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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 740-746
RED CELLS
A novel endoproteolytic processing activity in mitochondria of
erythroid cells and the role in heme synthesis
Vijole Dzikaite,
Arvydas Kanopka,
Jeremy H. Brock,
Arunas Kazlauskas, and
Öjar Melefors
From the Microbiology and Tumorbiology Center,
Karolinska Institutet, Stockholm, Sweden, and the Department of
Immunology, University of Glasgow, Western Infirmary, Glasgow,
Scotland.
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Abstract |
The erythroid isoform of aminolevulinate synthase (eALAS) protein is
a major control point in erythroid heme synthesis and hemoglobin
formation. Erythroid cells were extracted from mouse blood and bone
marrow and metabolically labeled with 35S-methionine. This
was followed by immunoprecipitation of eALAS protein products. The
results show that the N-terminus of the expected full-length 59-kd form
of the eALAS protein is truncated in bone marrow erythroid cells by
approximately 7 kd. More differentiated erythroid cells in the
peripheral blood exhibit very little of this protein truncation.
Erythroid cells from the bone marrow were isolated using monoclonal
antibody TER-119 and were shown to contain a unique endoprotease
activity that could cleave the eALAS protein to the shorter form in
vitro. With or without the mitochondrial signal sequence, the eALAS
protein could serve as a substrate for the cleavage. This cleavage
renders a functional eALAS protein and only removes a domain of unclear
function, which has previously been reported to vary in size as a
result of alternative RNA splicing. The protease activity was enriched
from the membranes of mitochondria from bone marrow cells and was shown
to be different from mitochondrial processing peptidase, medullasin,
and other known proteases. Apart from the mitochondrial processing
peptidase that cleaves the import signal sequence, this is the first
description of a mitochondrially located site-specific processing
protease activity.
(Blood. 2000;96:740-746)
© 2000 by The American Society of Hematology.
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Introduction |
Adult hemoglobin is made up of 2 - and 2 -globin
polypeptides, each of which contains a prosthetic heme group.
Heme (a porphyrin chelate of iron known as
Fe-protoporphyrin IX) controls translation of the globin messenger RNAs
(mRNAs) by using a heme-controlled kinase1 to phosphorylate
the translation initiation factor eIF2-alpha as well as the expression
of other erythroid genes.2,3 Formation of heme itself
initially requires the protoporphyrin IX skeleton, which is made in 7 enzymatic steps, starting with the rate-limiting
aminolevulinate synthase (ALAS) [Enzyme Catalogue 2.3.1.37] catalyzed
reaction between glycine and succinyl-coenzyme A (succinyl-CoA).
Erythroid cells express an isoform of ALAS (eALAS or ALAS2)
in addition to the ubiquitously expressed housekeeping form of ALAS
(hALAS or ALAS1).4-9
eALAS and other erythroid-specific genes are induced in nucleated
erythroid progenitor cells via specific transcription factors. Even
after expulsion of the nucleus, a tight posttranscriptional control of
heme synthesis maintains iron and protoporphyrin compounds at nontoxic
levels despite the fact that the rate of heme production is at least 1 order of magnitude greater in erythroid cells than in nonerythroid
cells. eALAS is believed to be the key control point for regulating
heme synthesis in reticulocytes in response to heme, iron, or other
physiological stimuli.8 Part of the regulation of eALAS and
heme synthesis has been attributed to the iron-responsive element (IRE)
in the 5' untranslated region of the eALAS
mRNA.10,11 Under low-iron conditions, iron regulatory proteins (IRP 1 and 2) will bind to the IRE in the eALAS mRNA and
repress translation. However, increased iron or heme will convert IRP
into a nonbinding form and allow translation.12,13 IRP has
also been shown to respond to other physiological stimuli in mammalian
erythroid cells, such as nitric oxide and
erythropoeitin,14,15 making it an important control point
in hemoglobin production. Heme also exerts a negative feedback effect
on its own synthesis; increasing heme levels appear to block import of
the eALAS protein into the mitochondria by binding to a specific
cysteine-proline rich sequence in the signal peptide.16
Heme may also exert a negative effect on iron release from transferrin
in red cells.8
Previous work on hemin and iron regulation of erythroid heme synthesis
has been mainly limited to erythroid cell lines. The levels of
hemoglobin are considerably lower in these cell lines, and the genes of
the heme synthesis pathway are not fully induced compared with natural
hemoglobin-producing cells in the body.17 During studies on
eALAS regulation in erythropoietic cells, we observed that 2 forms of
the protein were sometimes detected by immunoprecipitation. Because
there is no previous evidence of eALAS heterogeneity, we decided to
investigate the origins of these 2 forms.
