DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial
nucleoid of Physarum polycephalum
Kie Itoha,b,c,⁎, Akiko Izumic, Toshiyuki Morid, Naoshi Dohmaee, Ryoko Yuia, Katsura Maeda-Sanoc,f,
Yuki Shiraic, Masahiro M. Kanaokaa, Tsuneyoshi Kuroiwag, Tetsuya Higashiyamaa, Mamoru Sugitaa,
Kimiko Murakami-Murofushic, Shigeyuki Kawanob, Narie Sasakia,c,⁎⁎
aDivision of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, Japan
bDepartment of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan
cDepartment of Biology, Faculty of Science, Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan
dMiyagishima Initiative Research Unit, Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
eBiomolecular Characterization Team, Advanced Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
fDepartment of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
gResearch Information Center for Extremophiles, Graduate School of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo 171-8501, Japan
a b s t r a c t a r t i c l e i n f o
Received 6 October 2010
Received in revised form 3 February 2011
Accepted 4 March 2011
Available online 13 March 2011
DNA packaging protein
Mitochondrial DNA (mtDNA) is generally packaged into the mitochondrial nucleoid (mt-nucleoid) by a high-
mobility group (HMG) protein. Glom is an mtDNA-packaging HMG protein in Physarum polycephalum. Here
we identified a new mtDNA-packaging protein, Glom2, which had a region homologous with yeast Mgm101.
Glom2 could bind to an entire mtDNA and worked synergistically with Glom for condensation of mtDNA
in vitro. Down-regulation of Glom2 enhanced the alteration of mt-nucleoid morphology and the loss of
mtDNA induced by down-regulation of Glom, and impaired mRNA accumulation of some mtDNA-encoded
genes. These data suggest that Glom2 may organize the mt-nucleoid coordinately with Glom.
© 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
The mitochondrion is an important organelle that produces the
energy for cell viability. This organelle possesses its own DNA, termed
mitochondrial DNA (mtDNA), which encodes essential components of
the respiratory chain for the synthesis of ATP. Faithful maintenance of
mtDNA is necessary for the cell. MtDNA is packed into highly organized
structures called mitochondrial nucleoids (mt-nucleoids) containing
many proteins (Kuroiwa, 1982; Kucej and Butow, 2007; Spelbrink,
2010). In the eukaryotic nucleus, genomic DNA is organized into
repeating nucleosomal units composed of histones and their 1.7-turns
mt-nucleoid is packaged and organized is not known.
are major protein components of the mt-nucleoid in many species,
including animals, fungi, and slime mold (Diffley and Stillman, 1991;
Antoshechkin and Bogenhagen, 1995; Larsson et al., 1996; Takamatsu
et al., 2002; Sasaki et al., 2003). An exception has only been reported in
mitochondria (kinetoplasts) of the tripanosomatid Crithidia fasciculata,
the mt-nucleoid (Xu et al., 1996; Hines and Ray, 1998). These proteins
are highly basic and have the ability to directly bind to mtDNA with no
sequence specificity. Studies have estimated that sufficient Abf2 and
TFAM, which are mitochondrial HMG proteins in yeast and animals,
respectively, exist to bind to every 15–30 and 10–20 bp of mtDNA
2004). These HMG proteins can entirely cover mtDNA because one
molecule occupies 25–30 bp of mtDNA (Fisher and Clayton, 1988;
Diffley and Stillman, 1992; Antoshechkin and Bogenhagen, 1995). Abf2
and TFAM can bend and wrap DNA, and package it into a condensed
2007). These properties indicate that mitochondrial HMG proteins play
an important role in mtDNA packaging, similar to histones in the
nucleus. In addition to their role in organizing the mt-nucleoid, mtDNA
packagingproteinsparticipate in theoverall regulation of DNA function
by binding to the entire mtDNA. For example, knockout of Abf2 and
knockdown of TFAM decrease mtDNA (Diffley and Stillman, 1991;
Mitochondrion 11 (2011) 575–586
high-mobility group; MO(s), morpholino antisense oligomer(s); inv., inverted oligomer.
⁎ Correspondence to: K. Itoh, Division of Biological Science, Graduate School of
Science, Nagoya University, Furo-cho, Nagoya, Aichi 464-8602, Japan. Tel.: +81 52 789
3087; fax: +81 52 789 3081.
⁎⁎ Correspondence to: N. Sasaki, Division of Biological Science, Graduate School of
Science, Nagoya University, Furo-cho, Nagoya, Aichi 464-8602, Japan. Tel./fax: +81 52
E-mail addresses: email@example.com (K. Itoh), firstname.lastname@example.org
1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/mito
MegrawandChae,1993;Larssonet al.,1998; Zelenaya-Troitskaya etal.,
1998; Goto et al., 2001; Kanki et al., 2004). Abf2 also functions in
recombination of mtDNA (MacAlpine et al., 1998). Moreover, TFAM is
involved in the transcription of mtDNA (Parisi and Clayton, 1991;
McCulloch and Shadel, 2003) and possibly also in repairing mtDNA
(Yoshida et al., 2003).
In the nucleus of the cell, various architectural proteins, including
core and linker histones and HMG proteins, are involved in structural
features of the chromosome (Khorasanizadeh, 2004). In bacteria, the
nucleoid is also organized not only by the histone-like protein called
HU but also by other architectural proteins including IHF, FIS, and
H-NS (Schmid, 1990). Recently, in mt-nucleoids, more than 50 kinds
of protein associated with mt-nucleoids, other than HMG proteins,
were identified in several species using mass spectrometry (Kaufman
et al., 2000; Chen et al., 2005; Wang and Bogenhagen, 2006;
Bogenhagen et al., 2008). Many of them were involved in mtDNA
maintenance, including mitochondrial DNA polymerase (Foury,
1989), mitochondrial RNA polymerase (Greenleaf et al., 1986), DNA
helicase (Foury and Lahaye, 1987), mitochondrial single-stranded
DNA-binding protein (Van Dyck et al., 1992), heat shock proteins
(Kaufman et al., 2000), aconitase (Chen et al., 2005), subunits of α-
ketoglutarate dehydrogenase (Kaufman et al., 2000), prohibitin
(Berger and Yaffe, 1998), acetohydroxyacid reductase (Zelenaya-
Troitskaya et al., 1995), and Mgm101 (Chen et al., 1993). However,
except for mitochondrial HMG and kinetoplast H1-like proteins, no
architectural proteins involved in the packaging of the entire mtDNA
have yet been identified.
In this study, we searched for a new mtDNA-packaging protein
that organizes the whole mt-nucleoid using a true slime mold,
Physarum polycephalum. P. polycephalum has a simple and extraordi-
narily large rod-shaped mt-nucleoid (Kuroiwa, 1974). We previously
established a method to isolate highly purified mt-nucleoids from
P. polycephalum that retain the same structure and gene expression
level as found in vivo (Suzuki et al., 1982; Sasaki et al., 1998). Isolated
mt-nucleoids consist of more than 70 proteins. Among these proteins,
Glom was identified as the most abundant basic protein, and in vivo
constitutes one-fifth of the total protein amount of mt-nucleoids
(Sasaki et al., 2003). Glom possesses two HMG boxes, like Abf2 and
TFAM, and a lysine-rich region. It has high DNA-binding and DNA-
agglomeration ability. Glom was named as a protein inducing
agglomeration of mitochondrial chromosomes. Glom has polyproline
tracts in the lysine-rich region, which are required for intense
condensation without suppressing DNA function. When isolated mt-
nucleoids are disassembled by a gradual increase in concentration of
NaCl in buffer, several proteins are released from the mt-nucleoids
with Glom, correlating with dispersion of the nucleoid structure
(Sasaki et al., 2003). This suggests the possibility that these proteins
might also be involved in DNA packaging.
Here, we report a second mtDNA-packaging protein, Glom2,which
organizes the entire mt-nucleoid together with Glom. Moreover, we
report a new down-regulation method using morpholino antisense
oligomers (MOs) in P. polycephalum. We suggest coordination of the
two mtDNA-packaging proteins in the organization and function of
2. Materials and methods
2.1. Strains and culture methods
Microplasmodia of P. polycephalum, Colonia isogenic strain
KM182×KM187, were used for isolating mitochondria and for
microinjection of MOs. Microplasmodia were cultured in semi-defined
liquid medium as described by Daniel and Baldwin (1964) at 23 °C.
