalytic center and the
site for cofactor binding, but lacks the carboxy terminal region that is supposed to confer substrate specificity (see
Discussion).
Developmental and Tissue Expression of scully
Expression of scu transcripts was analyzed by Northern blot and in situ hybridization to paraffin sections. Northern analysis detected a single 1.0-kb band at all developmental stages studied that displayed developmental quantitative differences. mRNA was low in unfertilized oocytes, and increased during embryonic development (Fig. 3 a). In adults, expression was higher in females than in males (Fig. 3 a). mRNA in situ hybridization to late embryos showed a very intense signal in the gonadal primordium (Fig. 3 b). The high expression in gonads persisted through all larval stages, during which other tissues, such as third instar larvae imaginal discs, central nervous system, and salivary glands also showed scu expression (Fig. 3 c). A weaker signal is also observed in the gut epithelial cells and malphigian tubules (not shown). In adults, the transcript was especially abundant in female abdomen where signal appeared associated with nurse cells in the ovaries (Fig. 3 d). In males, testes also maintained high levels of mRNA (not shown).
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scully Mutant Phenotypes
The developmental effects of scu mutations were analyzed in different cell types in either entirely mutant or mosaic flies. Analysis of the lethal phases revealed interallelic differences (Table I). These differences were studied in detail for three alleles: scu174, scu4058, and scu3127. scu174, which encodes a protein with a single amino acid substitution, and scu4058, which contains a small carboxy-terminal deletion, exhibit an early lethal phase during the embryo stage, followed by a major period of lethality around the onset of metamorphosis. Interestingly, scu3127, which encodes a protein truncated at its amino terminus, is completely viable during embryonic development, and becomes lethal only when larvae reach the late third instar. The molecular alteration of scu3127 indicates that this is a functionally null allele, and mutant survival during embryonic and larval stages must result from the maternal normal product accumulated during the oogenesis. By contrast, scu174 and scu4058 behave as dominant negative (antimorphic) alleles; they show earlier lethality that cannot be completely rescued by maternal protein.
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We analyzed the in vivo effects of scully mutations in
mosaic adult flies with patches of mutant cells. Previously
it was reported that recovery of gynandromorphs for scu174
was extremely rare (10%), and male/mutant territories
were small and with few and short bristles (Ferrús et al.,
1990). Small somatic mosaics were produced in the cuticle
of individuals heterozygous for scu4058 or scu3127. Bristle
markers allowed to recognize the wild-type (+/+) and homozygous mutant (scu*/scu*) areas derived from a single
heterozygotic cell (twin mosaic analysis; see Materials and
Methods). Table II shows that the number of mutant
clones obtained was reduced with respect to the wild-type
controls in both alleles tested. This result indicates that
scully activity is required for cell survival, and that this requirement is cell-autonomous. As in the previous trait,
there are differences between the two alleles. scu4058 was
incapable of generating mutant clones with more than one cell, and, similar to scu174, the bristles were short and thin.
scu3127, however, was able to generate larger clones of normal morphology. Thus, the null mutation (e.g., scu3127)
also showed the mildest phenotype in mosaic analysis.
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To analyze the effects of scully mutations on cell morphology, we attempted to obtain larger patches of mutant
cells in the form of Minute+ clones (see Materials and
Methods). Eye mosaics of the allele scu3127 were recovered
at a frequency three times lower than that of controls.
Photoreceptor cells did not differentiate normally, and
ommatidia were not recognizable in the mutant territory
(Fig. 4 a). Ultrastructural analysis of these mosaics revealed putative photoreceptor cells that were unable to
form a proper rhabdomere, and usually contained large
multilayered accumulations of membranous material (Fig.
4 c). Particularly interesting was the abnormality detected
in the mitochondria. They appeared smaller and with
fewer and swollen crestae when compared with neighboring wild-type cells. This mitochondrial phenotype (pleomorphism) is a typical pathology in humans with inherited
oxidation deficiencies (reviewed in Roe and Coates, 1995
).