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Materials and methods |
Preparation of cell lysates
Balb/C mice (6-8 weeks old) were killed by cervical dislocation
following local ethical guidelines. Bone marrow cells were extracted
from the femurs and added to minimal essential medium (MEM) with Earle
salts or Dulbecco modified Eagle medium (DMEM) without methionine
(Gibco BRL, Paisley, UK) using a 27-and-3/4-gauge needle.
Suspensions of spleen or liver cells were prepared by gently mashing
the nonperfused excised organ on a nylon net and discarding sedimented
debris. Peripheral blood was collected into heparinized plastic tubes.
All cells were washed by low-speed centrifugation in serum-free medium
and routinely applied to a Ficoll-Hypaque gradient (Ficoll-Hypaque
Plus; Pharmacia, Uppsala, Sweden) to enrich reticulocytes.
The cells were diluted to 3 mL in MEM, carefully overlaid
on an equal volume of Ficoll-Hypaque in a 15-mL plastic tube, and
centrifuged at 30g for 90 minutes at 4°C. The high- and
low-density fractions of cells were removed with a Pasteur pipette,
washed 3 times, and resuspended in IMDM without methionine with 10%
fetal calf serum (FCS), L-glutamine, and streptomycin/penicillin G (Gibco).
The cells from blood and bone marrow were stained with
May-Grünwald Giemsa (MGG) or methylene blue stain and examined by microscopy. Peripheral blood contained 2.7% reticulocytes. Following Ficoll-Hypaque separation, the lower fraction contained 4.0%
reticulocytes, whereas the upper fraction was virtually depleted of
reticulocytes. Bone marrow cells contained 32% erythropoietic cells;
of the erythroblasts, 30% were proerythropoietic; 49%,
basophilic; 12%, polychromatic; and 9%, orthochromatic. Although we
routinely used the lower fraction from the Ficoll-Hypaque gradient in
our experiments, separation of bone marrow cells does not give a clear
enrichment of the protease activity, which may also be present in
several erythroblast stages that separate into the upper Ficoll-Hypaque
fraction. To acquire labeled extracts, we used the following
concentrations of 35S-L-methionine as indicated:
3.7 × 1013 Bq or 0.37 MBq/µL
(1000 Ci/mmol or 10 µCi/µL) (NEG-009A; New England Nuclear, Boston, MA). Labeled cells were washed twice in ice-cold
phosphate-buffered saline (PBS) and resuspended for 30 minutes on ice
in ratio immunoprecipitation assay (RIPA) detergent
buffer containing 0.15 mol/L sodium chloride (NaCl); 1% Nonidet P-40;
0.5% deoxycholate; 0.1% sodium dodecyl sulfate (SDS); 50 mmol/L Tris
HCl (trishydroxymethyl aminomethane hydrochloride) (pH 7.5); and either
the LPPA protease inhibitors, 1 µmol/L leupeptin, 1 µmol/L
pepstatin, 2 mmol/L Pefabloc, and 0.2 µmol/L aprotinin, or Complete
Mini tablets (all from Boehringer-Mannheim, Mannheim, Germany). After
suspension on ice, the cells were centrifuged at 15 000g at
4°C to remove debris. Nonlabeled lysates of tissues were prepared
without prior labeling. The protein content of lysates was analyzed by
standard methods, and comparable amounts were used in experiments.