Cultures at the middle exponential phase were used in the study. For
microinjection of MOs, microplasmodia (cultured in liquid medium)
were collected by brief centrifugation, and then the 150 μl of pelletable
peptone, 1.5% bacto-agar, and 2.5 μg/ml hemin) (Kawano et al., 1987)
and cultured at 23 °C in the dark. Twenty-four hours later, micro-
plasmodia were fused into one plasmodium and used for injection.
Myxamoebae of P. polycephalum, Colonia isogenic strain KM182,
were used for immunolocalization analysis. Myxamoebae were
cultured on PGY plates (Ohta et al., 1993) with live bacteria (Klebsiella
aerogenes) as food at 23 °C.
2.2. Preparation of mt-nucleoids
P. polycephalum as described in Sasaki et al. (1998). In this study, we
developed a method for isolating mt-nucleoid to remove the slight
amount of remaining mitochondrial membrane. At first, we isolated the
mt-nucleoid as described in a previous report (Sasaki et al., 1998): the
isolated mt-nucleoids were resuspended in NE1-S buffer [0.5 M sucrose,
20 mMTris–HCl(pH7.7),1 mMEDTA(pH7.5),7 mM2-mercaptoethanol,
0.4 mM spermidine, and 0.4 mM phenylmethylsulfonyl fluoride (PMSF)]
toa concentrationof2 μg/μl.Theisolatedmt-nucleoidsweretreatedwith
0.5% Nonidet P-40 (NP-40) for 1 min and centrifuged at 18,500×g for
10 min. The sedimented mt-nucleoids were washed twice, resuspended
in NE1-S buffer, and stored on ice until use.
2.3. In vitro disassembly of mt-nucleoids by NaCl or DNase
The isolated mt-nucleoids (8 μg) were suspended in NE1-S buffer
to a concentration of 0.8 μg/μl. For disassembly of the mt-nucleoid by
NaCl, an equal volume of NE1-S buffer containing NaCl at twice the
final concentration was added to the suspension and incubated at
25 °C for 3 h. For disassembly of mt-nucleoids by DNase I, DNase was
added in NE1-S buffer containing 10 mM MgSO4at a final concentra-
tion of 0.01 mg/ml and incubated at 25 °C for 2 h. Each disassembled
mt-nucleoid fraction was centrifuged at 18,500×g for 20 min at 4 °C.
The pelletable fraction and supernatant fraction were precipitated
with tricarboxylic acid (TCA) and subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
2.4. DNA-cellulose column chromatography
Mt-nucleoids (80 μg) were incubated for 2 h at 25 °C in high salt
buffer [1 M NaCl, 100 mM Tris–HCl (pH7.0), 1 mM EDTA, and 10 mM
mercaptoethanol] to extract DNA-associated proteins. The extracted
fraction was centrifuged at 18,500×g for 20 min. The supernatantwas
dialyzed with equilibration buffer [50 mM NaCl, 100 mM Tris–HCl
(pH7.0), 1 mM EDTA, and 10 mM mercaptoethanol], and adsorbed to
the native DNA-cellulose column (GE Healthcare, Milwaukee, WI,
USA). Proteins were eluted with various concentrations of NaCl buffer
[0.1, 0.2, 0.3, 0.4, 1.0, or 2.0 M NaCl, 10 mM Tris–HCl (pH7.7), 1 mM
EDTA, and 10 mM mercaptoethanol]. The eluted fractions precipitated
with TCA were subjected to SDS-PAGE.
2.5. Cloning of Pmn56 cDNA
fluoride (PVDF) membrane, and bands of 56 kDa were sequenced from
the N-terminus using a protein sequencing system (model procise,
494/492cLC); the following 22-amino acid sequence was obtained:
TTETEVAFPVEAPNAYPTRTSD. To obtain internal peptide sequences, the
SDS, 1 mM EDTA, and 100 mM Tris–HCl (pH9.0) at 37 °C overnight. The
digested peptides were separated by reverse-phase high-performance
liquid chromatography (HPLC) with a linear gradient of acetonitrile
from 0% to 50% in 0.085% or 0.075% TFA. Two peptides were
sequenced using a protein sequencer, and the following 12 and 25
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
amino acid sequences were obtained: EIRAFTTTSQPK and
Using cDNA of P. polycephalum, reverse transcription (RT)-PCR was
performed with four degenerate primers based on the amino acid
sequences obtained; first PCR: 5′-GARACIGARGTIGCTTYCC-3′
PCR: (Pmn56F1) and 5′-GARACICARGCICCIGU-3′ (Pmn56R2). The
resulting PCR fragment was cloned and sequenced. For cloning of the
full-length Pmn56, 5′- and 3′-rapid amplification of cDNA ends (RACE)
PCR analyses were carried out (GeneRacer Kit, Invitrogen, Carlsbad, CA,
USA). For 5′-RACE, we used 5′-GCCAATGCCCAAATCCTTGCAG-3′
(Pmn56-5race1) and GeneRacer 5′-primer in the first PCR, and 5′-
GCTCCAGGGCCAAATGCACGATT-3′ (Pmn56-5race2) and GeneRacer 5′-
nested primer in the second PCR. For 3′-RACE, we used 5′-GCCTTTA-
GAGACTGGTGTTG-3′ (Pmn56-3race1) and GeneRacer 3′-primer in the
first PCR, and 5′-ATTTGGCCTCCAGATTGCCA-3′ (Pmn56-3race2) and
GeneRacer 3′-nested primer in the second PCR. The Genbank accession
number of Glom2/Pmn56 is AB537161.
2.6. Phylogenetic analysis
Phylogenetic analysis (neighbor joining) wasdone using thedefault
for sequences for phylogenetic analysis are XP_642651 (Dictyostelium
discoideum), XP_002111538 (Trichoplax adhaerens), NP_596277
(Schizosaccharomyces pombe), XP_572928 (Cryptococcus neoformans),
XP_755290 (Aspergillus fumigatus), CAA48502 (Saccharomyces
cerevisiae), XP_002416920 (Candida dubliniensis), XP_001623521
(Nematostella vectensis), XP_002111538 (Trichoplax adhaerens), and
CAJ75143 (Kuenenia stuttgartiensis).
2.7. Antibody preparation
in Escherichia coli BL21 (DE3) CodonPlus (Novagen, Darmstadt,
Germany) as a polyhistidine (His)-tagged protein using vector pET-
21a (Novagen). This protein was purified using Ni-chelating affinity
column chromatography (HisTrap FF column; GE Healthcare) and then
used to raise polyclonal rabbit antisera. An anti-Pmn56c antibody was
purified from serum by Protein G affinity chromatography (MABTrap
Kit; GE Healthcare).
2.8. Immunoblot and immunofluorescence
(cell, mitochondria, mitochondrial sup, mt-nucleoid) was electropho-
resed and blotted onto PVDF membranes (Immobilon-P; Millipore,
Billerica, MA,USA). The primaryantibodywas anti-Pmn56C ata dilution
of 1:20,000 (v/v) in gelatin blocking buffer [1% gelatin in Tris-buffered
saline (TBS) containing 0.05% Tween-20], and the secondary antibody
was peroxidase-conjugated goatanti-rabbitIgG(KPL,Baltimore,USA)at
a dilution of 1:20,000 (v/v) in gelatin blocking buffer. For immunoblot-
ting analysis of the MO sample, 12 μg of each total cell extract was
electrophoresed and blotted onto PVDF membranes. The primary
antibodies were anti-Glom andanti-ANT, diluted 1:5000 (v/v) in gelatin
blockingbuffer, and thesecondary antibody was peroxidase-conjugated
goat anti-rabbit IgG at a dilution of 1:20,000 (v/v) in gelatin blocking
For immunofluorescence staining, amoebae cells were fixed onto a
coverslip with 3.7% formaldehyde in 25 mM potassium phosphate
buffer (KPB) for 20 min, and then permeabilized with 0.1% Triton
X-100 in distilled water for 12 h at 4 °C. The amoebae cells were
treated for 1 h with the anti-Pmn56C antibody at a dilution of 1:500
(v/v) in bovine serum albumin (BSA) blocking buffer (5% BSA in KPB).