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We also examined the morphological phenotypes in scu
mutant males before onset of lethality. In third instar larvae, we observed a clear reduction of the salivary glands
(not shown). Most striking, however, was the dramatic reduction of testes size in all four scu alleles. The degree of
reduction varied in the following order: scu174 > scu4058 > scuS152 > scu3127. This striking phenotype in testes
prompted a functional assay for HADH activity in these
organs. We analyzed crude extracts from wild-type and
mutant-dissected male gonads, and we detected up to 102
U/mg of total protein (see Materials and Methods) in the
wild-type extracts, while scu3127 yielded no detectable activity. This observation is further evidence that scully encodes a HADH activity, and that testes have a specific requirement for this enzyme. To further study the morphological features of the mutant phenotype, we analyzed
the ultrastructure of testes from either mutant or rescued
early pupae. In wild-type pupal testes when early stages of
spermatogenesis are occurring, spermatocytes are organized in cysts of 16 cells derived from four mitotic divisions (Fuller, 1993
). However, in all scu mutants, spermatocytes were generally organized in smaller groups of
~2-6 cells. Normally, the first cohort of developing spermatocytes enters meiosis at about the white prepupal stage, and
forms sperm bundles. Except for scu3127, which contained
few sperm bundles (Fig. 5 a), we did not detect any bundle
of maturing sperm in any of the other scu alleles, even at
the later brown pupal stage. Degenerating spermatocytes are only occasionally observed in normal testes, but we detected numerous degenerating necrotic cells in scu174 (Fig.
5 c). However, the most remarkable phenotype at the light microscope was the high amount of clear cytoplasmic vesicles observed in mutant testes cells (Fig. 5, a and c). The
lipid nature of these inclusions was demonstrated after oil
red staining of cryostat sections (Fig. 5 d). The large accumulation of small fat-containing vesicles in the cytoplasm
of mutant spermatocytes was more clearly observed at the
electron microscope (Fig. 6). At this magnification, the
normal eccentric position of nuclei in primary spermatocytes was conserved in scu3127 mutants, but the nucleoli of
mutant cells were not as compact as in wild-type cells, and
had many cavities. Moreover, the cytoplasm of the spermatocytes normally has a large number of mitochondria, whereas this number was dramatically reduced in all scu
mutants (Fig. 6 a). Rescued scu3127; T(2)422E1/T(2)422E1
displayed wild-type characteristics. Most strikingly, mitochondria were abundant, and steatosis was absent (Figs. 5
b and 6 b).
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By mosaic analysis, scu174 has been shown to be lethal in
the female germ line (Ferrús et al., 1990). This together
with the high level of scully expression in the nurse cells of
the ovaries, indicates a requirement for this protein during
oogenesis.
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Discussion |
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scully as a New Member of the Type II HADH Family
In this study we present the molecular and phenotypical
characterization of a Drosophila gene named scully. The
high degree of structural homology between the encoded
product and the biochemically characterized bovine mitochondrial 3-L-hydroxyacyl-CoA dehydrogenase type II
(type II HADH) strongly suggests a functional conservation between these two proteins. The involvement of
Scully in -oxidation of fatty acids is further supported by
the absence of HADH activity in mutant testes extracts
and the steatosis trait of the mutant phenotype. Other sequences of still uncharacterized function in human, mouse,
and Caenorhabditis elegans show strong identity with both
Scully and its bovine homologue, indicating that they may
be counterparts of this enzyme. The human protein
ERAB has been related with Alzheimer's disease, although its pathological mechanism remains unknown (Yan
et al., 1997
). The primary sequence of Scully and its homologues identifies these proteins as members of the SDR
family. This family includes a growing number of proteins from prokaryotes to mammals, with diverse substrate
specificity but with conserved tertiary structure (reviewed
in Krozowski, 1994
and Jörnvall et al., 1995
). Two other
known members of the SDR family in Drosophila melanogaster are encoded by the Adh and the Fbp2 genes. While
these genes are closely related to one another, scully displays low homology with them, indicating a very distant evolutionary relationship.
scu Mutations and Their Structural Implications
The amino-terminal 16 amino acids of bovine type II
HADH has been proposed as a noncleaved signal that targets the enzyme into mitochondria (Furuta et al., 1997) by
a mechanism similar to that used by another enzyme of
mitochondrial
-oxidation, the short-chain 3-oxoacyl-CoA
thiolase (Arakawa et al., 1990
, Amaya et al., 1988
). Comparison of the amino-terminal sequence of these proteins
reveals only a loose consensus, but a high number of hydrophobic and basic amino acids and a lack of acidic residues are in common. Although the putative import signal
of Scully is significantly shorter than that of its homologues (10 vs. 15-19 aa, see Fig. 2), it is rich in basic and
hydrophobic residues, suggesting that it might have a similar function. Furthermore, morphological defects observed in mitochondria of scu mutants support a mitochondrial localization of Scully.