To isolate erythroid and granulocyte subpopulations, respectively, from
bone marrow cell suspension, we used the rat monoclonal antibodies
(mAbs) TER-11918 (PharMingen, San Diego, CA) and CL8991AP19 (Cedarlane, Quebec, Canada). Bone
marrow cells from 2 mice (60 × 106 cells) were
diluted in complete DMEM and incubated with 4 µL TER-119 (0.5 mg/mL)
or CL8991AP (1 mg/mL) for 60 minutes at 4°C in a rotator. A
120-µL aliquot of a 50% suspension of Dynabeads (No. 110.07; Dynal
AS, Oslo, Norway) that recognize rat immunoglobulin G
(IgG) were added, and the samples were incubated for another 30 minutes at 4°C. The beads with attached cells were
isolated and washed 3 times in complete DMEM using the supplied
magnetic device, and the lysates were prepared as described above.
Aliquots of separated cell populations were stained with MGG, and
microscopic inspection confirmed a strong enrichment of the specific
cell type.
Immunoprecipitation
Lysates were routinely precleared of endogenous protein A-binding
Ig chains by incubation on a rotator with 50-µL of a 50% suspension
of protein A CL-4B Sepharose beads (Pharmacia) for 1 hour at 4°C in
a total volume of 500 µL. Titrated amounts of specific antisera
against eALAS20 were rotated for 3 hours at 4°C in a
total volume of 500 µL. This was followed by incubation with 50 µL
beads for 1 hour. In the indicated experiments, cold lysates were
included in the reaction. Thereafter, beads were washed
at low speed 3 times in 1 mL RIPA, resuspended in Laemmli loading
buffer, heated at 95°C for 10 minutes to elute proteins, and loaded
on 12% SDS-PAGE (polyacrylamide gel electrophoresis), which was fixed
and exposed to X-ray film or phosphor imager screens. The protein sizes
were determined by parallel electrophoresis of commercial molecular
weight standards. The presence of the shorter form of eALAS could not
be explained by nonspecific protein degradation. No changes in the
ratio between the different eALAS forms were observed when parallel
reactions were subjected to different lengths of incubation, different
temperatures, or the addition of protease inhibitors.
Using this antiserum, we did not detect cross-reaction with hALAS or
any other proteins of nonerythroid cells.20
Subfractionation of mitochondria
Bone marrow or blood cells were separated on 3-mL Ficoll-Paque
cushions (Pharmacia) by centrifugation at 900g for 1 hour at 4°C. The high-density fraction was washed 3 times and resuspended in 1 mL methionine-free DMEM. During labeling experiments, the cells
were incubated with 35S-methionine for 3 hours at 37°C,
and the cells were then washed in PBS. The cells were subsequently
washed twice in ice-cold buffer A (250 mmol/L sucrose, 50 mmol/L Tris
HCl [pH 7.4], and 1 mmol/L EDTA [ethylenediamine tetraacetic acid])
with protease inhibitors. The cells were resuspended in 3 mL buffer A
and then lysed by sonication (Branson Sonic-Power B-15P Sonifier,
Danbury, CT) with 2 bursts of 10 seconds each (setting,
5). Lysis was confirmed by microscopic inspection. To remove debris,
the lysate was centrifuged twice at 900g for 20 minutes at
4°C. To pellet mitochondria, the supernatant was subsequently
centrifuged at 12 000g for 25 minutes at 4°C. The
resulting supernatant was defined as "cytosol fraction." The
mitochondrial pellet was washed twice in ice-cold buffer B (250 mmol/L
sucrose, 10 mmol/L Tris HCl [pH 7.4], and 1 mmol/L EDTA) and
resuspended in RIPA ("crude mitochondrial lysate").