The secondary antibody was Alexa Fluor 488-conjugated anti-rabbit
in BSA blocking buffer.
2.9. Purification of recombinant proteins
Full-length mature Pmn56 (amino acids 44–458) was expressed as
a His-tagged protein using vector pET-28 (Novagen) in E. coli BL21
(DE3) CodonPlus. This expressed protein was purified using a
Ni-chelating affinity chromatography column, and then refolded
with a 6–0 M urea gradient and dialyzed against buffer A [50 mM
Tris–HCl (pH7.5), 150 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol
(DTT)]. The recombinant Glom was expressed and purified as
described by Sasaki et al. (2003).
2.10. DNA mobility shift assay
DNA-binding activity using electrophoretic mobility shift assays with
XbaI-digested mtDNA of P. polycephalum. Two microliters of purified
Pmn56m or BSA in buffer A wasadded to 8 μl of reaction buffer[50 mM
Tris–HCl (pH7.0)] containing 200 ng of DNA and incubated at 26 °C for
1 h.Thereaction mixtureswere applied to 1%agarose gels inTAE buffer
[40 mM Tris-acetate (pH8.0) and 2 mM EDTA]. After electrophoresis,
gels were stained with ethidium bromide.
2.11. RNA mobility shift assay
Purified full-length mature of Pmn56 (Pmn56m) was analyzed for
its RNA-binding activity using electrophoretic mobility shift assays
with total mitochondrial RNA of P. polycephalum. Total mitochondrial
RNA was extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden,
Germany) from isolated mitochondria. Each 2 μl of purified Pmn56m,
purified Glom, Ribonuclease (50 ng), or BSA (2 μg) in buffer A was
added to 8 μl of reaction buffer [50 mM Tris–HCl (pH7.0)] containing
200 ng of RNA and incubated at 26 °C for 1 h. The reaction mixtures
were applied to 1% agarose gels in TAE buffer. After electrophoresis,
gels were stained with ethidium bromide.
2.12. DNA condensation assay
Two microliters distilled water containing 450 ng mtDNA (not
digested)and1.8 μleach of purified Pmn56m,Glom,orBSA in buffer A
were added to 6.6 μl of reaction buffer [50 mM Tris–HCl (pH7.5)] and
then incubated at 26 °C for 1 h. The reaction mixtures were examined
by 4′,6-diamidino-2-phenyl-indole (DAPI) fluorescence microscopy.
2.13. Microinjection of MOs into the plasmodium
MOs conjugated with fluorescein 5-isothiocyanate (FITC) at the
3′-end were obtained from Gene Tools, LLC (Philomath, OR, USA). The
sequences of MOs against Glom or Glom2 mRNA located near the
ACA-CTT-G (Glom) and ATG-TAC-ACT-AGG-GTG-GCC-AGG-GTT-T
(Glom2). As negative controls, inverted oligomers (inv.) and without
injection (W/O) were used. After dissolution in distilled water to a
concentration of 1 mM, each 1.5 μl of MO was mixed or diluted to 3.0 μl
and injected with a glass needle into macroplasmodia on MEA plates.
glassneedle, withanouterdiameteratthetip of 8–10 μm,wasmadeby
pulling glass capillaries (GD-1; Narishige, Tokyo, Japan) with PC-10
micropipette pullers (Narishige) and then trimming to a needlepoint
with the side of a glass slide.
Macroplasmodia were prepared as described above. Every 3 days
after the first injection, a 4-cm2section of the plasmodium, together
with the agar, was cut out and placed upside down on a fresh agar
plate as an inoculum. At 5.5 days after the first injection, the
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
plasmodium was re-injected with MOs. Then 12 days after the first
injection, a part of the plasmodium was scraped from the agar plate
and analyzed. In a single experiment, each MO was injected to one
multinucleate cell. We repeated this experiment 2–5 times indepen-
dently and respectively.
2.14. Microscopic observations
To observe the mitochondria and mt-nucleoid, cells were fixed
with 0.2% glutaraldehyde in 12 mM KPB (pH7.0) containing 0.1%
Triton X-100 and 0.25% Tween 20, and DNA was then stained with
DAPI. Samples were observed with a BX51 microscope (Olympus,
Tokyo, Japan). Image processing and analysis were performed with
the following software: Adobe Photoshop 7.0.1 (Adobe systems, CA,
USA) and ImageJ (http://rsb.info.nih.gov/ij/).
2.15. Real-time PCR
We applied real-time PCR to detect the mtDNA copy number and
mitochondrial RNA accumulation levels. The real-time PCR used the
StepOnePlus Real-Time PCR Detection System (Applied Biosystems,
time PCR (20 μl total volume) contained 9 μl of template genomic DNA
(50 ng)orcDNAdilutedindistilledwater,10 μlof2×PowerSYBRGreen
PCR Master Mix (Applied Biosystems), and 1 μl of each forward and
reverseprimer.Samples were denatured byheatingat95 °Cfor 10 min,
followed by 42 cycles of amplification and quantification (95 °C for 15 s
and 60 °C for 1 min). Each measurement was repeated at least three
times and normalized in each experiment against the control.
FormtDNAcopynumber analysis,totalcellular DNAfromcells was
extracted using the Plant DNeasy Mini Kit (Qiagen) according to the
manufacturer's instructions. As previously described, we compared
the relative amounts of mtDNA and nuclear DNA copy number.
MtDNA amplicons were generated from the mitochondrial rRNA
segment using mtDNA primers (rRNA: 5′-CACACTAGCTA-
AGGACCGCAATAA-3′ and 5′-CGTGAGCTGTAACGCTTTCTTTAA-3′,
fragment length 110 bp). The nuclear amplicon was generated by
amplifying the Glom segment, which occurs in a single copy, using
nuclear DNA primers (Glom: 5′-TGTCAGCCTTTTCCATCTTCGT-3′ and
5′-TGCTTGGACAATTGCTTCATG-3′, fragment length 100 bp). The
threshold cycle number (Ct) values for Glom and mtDNA were
determined for each individual quantitative PCR run. For mitochon-
drial RNA accumulation analysis, total RNA was extracted with the
RNeasy Plant Mini Kit (Qiagen) with the RNase-Free DNase Set
(Qiagen). Reverse transcription was performed with random primers
(6mer) and SuperScript III Reverse Transcriptase (Invitrogen). The
primers for GAPDH as an internal standard were 5′-TGGCTAAGAT-
CATCCACGAGAAG-3′ and 5′-TGCCAGAGGGACCATCTACAG-3′ (frag-
ment length 102 bp). In P. polycephalum, nearly all mitochondrial
transcripts require a large range of RNA editing activity (Byrne et al.,
2007). The primers for three genes on the mitochondrial genome
were designed with reference to Gott et al. (1993) and Takano et al.
(2001). The primers for cox1 were 5′-TGGGTTTTATAGTGTGGGCTCAT-
3′ and 5′-CGTTGGCACAGCAATATCATAGTAG-3′ (fragment length
101 bp). The primers for nad6 were 5′-TACATTTACGCTCATACACGC-
3′ and 5′-GGTATCGCTAAATAAAAAAGTC-3′ (fragment length 104 bp),
and for nad7 were 5′-TCATAAAACGGTCATAACAATC-3′ and 5′-
CTGGTGTTATGGTTCGAGGTAG-3′ (fragment length 129 bp).
2.16. Northern blot analysis
Total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen)
with the RNase-Free DNase Set (Qiagen). For northern analysis, RNA
samples (2, 6, and 3.6 μg for cox1, nad6, and nad7 respectively) were
separated on 1.4% agarose gels in 10 mM sodium phosphate (pH7.0)
and transferred to Hybond-N+ membranes (GE Healthcare) by
capillary elution with 20× SSC buffer (3 M NaCl and 0.3 M sodium
citrate, pH 7.0). Membranes were hybridized with gene-specific
probes and detected using the AlkPhos Direct Labelling and Detection
System with CDP-Star (GE Healthcare). The primers for cox1 probe
were 5′-ATACCTTCGCAGATCGTTGG-3′ and 5′-CCTGCTAAGACAGG-
CAGTGA-3′. The primers for nad6 probe were 5′-TGCTTTTAAGCTTTA-
TATTTTTCTCC-3′ and 5′-ATAGCAGTTGTTTTATTATATCCGACA-3′. The
primers for nad7 probe were 5′-GGCTTTTGAAGAGCGAGAAA-3′ and
5′-TCCTTTTGGAGCTTCGACAC-3′. Hybridization signals were visual-
ized on a luminescent image analyzer (LAS4000mini, GE Healthcare).