Downstream from the targeting signal in the primary sequences of all members of the SDR family, there is a segment with three glycine residues. These residues are characteristic of the cofactor (NAD[P]+)-binding fold that is
common to dehydrogenases in general (Rossman et al.,
1975). Structural and mutational analysis indicates that the
adjacent region is also important for cofactor affinity and
specificity (Grimshaw et al., 1992
; Tanaka et al., 1996
; Nakanishi et al., 1997
). Interestingly, the scu174 substitution
L33N is located in this area, most likely affecting the
cofactor-binding capacity of the enzyme, and therefore
strongly diminishing its activity. Alternatively, the substitution in scu174 might have a more general effect by disrupting the tertiary structure of the protein. In fact, crystallographic studies of two members of the SDR family
have shown that these proteins form almost identical one-domain structures in which single substitutions might have profound effects (Ghosh et al., 1991
; Ghosh et al., 1994
;
and Varughese et al., 1992
).
Several members of the SDR family are known to form
homomultimers. Two -helices have been identified in
each of two SDR proteins as the subunit-interacting area
that gives strong dimer interactions (Ghosh et al., 1991
;
Varughese et al., 1992
). Kobayashi et al. (1996)
have
shown that bovine type II HADH is a tetramer. The antimorphic behavior of three scu mutations (scu174, scu4058,
and scuS152) is consistent with the Scully protein also forming homomultimers. Mutation scu3127 gives rise to a truncated transcript encoding a peptide that contains only the
86 amino-terminal amino acids. This peptide includes the
coenzyme-binding regions, but lacks both the putative interacting domain and the catalytic region. However, although scu3127 should be a functionally null mutation, it
displays the mildest phenotype: absence of embryonic lethal phase, larger somatic clones, and weaker testes reduction. In contrast, the other scu alleles, two of which have
only a single amino acid substitution, and the other of
which has only a small carboxy-terminal deletion, show more extreme phenotypes. In these latter cases, the hypothetical formation of multimers composed of mutant and
wild-type maternal subunits would result in effective sequestering of functional monomers into enzymatic complexes with reduced or null activity. The amino acid
change F120I in scuS152 affects an
-helix putatively involved in multimer formation. Therefore, this mutation
may impair formation of the multimer, and may explain
why scuS152 is the least antimorphic allele.
Photoaffinity labeling studies indicate that substrate
specificity of the SDR enzymes is determined to a large
extent by the carboxy-terminal portion of the protein
(Murdock et al., 1986). In fact, this portion is the most
variable region in the SDR family, reflecting the wide variety of substrates for the different enzymes. Biochemical
analysis of bovine type II HADH have shown that the specific activity of this enzyme is much higher with longer carbon chain substrates than that of type I enzyme (apparent Km values of type II enzyme for substrates other than 3-hydroxyhexadecanoyl-CoA are higher; Kobayashi et al., 1996
). The
strong similarity between Scully and the bovine type II
HADH in their carboxy-terminal regions suggests similar
substrate specificities. scu4058 generates a putative truncated protein lacking this region, and may have lost at
least substrate specificity.
Mutant Phenotype and -oxidation
One striking observation of the scully subcellular phenotype is its similarity with the human pathologies associated
with defective -oxidation of fatty acids (reviewed in Roe
and Coates, 1995
). One of the most characteristic of these
pathologies, mitochondrial pleomorphism, is also observed in mutant scu photoreceptors. Blockade of the
HADH activity leads to strong inhibition of mitochondrial
-oxidation by direct and feedback effects of the precursors on most enzymatic reactions of the chain (review in
Eaton et al., 1996
). As a direct consequence, depletion of
the mitochondrial CoA pool results in arrest of mitochondrial fatty acid import and accumulation of lipids in the cytoplasm. This phenotype is observed in the testes of all scu
mutants (Fig. 5), and resembles steatosis, another common
pathology caused by
-oxidation deficiencies. Studies in
both invertebrates (Geer et al., 1972
) and vertebrates (Risley, 1990
) have shown that there are important metabolic changes in testicular enzymatic activities during sexual maturation. In Drosophila, spermatogonia and spermatocytes obtain energy mainly from fatty acids oxidation,
while spermatozoa primarily use carbohydrates and amino
acids as energy sources (Geer et al., 1972
). The high requirement for
-oxidation during the first developmental
stages in testes accounts for the phenotypes observed
mostly in the spermatocyte stages in scu mutants. The requirement of Scully for gonad development is also consistent with the high mRNA expression observed in these organs since early developmental stages.