For further subfractionation, the mitochondrial pellet was resuspended
in 1 mL buffer B and sonicated on ice 3 times for 30 seconds each time (setting, 5) followed by a 12 000g
centrifugation for 15 minutes to sediment intact mitochondria. The
suspension or sonicate was centrifuged at 250 000g for 60 minutes at 4°C. The supernatant was recentrifuged to remove debris
and was defined as the "matrix fraction" (although it might also
include intermembrane space proteins). The pellet was washed in buffer
B and defined as the "membrane fraction." Fractions were tested
by Western blot analysis for purity by using antisera against the
membrane-bound succinate dehydrogenase21 or the
matrix-bound mitochondrial transcription factor A (MTA)
protein22 (data not shown). To further enrich the
mitochondrial population, we loaded the mitochondrial pellet (see
above) on a gradient containing 30% Percoll (Pharmacia, Uppsala,
Sweden), 0.25 mol/L sucrose, 5 mmol/L Tris (pH 7.4), and
0.5 mmol/L EDTA. This was followed by centrifugation at
60 000g for 60 minutes at 4°C, and then the specific
mitochondrial fraction ("pure mitochondria") was collected.
In vitro reconstitution
Plasmids pPre-ALAS-E-minor and pPre-ALAS-E-major
express 2 natural variants of murine pre-eALAS under the control of a
phage T3 promoter in the pBluescript vector.16 The protein
derived from pPre-ALAS-E-minor is identical to the protein derived
from pPre-ALAS-E-major except that it lacks amino acids 61-75 of the pre-eALAS sequence. Plasmids were linearized with BamHI and transcribed and translated in vitro in the presence of 35S-methionine
in the Flexi Rabbit reticulocyte lysate system (Promega, Madison, WI)
according to the manufacturer's instructions. The products were
dialyzed against 10 mmol/L HEPES-KOH
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.4) and 0.2 mmol/L DTT. We mixed 0.2 µg dialyzed 40%-60% ammonium sulfate
precipitation fraction of a bone marrow crude mitochondria lysate with
9 µL in vitro translated eALAS protein in the above buffer (with 0.5 mmol/L MnCl2). This was completed in a total
volume of 20 µL and incubated at 27°C for 60 minutes before gel separation.
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Results |
To study eALAS during differentiation, we metabolically labeled ex
vivo cells from mouse peripheral blood and bone marrow with
35S-methionine. As expression of eALAS is limited to
erythroid cells, detergent lysates were prepared (in the strong ionic
detergent RIPA) and used directly, without further enrichment of
erythroblasts, for immunoprecipitation with an antiserum against murine
eALAS. Immunoprecipitation of eALAS from lysates of peripheral blood (Figure 1) revealed an expected 59-kd
double band (see below) representing the full-length forms of eALAS. A
weaker 65-kd band, presumably corresponding to the precursor before
cleavage of the mitochondrial signal peptide, was also revealed. Both
have been previously demonstrated in mouse erythroleukemia (MEL) cells
with this antiserum.20
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| Fig 1.
eALAS protein synthesis in different erythroid tissues.
Peripheral blood and bone marrow were diluted with DMEM
without methionine to 3 mL, separated on 3 mL Ficoll-Hypaque gradients,
resuspended in 1 mL medium (blood, 1.5 × 108 cells;
bone marrow, 1.9 × 107 cells), and incubated with
7.4 MBq (200 µCi) 35S-methionine for 3 hours. Detergent lysates were prepared, and eALAS and gel separation
were immunoprecipitated. The arrows indicate the presumed pre-eALAS,
the 59-kd form, and the herein described 52-kd eALAS-short.