The regions hybridized with DNA probes in mRNA were 679 base
(cox1), 487 base (nad6), and 610 base (nad7) respectively.
3.1. Identification of 56-kDa protein from isolated mt-nucleoids as a
candidate mtDNA-packaging protein
The mt-nucleoid of P. polycephalum consists of more than 70
proteins (Sasaki et al., 2003). To search for mtDNA-binding proteins
required for the packaging of mtDNA, we compared the results of
three parallel experiments, namely, disassembly of mt-nucleoids by
NaCl treatment, disassembly of mt-nucleoids by DNase I treatment,
and affinity chromatography of mt-nucleoid proteins using a DNA-
cellulose column. Candidate mtDNA packaging proteins were
expected from all three experiments.
First, we investigated which proteins were released from the
mtDNA along with Glom on disassembly of the isolated mt-nucleoid
structure. Mt-nucleoids were treated with NaCl of increasing ionic
strength to induce their disassembly. These were centrifuged and the
supernatant and pellet fractions analyzed by SDS-PAGE (Fig. 1A). The
condensed nucleoid structure began to disassemble at 0.2 M NaCl, as
previously reported (Sasaki et al., 2003). At 0.2 M NaCl, proteins of
38-, 56-, 60-, 68-, 70-, 90-, 130-, and 170-kDa began to be released
into the supernatant fraction and were almost completely released at
0.5 M NaCl, together with Glom (Fig. 1A and Table 1). These proteins
are possibly involved in organization of the mt-nucleoid structure.
Second, we investigated which proteins were released from mt-
nucleoids with Glom on disassembly of its structure with DNase I,
which induces partial digestion of mtDNA. When mt-nucleoids were
treated with 0.01 mg/ml DNase I at 25 °C for 2 h, DAPI staining
showed that the rod-shaped structure was dispersed, as seen with the
NaCl treatment (Supplementary information, Fig. S1). The extract was
centrifuged and the supernatant and pellet fractions analyzed by SDS-
PAGE (Fig. 1B). DNA-binding proteins are predicted not to be released
to the supernatant without DNase I treatment (−), but will be
released after DNase treatment (+). As well as Glom, proteins of 20-,
31-, 34-, 38-, 40-, 40.5-, 52-, 56-, and 90-kDa were released to the
supernatant after the DNase I treatment (Fig. 1B and Table 1). These
proteins are the second candidates involved in the organization of the
mt-nucleoidstructure. In these proteins,34-kDaprotein (Pmn34)was
identified as a protein which has an exonuclease motif and localizes in
the mt-nucleoid periphery (Itoh et al., 2009).
Third, a DNA-cellulose chromatography was used to identify
candidate proteins that bind to DNA. Mt-nucleoid proteins were
prepared usinga 1.0 MNaCl treatmentand wereadsorbedontoa native
DNA-cellulose column. Proteins bound to the column were eluted with
56-, 70-, and 95-kDa proteins were mainly eluted with 0.3 M NaCl and
that a 56-kDa protein was mainly eluted with 0.4 M NaCl, together with
tobindmore stronglyto DNAthan the othercandidate proteinsbecause
they remained bound to the DNA column even at higher NaCl
concentrations. As summarized in Table 1, a 56-kDa protein was found
of the most abundant proteins of the mt-nucleoid (Fig. 1A–C). Thus, in
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
further analysis, we focused on the 56-kDa protein, hereafter called
Pmn56 (P. polycephalum mitochondrial nucleoid protein 56).
3.2. Primary structure of Pmn56 has an N-terminal Mgm101 half and a
unique C-terminal half
Partial amino acid sequences of the N-terminal and internal
structure of Pmn56 were determined using the Edman degradation
method. After a partial coding sequence of cDNA was determined by
degenerate PCR analysis, the sequence of the full-length cDNA was
determined by 3′- and 5′-RACE analysis. The primary sequence of
Pmn56 suggests that it encoded a positively charged protein of 458
amino acids. The mature amino terminus of the protein is located at 44
acids are assumed to be a mitochondrial transit peptide resulting in a
mature protein of 415 aminoacids. This isconsistent with Pmn56 being
encoded by thenuclear genome, asthe mitochondrial genome does not
contain the Pmn56 gene (Takano et al., 2001). The molecular mass of
mature Pmn56 was estimated at 45,860. The difference from the
apparent molecular mass, 56-kDa, might be due to a positive charge in
mature Pmn56 (isoelectric point: 8.15).
BLAST search analysis revealed that Pmn56 has an Mgm101 domain
in the N-terminal half (amino acids 70–239), which has been identified
as a mitochondrial nucleoid protein involved in mtDNA maintenance
protozoa, fungi, and animals (Fig. 2A–C). The C-terminal half of Pmn56
(amino acids 240–458) showed no sequence homology with other
the lysine-rich, N-terminal half of Glom (Sasaki et al., 2003). Both the
C-terminal half and N-terminal half of Pmn56 are rich in basic amino
acids. No specific DNA-binding motif was found.
3.3. Pmn56 is localized to the entire mt-nucleoid
We raised a polyclonal antibody against the C-terminal half of
Pmn56 expressed in E. coli. To examine the intracellular localization
of Pmn56, we performed Western blotting using various fractiona-
tions of the cell: total cell extract, mitochondria, mitochondrial-sup
(matrix and membrane), and mt-nucleoids. Pmn56 was specifically
recognized by the antibody and was concentrated in the mt-nucleoid
fraction (Fig. 3A), but was scarcely detected in the mitochondrial-
sup fraction. We next performed immunostaining to visualize
Pmn56 in the mt-nucleoid of amoeba cells (Fig. 3B). We found that
Pmn56 was localized to all mt-nucleoids but not to the nucleus.
Moreover, Pmn56 was localized uniformly to the entire mt-nucleoid.
By SDS-PAGE and Western blotting analysis, we investigated the
physiological concentration of Pmn56 and Glom in the mt-nucleoid.
Both results showed that 5 μg of mt-nucleoid contained approxi-
mately 0.25 μg of Pmn56m and 1 μg of Glom (data not shown).
Because it has been reported that 5 μg of mt-nucleoid contains
450 ng of mtDNA in vivo (Sasaki et al., 2003), it was calculated that
the mt-nucleoid contained about 0.56 μg of Pmn56m per μg of
3.4. Pmn56 has the ability for mtDNA and mitochondrial RNA binding
To determine whether Pmn56 directly binds to mtDNA and
mitochondrial RNA, we performed DNA and RNA mobility shift assays
using a recombinant protein of Pmn56m, a mature form lacking the
transit peptide (Fig. 4A). The purity of the recombinant proteins was
checked by SDS-PAGE (Fig. 4B). Isolated mtDNA of P. polycephalum
was digested by XbaI and mixed with Pmn56m (Fig. 4C). Electropho-
resis of the protein–DNA mixture showed that mtDNA was trapped in
wells as the concentration of Pmn56m increased. At 4 μM of Pmn56m,
all fragments of 200 ng mtDNA were completely trapped in these
wells. The possibility exists that Pmn56m-bound DNA could not enter
the gel. This result suggests that Pmn56m was directly bound to
mtDNA without sequence specificity and induced DNA condensation.