Differential metabolic patterns and energy demands can
also explain both the tissue distribution of the scu transcript and the different phenotypes of various cell types.
For example, scu photoreceptors do not show lipid inclusions; instead, rhabdomeres are absent, and cytoplasmic
multi-lamellar bodies are often observed. During photoreceptor differentiation, formation of rhabdomeres requires
very active synthesis of lipids; this process needs ATP and
acetyl-CoA, both produced by the -oxidation pathway.
In this case, the primary defect of scu deficiency could be
related more to phospholipid synthesis alteration than to
energy imbalance.
Several human inherited disorders have been related to
alterations in mitochondrial -oxidation (see Roe and
Coates, 1995
). They are a common cause of exercise-
induced rhabdomyolisis and myoglobinuria (Tonin et al.,
1990
). These pathologies, together with other peripheral
neuropathies, have been associated with alterations of the
long-chain HADH (Schaefer et al., 1996
). Hepato and cardiomegalies (Tyni et al., 1997
), and retino and acute encephalopathies (Pons et al., 1996
) have also been related
with deficiencies of this enzyme. Short-chain type I
HADH alterations have been described in a few cases
(Bennett et al., 1996
, Tein et al., 1991
), but the enzymatic
defect in several cases with altered oxidation remains uncharacterized. Our results indicate a more stringent requirement of Scully function. Scully is absolutely necessary for normal development in Drosophila, and point
mutations in this protein can have dominant negative effects. Studies in mammals will be required to understand
the role of this enzyme in mammalian development and
physiology. In this regard, it was recently shown that the human homologue ERAB mediates neurotoxicity induced
by amyloid-
peptide, and is overexpressed in neurons affected by Alzheimer's disease (AD; Yan et al., 1997
). Defects in mitochondrial function have been reported in AD,
and it has been proposed that mitochondrial dysfunction is
a primary causal factor for AD (Sims, 1996
). Generation of reactive oxygen species and disturbances in calcium homeostasis can ultimately lead to cell damage and neuronal
loss in AD. Also, the reduction in the activity of mitochondrial pyruvate dehydrogenase observed in AD has been
suggested as a possible cause for the disease-associated
cholinergic deficit through a decrease in acetyl-coA production (Hoshi et al., 1997
). Therefore, an attractive
model for the role of ERAB in Alzheimer's pathology is
that the effect of amyloid-
peptide on its enzymatic activity may result in energy imbalance and/or acetyl-coA depletion, leading to mitochondrial dysfunction and, finally,
neuronal damage.
An intriguing question is that the activity of several enzymes of the -oxidation of fatty acids in the brain increase during the postnatal period in mammals (Reichmann et al., 1988
; Kelly et al., 1989
), even though there are
no evidences that the brain can produce energy from systemic fatty acids. Analysis of scu phenotypes demonstrates
a role of this enzyme, at least in the development of the
photoreceptors, and findings about human ERAB also
support a role in neuronal physiology. Further analysis of the function of this enzyme in neurons will help us to understand the role of fatty acid
-oxidation in brain and to
gain new insights into the pathology of some neurodegenerative diseases.
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Footnotes |
---|
Received for publication 20 January 1998 and in revised form 31 March 1998.
Generation of transgenic flies was done in the laboratory of Dr. Kalpana White. We are especially grateful to her for her scientific advice and experimental support. The authors wish to thank Dr. A. Prado for generating reduced genomic duplications that first located the scully mutants. We are indebted to Drs. A. Rodríguez-Tébar, P. Bovolenta, G. Marqués, and M. Gordon, who criticized and improved this manuscript. D. Ortuño- Sahagún was on leave of absence from Universidad de Guadalajara (México).This work has been funded by grants PB93-149 and PB96-006 from the Dirección General de Ciencia y Tecnologia.
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Abbreviations used in this paper |
---|
AD, Alzheimer's disease; CS, Canton-S; EMS, ethyl methanesulphonate; HADH, 3-hydroxyacyl-CoA dehydrogenase; SDR, short-chain dehydrogenase/reductase.
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