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In addition, a specific shorter form of the eALAS (eALAS-short)
protein, approximately 52 kd in size, was precipitated from blood
cells. This was consistently the only form to be expressed in bone
marrow cell lysates. In blood cell lysates the relative amount of the
eALAS-short protein varied between experiments, ranging from
undetectable to being equimolar to the 59-kd form. This observed
pattern thus suggested a loss of eALAS-short and the appearance of the
59-kd form with the maturation of the erythroid cells. It is important
to note that the ex vivo metabolic labeling enables us to detect only
de novo formation of proteins and not steady state levels of the 2 forms of eALAS in the blood. However, a de novo pool of the 59-kd form
of eALAS in the peripheral blood reticulocyte could rapidly form,
provided the eALAS protein turns over at a similar speed as the
nonerythroid form of ALAS (half-life, approximately 35 minutes).23
There was no obvious difference in the eALAS mRNA pattern in blood and
bone marrow (data not shown), as seen using high-resolution Northern
blot gels and reverse transcriptase-polymerase chain reactions
(RT-PCRs). This indicates that the eALAS-short protein arose by a
posttranslational event. To determine whether posttranslational proteolytic cleavage might be responsible for the short form, we
coincubated a "cold" bone marrow lysate and a radiolabeled blood
cell lysate during immunoprecipitation. Figure
2 demonstrates that we can
immunoprecipitate a shorter form of eALAS, identical in size to the
52-kd eALAS-short protein observed in bone marrow cells, which
originates from the 59-kd form of the protein in the blood cells.
In addition, a shorter band of 5-10 kd accumulated, although it remains unclear if this product is related to the cleavage process.
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| Fig 2.
Cleavage of eALAS by a proteolytic activity in bone
marrow erythroid cells.
(A) Blood (1 mL) was separated on a 3-mL Ficoll-Hypaque gradient. The
high-density fraction was resuspended in medium and divided into 2 tubes to which 7.4 MBq (200 µCi)
35S-methionine was added. A 300-µL nonlabeled bone marrow
lysate (1.5 × 108 cells) (lane 2) or medium alone
(lane 1) was added. Both samples were incubated at 37°C for 3 hours, and eALAS proteins were subsequently immunoprecipitated and
gel-separated. (B) Erythroid and granulocytic cells were isolated from
the bone marrow by mAbs coupled to magnetic beads ("Materials and
methods"). We incubated 80-µg lysates from these fractions with a
labeled blood lysate for 30 minutes at 37°C, and eALAS proteins
were subsequently immunoprecipitated and gel-separated. The far-right
lane represents a lysate after depletion of erythroid cells.
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To test if this protease activity was present in erythroid cells, we
next isolated such cells from the bone marrow by employing magnetic
beads and the erythroid-specific mAb TER-119.18 In parallel
we isolated granulocytes with this technique using the mAb
CL8991AP,19 as such cells contain potent lysosomal
proteases that could be released during lysis and cleave other
proteins. When we tested equal amounts of lysates from erythroid and
granulocyte cells for protease activity, it clearly showed that
erythroid cells contained this protease activity, whereas there was
no activity detected in granulocytes (Figure 2B). The claim
of an erythroid-specific activity was further supported by the fact
that there was no activity retained in a lysate from bone marrow cells
depleted of erythroid cells.
To localize the protease activity to a cell compartment, bone
marrow cells were lysed by sonication and further subfractionated by differential centrifugation into a mitochondrial and a cytosolic fraction. To exclude the possibility that the protease activity resided
in other organelles cosedimenting with the mitochondria, crude
mitochondria were also separated on a density gradient, and the
specific mitochondrial fraction was collected. The testing of this and
other fractions for protease activity showed clearly that the activity
resides in the mitochondria (Figure 3A). We also sonicated the mitochondria and divided them into a membrane and a
matrix fraction. From testing these for the ability to cleave the 59-kd
form of the eALAS protein, we could further localize the protease
activity to the membrane fraction (Figure 3B). The mitochondrial
membrane was additionally washed with up to 1 mol/L of chaotrophic
salts without loss of protease activity, indicating that it was an
integral part of the membranes (data not shown).
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| Fig 3.
Fractionation of protease activity.
(A) Lysates of 35S-methionine-labeled blood cells were
prepared ("Materials and methods"). Equal amounts were incubated
alone or mixed with nonlabeled lysates of bone marrow cells and
subfractions of 80 µg each. The mixtures were immunoprecipitated for
3 hours at 4°C with standard protease inhibitors before gel
separation. (B) Bone marrow crude mitochondria (mit.)
were further subfractionated (80 µg each) into a membrane and matrix
fraction and were analyzed the same way. (C) Nonlabeled 80-µg lysates
each of bone marrow, spleen, and liver tissue were mixed with the
labeled blood lysate and analyzed the same way.