No aggregation of Pmn56m was observed at 4 μM without DNA. This
characteristic of Pmn56 is different from that of Glom, which causes
shifts in mtDNA mobility but not trapping of mtDNA in wells of the gel
(Sasaki et al., 2003). Isolated mitochondrial RNA of P. polycephalum
was mixed with Pmn56m or Glom, and analyzed by agarose gel
electrophoresis (Fig. 4D). The mitochondrial RNA was not shifted
when it was incubated with a high amount of BSA, which was used as
a negative control. All mitochondrial RNA was clearly shifted at high
amounts of Pmn56m and Glom. The level of RNA fragment shift with
2 μM of Pmn56m was equivalent to that with 1.5 μM of Glom,
indicating that Pmn56m and Glom could bind to all mitochondrial
RNA, and the ability of RNA binding of Glom is much higher than that
0.1 0.2 0.3 0.4 1.0 2.0 MN
0 0.1 0.2 0.5 1.00 0.1 0.2 0.5 1.0
Fig. 1. Analysis of mtDNA-binding proteins in the isolated mt-nucleoid. (A) SDS-PAGE analysis of mt-nucleoid proteins released from mtDNA by treatment with various
concentrations of NaCl (0, 0.1, 0.2, 0.5, and 1.0 M). (B) SDS-PAGE analysis of mt-nucleoid proteins released from mtDNA by treatment with (+) or without (−) DNase I (0.01 mg/ml).
The isolated mt-nucleoid after treatment with NaCl or DNase I was centrifuged at 18,500×g for 20 min at 4 °C. Proteins from pelletable and supernatant fractions of the mt-nucleoid
were subjected to SDS-PAGE. (C) SDS-PAGE analysis of mt-nucleoid proteins bound to the DNA-cellulose column. The proteins of the isolated mt-nucleoid were applied to a DNA-
cellulose column, and bound proteins were eluted with NaCl (0.1, 0.2, 0.3, 0.4, 1.0, and 2.0 M). Proteins from the mt-nucleoid (MN, lane 1) and eluted fractions (lanes 2–7) were
analyzed by SDS-PAGE. The band just under 34-kDa (*) was a degradation product of Glom.
The candidates for mtDNA-binding proteins in the isolated mt-nucleoid.
Types of experimentsIdentified protein (kDa)
NaCl treatment of mt-nucleoids
DNase treatment of mt-nucleoids
DNA cellulose column chromatography
(0.3 M NaCl)
(0.4 M NaCl)
Glom, 38, 56, 60, 68, 70, 90, 130, 170
Glom, 20, 31, 34, 38, 40, 40.5, 52, 56, 90
Glom, 34, 38, 56, 70, 95
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
3.5. Pmn56 is an mtDNA-packaging protein with an ability for mtDNA
To evaluate the DNA condensation ability of Pmn56m using
isolated mt-nucleoids, we performed the in vitro reassembly assay as
described by Sasaki et al. (2003). The isolated mt-nucleoids were
disassembled by treatment with 0.3 M NaCl. This disassembly process
was reversible: subsequent dialysis of the high-salt treated sample
induced reassembly of the mt-nucleoids intoa condensed,rod-shaped
form (Fig. 4Ec). Thus, the factors required for DNA condensation can
re-associate with mtDNA to reassemble mt-nucleoid structure upon
dilution from high-salt solution. Conversely, when the high-salt
sample was depleted by centrifugation and the supernatant fraction
removed before dialysis, reassembly was severely inhibited (Fig. 4Ed).
Consistent with a previous work, the addition of purified Glom into
the 0.3 M NaCl supernatant-depleted fraction at physiological
protein/DNA ratio induced the reassembly of the mt-nucleoids
(Fig. 4Ee, Sasaki et al., 2003). The addition of purified Pmn56m at
physiological protein/DNA ratio also induced the reassembly of the
mt-nucleoids like Glom (Fig. 4Ef). As the concentration of Pmn56m
increased, the condensed level also increased (Fig. 4Ef–Eh). These
results demonstrated that Pmn56 is able to pack the mt-nucleoid into
a highly condensed state like Glom in vitro. Therefore, we named
Pmn56 as Glom2 (a protein inducing agglomeration of mitochondrial
Then we analyzed the DNA condensation ability of Glom2 using
isolated mtDNA. Isolated mtDNAwas incubated with various amounts
of purified Glom2 and/or Glom, and observed after DAPI staining
(Fig. 4F). When 450 ng of mtDNA was incubated with 0.25 μg of
Glom2 at a physiological concentration (0.5 μM), Glom2 induced
slight DNA condensation after DAPI staining (Fig. 4Fa). As the
concentration of Glom2 increased, the condensed level also increased
(Fig. 4Fa–Fc). Although we did not find DNA condensation when
450 ng of mtDNA was added to 0.2 μg of Glom (0.5 μM) (Fig. 4Fd),
DNA condensation was promoted by more than 1 μg of Glom at
physiological concentration (2.5 μM) (Fig. 4Fe and Ff). This means
that Glom2 could condense DNA at lower concentration (0.5 μM)
than Glom, implying that Glom2 has a higher condensation activity
than Glom. Note that mixing Glom2 and Glom together at
physiological concentrations (0.5 μM and 2.5 μM, respectively)
Schizosaccharomyces 100 SFFGLSSQPFSKEICDLLTAPLEVDDIEIKPDGILYLPEIKYRRILNKAFGPGGWGLAPR 159
Cryptococcus 151 SFSGLSEKPFPKEAADELLKPLTPVDVEIKPDGLLYLPEIKYRRTLNAAFGPGGWGLAPR 210
Aspergillus 136 SFHGLSAAPFPKEVADILLAEVDPEEVEIKPDGILYLPEIKYRRILNRAFGPGGWGLVPR 195
104 SWYGLGMKPFEAKVQKDLIEPLDPKDIEIKPDGLIYLPEIKYRRILNKAFGAGGWGLVPR 163
103 SFHGLGSQPFSREIAEILLAPVSEEDIEIKPDGLLYLPEIKYRRVLNRAFGPGGWGLAPR 162
71 LYSGAGSQAFSSDAVKALMAPVNEQDVEIKPDGLIYLPEIKYRRILNVAFGPGGWALVPR 130
59 DFVGIAASPFPKESADILMAPVKAEDVEVKPDGLLYLPEIRYRRILNQAFGPGGWALMPR 118
485 204 7044
70 IYDGIAQEPFGTEIASVLMAPIDPMDIEIKPDGLIYLPEIRYRRILNRAFGPGAWALLPI 129
76 KYQGISKEPFSKEIVDTLLADLNPDDIEIKPDGLIYLPEIKYRRILNQAFGPGGWALKPF 135
Schizosaccharomyces 160 GNTN------VTSKSVSREYALVCHGRLVSVARGEQTYFDPEG---IATASEGCKSNALM 210
211 GETD------VGPRIVSREWGLVCLGRLVSVARGEQEYFDPSG---IATATEACKSNALM 261
Aspergillus 196 SESI------VTPRTVTREYALVCNGRLVSVARGEQDYFTPDG---IPTATEGCRSNALV 246
164 SQTI------VTSKLVTREYGLICHGQLISVARGEQDYFNEAG---IPTATEGCKSNALM 214
163 TESL------VTSGQISREYGLICHGRLVSIARGEQDYFGGEEK--LTTALEGCKSNALM 214
131 GDSLQFQSEKDNSQLIVREYALFCEGRFASQATGEHTFYSNNGNMVYGKAIESAKSNALM 190
119 GQPTMINNEGELSQLVIREFALYVGGRFVAQSVGEHLHYSKNEL-STGKALESAKSNALM 177
130 SPPI------VERTNLIRTYALYCHGRFVSEATGEQPFFEGVA--STATAAEAAKSNALV 181
136 GPPV------VEGKTLIRPYALYCLGRYVAESIGEQQYVPNSF-ISFATATESAKSNALV 188
Schizosaccharomyces 211 RCCKDLGVASELWDPRYIRVFKRENCVEVFVENV-LTKKRRKLW-RRKEDKFS--YPYKE 266
262 RCCKDLGIASELWDPTFIRDFKKQHCVEVFVEHA-VKKNKKKLWRKKNSDKFE--YPWKE 318
Aspergillus247 RCCKDLGIASELWDPRWIRKFKAQYTREVFVEHV-VNKKRTKIW-VRKDDQVS--YPWKE 302
215 RCCKDLGVGSELWDPVFIKKFKVDHCTEKFVEHV-TTKRKKKIW-LRKDRQVE--YPYK- 269
215 RCCKDLGIASELWDPSFIRRWKKKYCEEIFVEHV-NTKKKKKIWKLKSIKTVD--YPYRM 271
191 RCCKDLGVASELWDPQDYGANPSHYTLRSFYTPDIDKELRIFEGQRGCPRKVFFSHRAIS 250
178 RCCKDLGVASELWDPTFVYNFKKNHCVEVWCENARNSKEKRKLWRLKRQGNDVLPYPWKP 237
182 RCCKDLGIGSELWDPQFIFDWKKKYSVEVQCSNMKNTNDRRRLWRRKDR--PPFEWPWKE 239
189 RCCKDLGIGSSLWDPIFIRQWKSEYATERWCENSK-TKERRLFWFLSNRSENQLPYPWKE 247
Fig. 2. (A) Alignment of amino acid sequences of the N-terminal region of Pmn56 and Mgm101 homologs from five major species of fungi including yeast, Dictyostelium discoideum,
and two animal sequences. Identical residues are indicated by black shading, and residues in more than 50% are indicated by gray shading. (B) Pmn56 has an additional region in the
C-terminal including three polyprolines compared to yeast Mgm101s. Other organisms, except for yeast, tend to have similar additional C-terminal regions. TP, transit peptide.
(C) Molecular phylogeny of Pmn56 and Mgm101s of various organisms. Shown is a neighbor-joining tree based on the Mgm101 domain. Branch length reflects the number of amino
acid changes. Bootstrap values (%) for 100 replications are indicated on each node.
Anti Pmn56 DAPI
Fig. 3. Intercellular localization of Pmn56 to the mt-nucleoid. (A) Immunoblot analysis
of cell fractionation using the anti-Pmn56 antibody. After treatment of isolated
mitochondria with NP-40 and centrifugation, the mt-nucleoid fraction was obtained in
the pelletable fraction, and the mitochondrial membrane and matrix fraction were in
the supernatant fractions. One microgram of proteins of the whole cell (lane 1), isolated
mitochondria (Mt, lane 2), the mitochondrial supernatant fraction after treatment with
NP-40 (Mt-sup, lane 3), and isolated mt-nucleoids (MN, lane 4) were separated by SDS-
PAGE and analyzed by immunoblotting using anti-Pmn56 antibody. (B) Immunoflu-
orescence micrograph showing the localization of Pmn56 to mt-nucleoids. The amoeba
cell was stained with DAPI (left). The same cell was also stained with anti-Pmn56
antibody and Alexa-conjugated second antibody (right). Scale bar, 5 μm.
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
considerably enhanced the condensation activity (Fig. 4Fg). This was
not due to an increase in the amount of proteins (3 μM in total)
because incubation with 3 μM of Glom caused less condensation,
which suggests that Glom2 and Glom work synergistically for
condensation of mtDNA.
3.6. Development of a down-regulation method for targeted genes using
To investigate the function of Glom2 in vivo, we developed a
down-regulation technique in P. polycephalum. Since plasmodium is a
single multinucleate cell, materials injected into a part of it can spread
to the entire plasmodium. We first tried an RNAi method by injecting
siRNA, as described previously, in P. polycephalum (Haindl and Holler,
2005; Pinchai et al., 2006); however, expression of the targeted genes
did not sufficiently decrease in our system. Therefore, we used MOs,
which are nucleic acid analogs resistant to nucleases that are able to
sustainably inhibit the expression of targeted genes by blocking the
access of other molecules for translation and splicing in many
organisms (Karkare and Bhatnagar, 2006). We designed MOs against
the translation initiation site of mRNA so that translation of targeted
genes was inhibited. When FITC-labeled MOs were injected into one
of the main veins of the plasmodium, the fluorescence of MOs spread
to the periphery instantaneously and then gradually spread through-
out the entire cell within 2 h (Fig. 5A). We injected 3 μl of MO into
each cell, which is less than 1% of the total cell volume.
We first tested a Glom2 MO to specifically down-regulate the
Glom2 gene. The expression of Glom2 began to decline 1 day after
injection, began to recover 7 days after injection, and fully recovered
to the original level 11 days after injection (Supplementary informa-
tion, Fig. S2). Note that such a sequential analysis in a single cell was
possible in P. polycephalum because we could collect part of a cell
without killing it. For more continuous and severe down-regulation,
MO was re-injected to the plasmodium 5.5 days after the first
injection (Fig. 5B). At 12 days after the first injection, the expression
of Glom2 was considerably reduced compared to negative controls,
i.e., W/O or inv. cells (Fig. 5C). Expression of neither Glom nor ANT
(mitochondrial outer membrane protein; Wada et al., 2007) changed
after the injection of Glom2 MO. We repeated the experiments at least
twice for each MO, and similar down-regulation was seen in all
Specific down-regulation of Glom wasalso observedafter injection
of Glom MO. Moreover, when we simultaneously injected MOs for
both Glom and Glom2, their proteins were reduced at the same time
(Fig. 5C). We found defects in proliferation of injected cells (Fig. 5D).
The injection of Glom2 MO did not cause an apparent defect in the
growth speed of cells, but we detected a significant delay in the cell
cycle when measured from days 12 to 15 after the first injection. The
time required for one nuclear division cycle from one M phase to the
1 44 70
Fig. 4. In vitro functional analyses of Pmn56 for nucleotides. (A) Constructs of recombinant Pmn56. Pmn56m is a mature form of Pmn56 that had the mitochondrial targeting peptide
removed. (B) Coomassie-stained gel after SDS-PAGE of the purified Pmn56m (1 μg). (C) DNA mobility shift assay of Pmn56m using XbaI-digested mtDNA. Each digested mtDNA
(200 ng) was incubated without (−) or with various concentrations (4.0, 2.0, 1.0, and 0.5 μM) of Pmn56m and BSA as described in Materials and methods. (D) RNA mobility shift
assay of Pmn56m and Glom using mitochondrial RNA. Each mitochondrial RNA (200 ng) was incubated without (−) or with various concentrations (8.0, 4.0, 2.0, 1.0, and 0.5 μM) of
Pmn56m and Glom, RNase, and BSA as described in Materials and methods. (E) Reassembly analysis of mt-nucleoids with Pmn56m and Glom. (a, b) DAPI-fluorescence micrographs
showing the isolated mt-nucleoids (a) and the isolated mt-nucleoids treated with 0.3 M NaCl (b). (c–h) DAPI-fluorescence micrographs showing the reassembly of mt-nucleoids by
dialysis. The isolated mt-nucleoids (5 μg) were disassembled at 0.3 M NaCl and then 0.3 M NaCl supernatant-depleted fractions were prepared as described by Sasaki et al. (2003).
0.3 M NaCl disassembled mt-nucleoids fraction (c), 0.3 M-NaCl supernatant-depleted fraction (d), and 0.3 M NaCl supernatant-depleted fraction with 1 μg of Glom (e), 0.25 (f), 1 (g),
or 2 μg (h) of Pmn56, were dialyzed against NE1-S buffer. Scale bar, 2 μm. (F) DNA condensation ability of Pmn56 and Glom. Each mtDNA (450 ng) was incubated with Pmn56m (a–
c) or Glom (d–f) at protein concentrations of 0.5 μM (a, d), 2.5 μM (b, e), or 5.0 μM (c, f). Incubation with Pmn56 and Glom at concentrations of 0.5 μM and 2.5 μM, respectively,
induced high condensation of mtDNA (g). BSA (h) and free protein (i) were used as controls. Resultant DNA was examined by DAPI fluorescence microscopy. Scale bar, 5 μm.
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
next was 17 h in injected cells but 13 h in cells without injection.
Moreover, the growth speed was severely decreased by down-
regulation of Glom or both Glom and Glom2. The apparent difference
in growth speed appeared from 4 days after injection, and the nuclear
division cycle was prolonged to more than 20 h. In most cases, down-
regulation of both Glom and Glom2 appeared to cause a severe defect
in the growth speed of cells. We repeated experiments more than five
times for each MO, and a similar defect in growth speed was always
observed, suggesting a high success rate and reproducibility of our
system. These results for MO injection together suggest that Glom and
Glom2 were both required for normal cell proliferation.