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We also prepared lysates of cells from the liver and spleen and tested
them for the presence of proteolytic activity in a similar way. No
protease activity could be detected in these tissues under the
conditions we used (Figure 3C). The bone marrow protease activity was
potent in a strong detergent (RIPA) and insensitive to several
inhibitors of serine, cysteine, and aspartic proteases or
metalloproteases as well as high levels of EDTA when these agents were
added to lysates at the indicated concentrations. We could only observe
inhibition of cleavage with high amounts of alpha-1-antitrypsin (Figure
4A). A dose response to
alpha-1-antitrypsin, which showed a proportional increase in the
substrate and a decrease in the product, was observed (Figure 4B). To
exclude the possibility that cleavage was due to a proteolytic activity
that was released from another cell type (or organelle) during lysis of
the bone marrow cells, we included alpha-1-antitrypsin in the lysis
buffer. But we could still only demonstrate the cleaved form (Figure
4B), which shows that the short form was not an artifact during
preparation. This conclusion was further consolidated by mixing labeled
blood and bone marrow cells before lysis in RIPA with
alpha-1-antitrypsin, as in the latter experiment. There was no observed
cleavage in the 59-kd blood eALAS protein, which served as an internal
probe in the lysate to exclude the presence of a protease (data not shown).
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| Fig 4.
Inhibition of protease activity.
(A) Bone marrow cells were aliquoted into 6 different tubes containing
RIPA and the indicated inhibitors (50 µg/mL elastatinal, 0.5 mg/mL
alpha-1-antitrypsin, 5 mmol/L EDTA, and
leupeptin/Pefabloc/pepstatin/aprotinin [LPPA]). A lysate of
35S-methionine-labeled blood cells (300 µL each) was
added, and the reaction was immunoprecipitated for 3 hours at 4°C
before gel separation. (B) The experiment was repeated with varying
amounts of alpha-1-antitrypsin (0.1, 0.25, 0.5, and 1 mg/mL, shown in
lanes 3-6, respectively). 35S-methionine-labeled bone
marrow cells were lysed in RIPA with or without 0.5 mg/mL
alpha-1-antitrypsin followed by immunoprecipitation for 1 hour at
4°C without any preclearing (lanes 7 and 8).
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Murine eALAS mRNA can be alternatively spliced to a longer form (85%
of the eALAS mRNA amount) and a shorter form. The shorter form results
in a preprotein that lacks amino acids 61-75.24 We have not
performed experiments specifically aimed to demonstrate these 2 mRNAs
in our system, but we believe that the double band observed in the
immunoprecipitations of the 59-kd form of eALAS on high-resolution gels
(Figure 1) is likely to correlate to this difference of 15 amino acids.
We translated in vitro these 2 pre-eALAS mRNAs in a rabbit reticulocyte
lysate in the presence of 35S-methionine and determined 2 slightly different 65-kd bands after SDS-PAGE (Figure
5). Both preproteins could be cleaved by
lysates from bone marrow mitochondria to the same size band,
identical to the shorter form of eALAS, because the 15 amino
acids are in the part which is cleaved off (Figure 5). This
demonstrated that cleavage occurs at the N-terminal region and not the
C-terminal end of the protein. The N-terminal region of the human eALAS
protein is also variable as a result of alternative
splicing25 (Figure 6). The
reason behind the alternative splicing in mouse and human has not been
elucidated, but it is notable that this region seems to be the subject
of more than one type of regulation. This N-terminal region is unique
to the erythroid form of ALAS, unlike the rest of the protein, which is
highly homologous with the housekeeping isoform. The N-termini of the
eALAS protein are, however, homologous between mouse, human, and rat
(Figure 6), which suggests that a similar type of cleavage could occur
in other mammals. A recent report demonstrated the presence of a fully
active shorter form of eALAS, approximately 49 kd, in human
reticulocytes, but this was not investigated further.26
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| Fig 5.
Proteolytic cleavage of in vitro translated eALAS.