3.7. Glom2 has different functions from Glom in the mt-nucleoid
We examined whether down-regulation of Glom and/or Glom2
caused changes in nucleoid structure (Fig. 6A and B). We could not
find any change in the mt-nucleoid at 12 days after injection of Glom2
MO. Down-regulation of Glom2 did not induce loss of mtDNA even
after more than 25 days after injection (approximately 50 cell
divisions) in P. polycephalum. However, in the case of injection of
Glom MO, smaller or thinner mt-nucleoids were found at 12 days
after injection that were not observed in cells without injection.
Furthermore, simultaneous down-regulation of both Glom and Glom2
led to the loss of the rod-shaped structure of mt-nucleoids; most
became smaller and completely round, with no difference in length
between the major and minor axes. Not only smaller but very large
mt-nucleoids emerged in this case. These results suggest that Glom,
but not Glom2, is essential for mt-nucleoid structure. Note that the
fluorescence intensity of DAPI staining did not change in these
mt-nucleoids, suggesting that mtDNA was still packed in a condensed
We investigated whether down-regulation of Glom and Glom2
caused changes in the total copy number mtDNA in the cell using real-
time PCR (Fig. 6C). Consistent with observations after DAPI staining
(Fig. 6A), the copy number of mtDNA did not change significantly in
cells injected with Glom2 MO (PN0.05, Student's t-test) but decreased
significantly in cells injected with Glom MO (33% against W/O;
Pb0.003, Student's t-test) or both Glom and Glom2 MOs (5.5% against
W/O; Pb0.001, Student's t-test). The difference between Glom MO
and both Glom and Glom2 MOs was also significant (Pb0.001,
Student's t-test). These results suggest again the primary role of Glom
and synergistic role of Glom2 in the maintenance of mtDNA.
We then investigated whether down-regulation of Glom and Glom2
caused changes in the transcript accumulation level of mtDNA by
genome, namely cox1, nad6, and nad7 (Fig. 6D). These three genes are
localized to different loci on the mitochondrial genome (Takano et al.,
Inv. Anti. Inv. Anti. Inv. Anti.
( 2, 5, 8 day )
( 0 day )
( 5.5 day )
FITC (MO) Light field
Fig. 5. Down-regulation method for targeted genes using morpholino antisense oligomers (MOs). (A) MOs labeled with FITC were injected into the plasmodium. The same
plasmodium immediately and 2 h after injection. Just after injection, FITC fluorescence appeared to spread along veins instantaneously. Two hours later, FITC fluorescence spread
throughout the cell. Scale bar, 1 cm. (B) The scheme of injection. MOs were re-injected to the plasmodium 5.5 days after the first injection, and the plasmodium was subcultured
every 3 days. (C) MOs (Glom MO, Glom2 MO, and Glom+Glom2 MOs) were injected or not injected (W/O) into the plasmodium. Twelve days after the first injection, cells were
sampled and analyzed. Amount of Glom2, Glom, and ANT protein (ANT: as a control of mitochondrial amount) in total cells (16 μg) was analyzed by Western blotting. Amount of
actin protein (as a control of cell amount) in total cells (12 μg) was analyzed by SDS-PAGE. The lower band detected by anti-Glom antibody was the degradation product of Glom.
(D) A 4-cm2area of the plasmodium was placed on a new plate 12 days after the first injection, and then 3-day cultured plates were observed.
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
of cox1 and nad7, but not nad6, decreased in cells injected with Glom2
MO against that in W/O cells. On the other hand, all transcript
accumulation of three genes decreased in cells injected with Glom MO
or both Glom and Glom2 MOs.
We next performed real-time RT-PCR to measure the transcript
accumulation level quantitatively. We found that the transcript levels
of cox1 and nad7 decreased to 41.8% and 41.1%, respectively, in Glom2
depressed cells against W/O cells (Fig. 6E). On the other hand, the
transcript level of cox6 was maintained. The expression level of cox1
and nad7 in cells injected with Glom2 MO tended to decrease to a
similar extent as with Glom MO, despite no significant decrease in the
copy number of mtDNA in the Glom2 depressed cells. We repeated
the experiments at least twice for each MO, and a similar change was
seen in all experiments. Thus, Glom2 is likely to play a different role
from that of Glom in the mt-nucleoid.
The mtDNA of many organisms is packed by the major HMG-type
mtDNA packaging proteins, Abf2, TFAM, and Glom (Diffley and
Stillman, 1992; Ghivizzani et al., 1994; Sasaki et al., 2003). In this
study, we identified a new Mgm101-type mtDNA packaging protein,
Glom2, which is involved in mt-nucleoid organization, genome
maintenance, and gene expression. Like major HMG-type mtDNA
packaging proteins, Glom2 is localized to the entire mt-nucleoid and
directly bound to mtDNA with no sequence specificity. Although the
in the mt-nucleoid. Quantitative analysis of Glom2 revealed that it
contained approximately 1/20 of the total mt-nucleoid proteins,
meaning that one molecule of Glom2 exists per 100 bp of mtDNA.
Because Glom binds to every 20 bp of mtDNA (Sasaki et al., 2003), the
molar ratio of Glom:Glom2 in the mt-nucleoid is 5:1.
We previously showed that Glom induced reassembly of mt-
nucleoids into a highly condensed state in vitro (Sasaki et al., 2003).
In these reassembled mt-nucleoids, poly-beaded-like structures
similar to non-treated mt-nucleoids were observed. In this study,
condensation in vitro (Fig. 4E, F). Although the DNA condensation
ability of Glom2 appeared higher than that of Glom, the condensed
DNA by Glom2 at a physiological ratio (Fig. 4Fa) was much less than
that by Glom (Fig. 4Fe). These results suggest that Glom is more
down-regulation of Glom, but not Glom2, induced morphological
changes in mt-nucleoids. However, a synergistic effect for DNA
Length of the minor axis (µm)
Length of the major axis (µm)
W/OGlom2 MO Glom MO
Glom + Glom2
mtDNA copy number
(% of W/O)
Fig. 6. Effect of down-regulation of Gloms on the mt-nucleoid morphology and the level of mtDNA and mtDNA transcripts. (A) Plasmodium injected or not injected with MOs were
stained with DAPI and observed by fluorescence and phase contrast microscopy. The lower panels were enlarged images of mitochondria. Scale bar, 5 μm. (B) Lengths of the major
and minor axes of mt-nucleoids were measured with ImageJ and plotted (n=200). (C) MtDNA copy number measured by quantitative real-time PCR. At 12 days after the first
injection, total genome was extracted from the plasmodium injected or not injected with MOs and subjected to real-time PCR. The copy number of mtDNA was estimated using
primers for the rRNA gene on the mitochondrial genome and was normalized using primers for the nuclear-encoded Glom gene. **Pb0.001, *Pb0.003 versus W/O control. Values are
mean±SE. (D) Northern blot analysis showing the level of mtDNA transcripts. At 12 days after the first injection, total RNA was extracted from the plasmodium injected or not
injected with MOs and subjected to Northern blot analysis. The blots were hybridized with a gene-specific DNA probe for cox1, nad6, and nad7. The ORF length of cox1, nad6, and
nad7 are 1785, 498, and 1206 base, respectively. Signals by cox1 or nad7 probe were detected as a similar size as their ORFs. Cox6 signal was detected larger than its ORF because it
was cotranscripted with adjacent genes. The lower panels are rRNAs stained with ethidium bromide as loading controls. (E) The level of mtDNA transcripts measured by real-time
RT-PCR. The transcript levels were estimated using primers for cox1, nad6, and nad7 mtDNA fragments individually and were normalized using primers for nuclear-encoded GAPDH
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
condensation was observed in vitro when the two proteins were
mixed (Fig.4Fg), anddown-regulationofboth Glomsinduceda more
severe morphological change of the mt-nucleoid (Fig. 6A). Although
it is not clear whether the effect in the double down-regulation is
direct or indirect, similar synergistic effect for DNA condensation
is also observed in histones in the nuclear chromatin. Nuclear DNA is
wrapped around a particle of core histone octamer, and then the
nucleosomal particles are folded and condensed into a higher-order
chromatin structure by linker histones (Allan et al., 1986; Thoma
et al., 1979; Wolffe, 1998). In this case, DNA-linker histone
interaction is important for facilitation of DNA condensation. It has
been revealed that the linker histone directly interacts with
nucleosomal DNA and linker DNA adjacent to the nucleosome core
to fold nucleosomal particles (Brown et al., 2006; Zlatanova et al.,
2008). Since Glom and Glom2 have DNA binding ability, DNA–Gloms
interactions may be important for mtDNA condensation. In addition,
it is possible that Glom–Glom2 interaction might also facilitate
mtDNA condensation. However, immunoprecipitation analysis could
not clarify the interaction between Glom and Glom2 because they
tended to bind to beads for analysis nonspecifically, whereas Far-
western blotting analysis showed no interaction between Glom and
Glom2 (data not shown). From these considerations, one possibility
is that Glom2 might act to assemble the fundamental Glom-DNA
complexes by its DNA binding ability and contributes to the
synergistic mtDNA condensation.
In wild type cell, the major axes' length of mt-nucleoids is
proportional to the copy number of mtDNA in mt-nucleoids, and the
minor axes' length of mt-nucleoids is maintained constant (Sasaki
et al., 1998; Fig. 6B). However, down-regulation of Glom reduced
the lengths of both major and minor axes, and simultaneous
down-regulation of both Glom and Glom2 induced a more severe
morphological change of the mt-nucleoid (Fig. 6A, B). It might be
possible to assume that some topological change in packaging and
coordination of mtDNA affects morphology of mt-nucleoids. Huge
mt-nucleoids also emerged in double down-regulated cells. These
results suggest that Glom and Glom2 are involved in the maintenance
of the architecture of whole mt-nucleoid.
In yeast cells lacking Abf2, the mt-nucleoid is diffuse and different
from that of the wild type (Newman et al., 1996). Our down-
regulation experiments with P. polycephalum suggested more redun-
dant mechanisms in the packaging of mtDNA. When we down-
regulated Glom, we could not detect a decrease in fluorescence
intensity after DAPI staining, even though the morphology of mt-
nucleoids apparently changed. No diffuse mt-nucleoid was observed
even after the down-regulation of both Gloms (Fig. 6A). These
observations indicate that mtDNA was still packed in a condensed
state. Low amounts of Glom and Glom2, which were hardly detectable
in our Western blotting analysis (Fig. 5C), may have been enough
for the packaging of mtDNA. However, we speculate that other
mt-nucleoid protein(s) might compensate for Glom and Glom2.
Miyakawaet al. (1995) reported in yeast that the packaging of mtDNA
by Abf2 was limited and that combination with other DNA-binding
proteins with molecular masses of 30-, 38-, 50-, 52-, and 67-kDa, was
important for DNA compaction in the mt-nucleoid. In our search for
DNA-binding proteins involved in the packaging of mtDNA, 38-, 70-,
and90-kDa proteins were alsogoodcandidatesalthoughtheyshowed
slightly lower affinity on the DNA-cellulose column than Glom and
Glom2 (Fig. 1C and Table 1). Detailed cytological analysis of the
nucleoidstructure, combinedwithdown-regulationof MO,will clarify
the possible existence of other DNA-packaging proteins.
The relationship in maintenance of mtDNA and HMG-type mtDNA
packaging proteins has been well characterized. In yeast, disruption of
Abf2 reduced mtDNA to 50% under respiration-dispensable conditions
or 0% under fermentable conditions (Diffley and Stillman, 1991;
Zelenaya-Troitskaya et al., 1998). In animals, homozygous disruption
of Tfam was lethal, while heterozygous disruption decreased mtDNA to
50% (Larsson et al., 1998; Matsushima et al., 2003). RNAi knockdown or
overexpression of TFAM showed a correlation between the amount of
mtDNA and TFAM (Goto et al., 2001; Matsushima et al., 2003; Ekstrand
et al., 2004; Kanki et al., 2004). These results indicate that the copy
number of mtDNA depends on the amount of Abf2 and TFAM. In
P. polycephalum, down-regulation of Glom reduced the copy number of
mtDNA to 33% (Fig. 6C), which is consistent with previous reports on
Abf2 and TFAM. Simultaneous down-regulation of both Gloms further
reduced mtDNA to 5.5% (Fig. 6C). Glom and Glom2 are suggested to
work synergistically in the maintenance of mtDNA.
In Glom depressed cells, transcript accumulation level of all three
genes decreased probably due to the decrease of mtDNA copy number
(Fig. 6C–E). However, although down-regulation of Glom2 only
did not cause apparent defects in the copy number of mtDNA and
the mt-nucleoid structure, the transcript level of cox1 and nad7 were
significantly impaired (Fig. 6D, E). Interestingly, the accumulation of
nad6 transcripts was not impaired by Glom2 down-regulation. Glom2
is suggested to be a novel DNA-packaging protein that effects on the
transcript level of some genes in the mt-nucleoid. This also implies
that Glom2 is not simply an accessory factor of Glom.Two possibilities
exist for the decreased mRNA accumulation: one is that stabilization
of mRNA was defective, and the other is that transcription was
suppressed. Since smear degradation products were not detected in
our Northern blot analysis (data not shown), Glom2 might not be
involved in the stabilization of mRNA. Therefore, Glom2 might be
involved in the transcriptional regulation of some mitochondrial
genes. Further investigation might provide a specific function of
Glom2 in the future.
Glom2 is partly homologous with Mgm101 of budding yeast, which
wasidentified in a genetic screen for mutants thatcauses loss of mtDNA
(Chen et al., 1993). Some properties of Mgm101 and Glom2 resemble
an abundant mtDNA-associated protein in yeast (Kaufman et al., 2000),
but its ability for mtDNA packaging is unknown. A lack of Mgm101 did
not cause significant changes in the nucleoid structure in yeast cells
(Meeusen et al., 1999). Mgm101 has been suggested to be involved in
the repair of oxidatively damaged mtDNA (Meeusen et al., 1999) and is
known as a putative replication initiator in budding yeast (Zuo et al.,
2002). In P. polycephalum, oxidative stress increased the expression of
Glom2 (Itoh, unpublished data), implying the possibility that Glom2
might alsobe involved inthe repairof oxidativelydamaged mtDNA,like
Mgm101. In contrast, Mgm101 has some different properties compared
to Glom2. Mgm101 is present only in a subpopulation of mt-nucleoids
of mtDNA (Chen et al., 1993). These different properties imply that
Mgm101 is not an ortholog of Glom2, although further analysis is
required toelucidatethispoint.No matter howGlom2 andMgm101 are
related, the wide distribution of Mgm101-like genes in eukaryotic cells,
possibly themitochondria of eukaryotic cells, maysuggestanimportant
function in mitochondria.
Down-regulation with MOs provided a powerful method to
investigate the function of mt-nucleoid proteins of P. polycephalum.
Since the plasmodium is a giant multinucleate cell, we could obtain
enough material for biochemical and molecular genetic analyses after a
wasalsopossible.Moreover, retaininga decreased expressionlevel was
possible by re-injecting MOs, and examining many genes at the same
time should also be possible by mixing MOs for the down-regulation of
multiple target genes. The mt-nucleoid of P. polycephalum consists of
more than 70 mt-nucleoid proteins (Sasaki et al., 2003). Analysis of all
two mitochondrial DNA-packaging proteins should provide the basis
for such future studies in P. polycephalum. Furthermore, our novel
model suggesting that plural DNA-packaging proteins organize the
K. Itoh et al. / Mitochondrion 11 (2011) 575–586
Supplementary materials related to this article can be found online
We thank Dr. Minako Ueda (Nagoya University) for the stimulat-
ing discussions and helpful advices, Dr. Kiyotaka Hitomi (Nagoya
University) for providing the anti-ANT antibody, and Mr. Hiroki
Tsutsui (Nagoya University) for the help with the phylogenetic
analysis. This work was supported by a Grant from the Japan Society
for the Promotion of Science Fellowships (07J11886 to KI), a Grant-in-
Aid for Creative Scientific Research (18GS0314-01 to NS), a Grant-in-
aid for Scientific Research (B) (19370017 to TH), JST ERATO
Higashiyama Live-Holonics Project (to TH), and Japan MEXT Grant-
in-Aid for Young Scientists (Start-up: 20870020 and B: 21770041 to
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