In vitro translated eALAS proteins representing the 2 natural splice
variants pre-ALAS-E minor and pre-ALAS-E major were
incubated alone (lanes 1 and 2) or with a bone marrow (b.m.)
mitochondrial lysate (lanes 3 and 4) for 60 minutes at 27°C before
gel separation. A labeled blood lysate was included as a control (lane
5).
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| Fig 6.
The N-termini of the pre-eALAS protein from mouse,
human, and rat.
The downward arrow indicates mitochondrial processing peptidase
(MPP) cleavage of the signal peptide.16 The reported
cleavage is estimated to be within the boxed region. Horizontal bars
show amino acids affected by alternative splicing: 15 amino acids
(positions 61-75) for mouse eALAS24 and 37 amino
acids (positions 102-138) for human eALAS.25 The bent arrow
marks the N-terminus of the functional protein expressed from
plasmid pTAD-ALAS2,27 and the upward
arrow marks the N-terminus for a functional rat eALAS in
vitro.28
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The 52-kd eALAS-short protein in the bone marrow must be
functional, as it is the only form of eALAS made in this tissue, and
bone marrow erythroid cells can synthesize heme. The catalytic activity
is confined to the C-terminal part of the mammalian eALAS protein.4-8 Deletion of the first 56 N-terminal amino acids
of the murine eALAS still allows an active protein in Escherichia coli,27 and truncation of the 83 N-terminal amino acids
of the rat eALAS protein does not impede function in
vitro28 (Figure 6). The observed cleaved form of eALAS is
indistinguishable in size from the truncated protein27
expressed in E coli on high-resolution gels (data not shown),
and it is therefore very likely to be enzymatically active. The
localization of the shorter form of eALAS to the mitochondria also
indicates that it may be functional.
The eALAS protein domains involved in the binding (or
catalysis) of the cofactor pyridoxal 5'-phosphate (Vitamin B6)
are not in the N-terminal region and should not be directly affected by cleavage.29 Although the part which is cleaved off is not
typically lipophilic, it may serve, directly or via another protein, as an anchor to the membrane. Electron microscopy of the housekeeping form
of ALAS suggests it is associated with the mitochondrial inner membrane
facing the matrix,30 possibly by being anchored to the
succinyl-CoA synthase beta-subunit.31 To investigate the
location of the 59-kd and eALAS-short forms of eALAS, we labeled erythroid cells from blood with 35S-methionine, ruptured
them by sonication, and isolated the mitochondria, which were divided
into a membrane and a matrix fraction. The subsequent
immunoprecipitation of eALAS clearly demonstrated that both forms of
eALAS were present in the mitochondrial membrane fraction (Figure
7). The location of both the protease
activity and its product in the membrane suggests that cleavage takes
place in the mitochondrial membrane in conjunction with the import of the preprotein. This would predict that cells harboring protease activity would not produce a 59-kd form of eALAS, and this could be the
reason that we were unable to detect any of this form in bone marrow
cells.
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| Fig 7.
The short eALAS is located in the mitochondrial membrane.
Blood (4 mL) was separated on a Ficoll-Hypaque gradient. The
high-density fraction was collected and labeled with 22.2 MBq (600 µCi) 35S-methionine for 3 hours. Suborganelles
were prepared, and the lysates were used for immunoprecipitation of the
eALAS protein followed by gel separation. Lysates of membrane and
matrix fractions contained 270 µg protein each.
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Discussion |
We describe a specific truncation of the erythroid isoform of ALAS
that takes place in erythroid cells in the bone marrow. The function of
the shorter form of eALAS has yet to be established, but one can assume
that an altered function of the eALAS protein will serve to modulate
heme synthesis.
Our data argue against a direct role of the cleaved N-terminus in
membrane anchoring, although it is still possible it could serve as a
binding site for other molecules. It is noteworthy that this sequence
contains a typical heme binding site,16 suggesting a
mechanism for end-product control of the eALAS protein. Such a
contention may also be supported by the fact that this heme-binding site is lost with the alternative splicing of eALAS mRNA.24 It could also be that cleavage of the N-terminus results in a loss of
the ability of the eALAS protein to homodimerize. Yet other reasons for
the shortening of eALAS may be the changes in eALAS protein stability
or enzyme activity.
We believe there could be reasons why heme synthesis is controlled
differently in bone marrow and peripheral blood. Excessive amounts of
iron or heme easily result in the formation of aggressive free radicals
via the Fenton reactions,32 which could lead to cell damage
and cell transformation. Nucleated erythroblasts in the bone marrow
could sustain an increased risk of erythroleukemic cell transformation.
Thus a more stringent control of heme formation is needed. However,
anucleate reticulocytes (which continue to synthesize proteins after
entering the bloodstream) cannot transform, but they need
a highly efficient hemoglobin synthesis and, as a result, might have
less need of avoiding oxidative stress damage.
Some types of sideroblastic anemias are caused by point mutations in
the eALAS gene,33 and 1 mutation evidently affects processing of the pre-eALAS protein.34 In other anemias,
the reduced hemoglobin formation and the unclear molecular basis may also be associated with eALAS. For example, in the anemia of chronic disease (ACD), lower hemoglobin synthesis in erythroblasts was correlated to a reduced eALAS activity.35
This is the first description of an endoproteolytic site-specific
processing activity in mitochondria, apart from proteases involved in
cleavage of the import signal peptide.36-39 The protease activity is active in a strong ionic detergent, has no clear dependence on chelatable metals, and is not affected by most common protease inhibitors. Together with tissue specificity and the specific localization to the mitochondrial membrane fraction, we conclude that
this protease activity must be different from known endoproteases in
the mitochondria, such as mitochondrial processing peptidase (MPP) and
mitochondrial intermediate peptidase (MIP), both of which are
instrumental in cleavage of the signal peptide.40 An
elastase-like protein (medullasin) from bone marrow was previously reported to inactivate aminolevulinate synthase.41
Medullasin was later shown to be identical in sequence to neutrophil
elastase.42-43 Unlike the present protease activity, it
resides in lysosomes of granulocytes and is readily inhibited by
elastatinal. Testing of purified medullasin and neutrophil elastase on
the eALAS substrates did not reveal any similar cleavage (data
not shown).
The protease activity seems to be tissue-specific, as we have not
observed it in lysates from tissues other than bone marrow. We have
further limited the activity to the erythroid cells in the marrow. This
argues that there must be a limited number of targets for this protease
activity. We also have not observed any changes in the protein profiles
in total lysates under conditions that allow cleavage of the eALAS protein.
Many disorders affect hemoglobin function, and eALAS plays a pivotal
role in the de novo formation of hemoglobin. A future challenge thus
lies in identifying the specific function of the eALAS-short protein.
Another issue will be the further characterization and regulation of
the tissue-specific protease activity, as this might enable us to
understand the switch between the long and short forms of eALAS.
| |
Acknowledgments |
We are grateful to Mike Timko for the pre-ALAS-E plasmids, Yosuke
Aoki for medullasin, Susanne Widell for cell analysis, Cristina Szigyarto for discussions, and Matthias Hentze for initial support.
| |
Footnotes |
Supported by grant 06593-304 from the Swedish
Natural Science Research Council, Sweden; the Swedish Association for
Medical Research, Sweden (von Kantzow stipend); the Swedish Society of Medicine, Sweden; and the Lars Hierta and Magnus Bergvall foundations (Ö.M.), Sweden. While on sabbatical leave from the EMBL,
Heidelberg, Germany, J.H.B was supported by CIBA-Geigy Switzerland and
the European Union, and A.K. received a Wenner-Gren postdoctoral
stipend. V.D. is on leave from the Vytautas Magnus University in
Kaunas, Lithuania.
Submitted June 21, 1999; accepted March 3, 2000.
Reprints: Öjar Melefors,
Microbiology and Tumor Biology Center, Karolinska
Institutet, SE-171 77 Stockholm, Sweden; e-mail: ojar.melefors{at}mtc.ki.se.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction