A icle
Sc een o mi ochond ial DNA copy numbe
main enance genes e eals essen ial ole o
ATP syn hase
A sushi Fukuoh
1,2,3,†
, Giuseppe Cannino
1,†
, Mike Ge a ds
1
, Suzanne Buckley
1
, Selena Kazancioglu
1
,
Filippo Scialo
1
, Ee o Liha ainen
4
, And e Ribei o
4
, E ic Du ou
1
& Howa d T Jacobs
1,5,*
Abs ac
The machine y o mi ochond ial DNA (m DNA) main enance is only
pa ially cha ac e ized and is o wide in e es due o i s in ol e-
men in disease. To iden i y no el componen s o his machine y,
plus o he cellula pa hways equi ed o m DNA iabili y, we
implemen ed a genome-wide RNAi sc een in D osophila S2 cells,
assaying o loss o luo escence o m DNA nucleoids s ained wi h
he DNA-in e cala ing agen PicoG een. In addi ion o p e iously
cha ac e ized componen s o he m DNA eplica ion and ansc ip-
ion machine ies, posi i es included many p o eins o he cy osolic
p o easome and ibosome (bu no he mi o ibosome), h ee
p o eins in ol ed in esicle anspo , some o he ac o s in ol ed
in mi ochond ial biogenesis o nuclea gene exp ession, >30
mainly uncha ac e ized p o eins and mos subuni s o ATP
syn hase (bu no o he OXPHOS complex). ATP syn hase knockdown
p ecipi a ed a bu s o mi ochond ial ROS p oduc ion, ollowed by
copy numbe deple ion in ol ing inc eased mi ochond ial u no e ,
no dependen on he canonical au ophagy machine y. Ou indings
will in o m u u e s udies o he appa a us and egula ion o
m DNA main enance, and he ole o mi ochond ial bioene ge ics
and signaling in modula ing m DNA copy numbe .
Keywo ds complex V; DNA eplica ion; mi ochond ial biogenesis; mi ochon-
d ial DNA; mi ophagy; nuclease; nucleoid; eac i e oxygen species
Subjec Ca ego ies Me hods & Resou ces; Me abolism
DOI 10.15252/msb.20145117 | Recei ed 12 Janua y 2014 | Re ised 2May
2014 | Accep ed 2May 2014
Mol Sys Biol. (2014)10:734
In oduc ion
Euka yo es ha use mi ochond ial oxida i e phospho yla ion
(OXPHOS) o gene a e ATP main ain a sepa a e mi ochond ial
genome (m DNA), encoding a subse o OXPHOS p o ein subuni s,
oge he wi h some componen s o he machine y o in ami o-
chond ial p o ein syn hesis. The main enance and exp ession o
m DNA is o he wise dependen on nuclea -coded gene p oduc s,
cons i u ing a sepa a e appa a us o genome main enance and gene
exp ession wi hin he cell (McKinney & Oli ei a, 2013).
The co e machine y o m DNA eplica ion is b oadly conse ed
among euka yo es. DNA eplica ion is assumed o depend on he
only DNA polyme ase consis en ly ound in mi ochond ia, DNA
polyme ase c(PolG; Kaguni, 2004), a membe o he amily A DNA
polyme ases (I o & B ai hwai e, 1991). I s closes p oka yo ic
homologue is he phage T7 DNA polyme ase, and i is assumed o
unc ion in conce wi h he mi ochond ial helicase Twinkle
(Spelb ink e al, 2001), a homologue o phage T7 helicase-p imase
(gp4). Twinkle is absen om yeas , whe e o he helicases a e
in ol ed in m DNA eplica ion. Main enance o m DNA equi es
also mi ochond ial ansc ip ion ac o s A (m -TFA o TFAM),
needed o m DNA compac ion and ansc ip ion (La sson e al,
1998; Kang & Hamasaki, 2005; Campbell e al, 2012), and B
(m -TFB2, TFB2M; Ma sushima e al, 2004). Th ee o he p o eins
a e essen ial o m DNA main enance, namely m SSB, he mi ochond ial
single-s anded DNA-binding p o ein (Maie e al, 2001), RNase H1
(Ce i elli e al, 2003), and, in some o ganisms, a second DNA
polyme ase, P imPol, ha addi ionally has p imase ac i i y
(Ga cı
´a-Go
´mez e al, 2013). A numbe o o he p o eins a e equi ed
o main ain no mal m DNA copy numbe o opology in di e en
o ganisms (Con amine & Pica d, 2000; Copeland, 2012). These
include he DNA-binding AAA p o ein ATAD3A (He e al, 2007),
some enzymes o nucleo ide me abolism and anspo (Saada,
2004), p o eins wi h oles in mi ochond ial memb ane dynamics
(Jones & Fangman, 1992; Wong e al, 2000; Elachou i e al, 2011;
Vielhabe e al, 2013), chape ones (Ciesielski e al, 2013),
exonucleases (Ko nblum e al, 2013), p o eases (He lan e al,
2003; Ma sushima e al, 2010; Sesaki e al, 2003), and e en
cy oskele al p o eins (Reyes e al, 2011). Mi ochond ia also con ain
1BioMediTech and Tampe e Uni e si y Hospi al, Uni e si y o Tampe e, Tampe e, Finland
2Depa men o Clinical Chemis y and Labo a o y Medicine, Kyushu Uni e si y G adua e school o Medical Sciences, Fukuoka, Japan
3Depa men o Medical Labo a o y Science, Junshin Gakuen Uni e si y, Fukuoka, Japan
4Depa men o Signal P ocessing, Tampe e Uni e si y o Technology, Tampe e, Finland
5Resea ch P og am o Molecula Neu ology, Uni e si y o Helsinki, Helsinki, Finland
*Co esponding au ho . Tel: +358 3 3551 7731,+358 50 341 2894; E-mail: howa d. .jacob[email p o ec ed]
†
These au ho s equally con ibu ed o his wo k.
ª2014 The Au ho s. Published unde he e ms o he CC BY 4.0license Molecula Sys ems Biology 10:734 |2014 1
Published online: June 21, 2014
opoisome ases, ligases, and o he nucleases, al hough hei speci ic
oles in m DNA me abolism a e unclea . While a c ude DNA
syn he ic machine y can be econs i u ed in i o om a minimal se
o hese p o eins, he ull complemen o p o eins equi ed o ai h-
ul m DNA eplica ion in i o emains o be de e mined.
Some componen s o he m DNA main enance machine y a e
sha ed wi h he nuclea compa men , including RNase H1
(Ce i elli e al, 2003) and many p o eins implica ed in base-excision
epai (Alexeye e al, 2013). Mos ly, hese a e syn hesized in wo
o mo e iso o ms ou ed o di e en cellula compa men s, o
example, ia di e en ial splicing, al e na i e ansla ional s a
si es (Suzuki e al, 2010), o ambiguous a ge ing signals (Ka niely
& Pines, 2005).
m DNA is packaged oge he wi h TFAM and some o he p o eins
in o disc e e in ami ochond ial s uc u es o a iable composi ion,
called nucleoids, by analogy wi h hose o bac e ia (Spelb ink, 2010;
Bogenhagen, 2012). They con ain a numbe o eplica ion p o eins
whose unc ional oles a e poo ly unde s ood, as well as p o eins
implica ed in o he cellula p ocesses, including me abolic enzymes
and chape ones, and p o eins in ol ed in in ami ochond ial p o ein
syn hesis (Hensen e al, 2014). The appa a us o mi ochond ial
ansla ion has been unc ionally implica ed in m DNA main enance
in yeas (Con amine & Pica d, 2000), hough no in me azoan cells
(S o ie & A a di, 1972).
The impo ance o m DNA main enance o cell physiology and
homeos asis is unde sco ed by he inding ha i s dys unc ion leads
o di e se ypes o human disease, including bo h in an ile and la e-
onse pa hologies, showing a bewilde ing a ie y o issue speci ici ies
(Shadel, 2008; Ro
¨ ig & Poul on, 2009; Ylikallio & Suomalainen,
2012). Loss o mi ochond ial genome in eg i y o ideli y is also
associa ed wi h aging (Oli ei a e al, 2010; B a ic & La sson, 2013).
Iden i ying he ull se o gene p oduc s in ol ed in ai h ul m DNA
main enance is hus o b oad in e es and impo ance.
To his end, we implemen ed a genome-wide (blinded) sc een o
D osophila S2 cells, using dsRNA-based RNA in e e ence (RNAi),
aking ad an age o he ac ha S2 cells ole a e loss o m DNA and
con inue o g ow wi hin he ime scale o a ypical expe imen ,
despi e dec eased OXPHOS capaci y. Fu he mo e, m DNA nucle-
oids may be iden i ied in hese cells on he basis o luo escence
signal om he opology-dependen DNA-in e cala ing dye Pico-
G een (Ashley e al, 2005). E ec s on m DNA copy numbe we e
hen p obed u he using quan i a i e PCR (QPCR), wi h addi ional
expe imen s conduc ed on he cellula pheno ypes p oduced by
knockdown o speci ic genes iden i ied in he sc een, no ably hose
encoding subuni s o ATP syn hase, in o de o es aspec s o he
mechanisms by which hey may ac .
Resul s and Discussion
Implemen a ion and ou come o he p ima y sc een
We se ou o sc een a genome-wide D osophila dsRNA lib a y in S2
cells, sco ing o disappea ance o he PicoG een signal o m DNA
nucleoids as indica i e o genes equi ed o m DNA main enance.
In ini ial ials, we ound i di icul o pick ou he nucleoid signal
agains backg ound cy oplasmic luo escence. Using a dsRNA
agains he PolG ca aly ic subuni ( amas) as a posi i e con ol,
and a dsRNA di ec ed agains GFP as a nega i e con ol, we
es ablished a p o ocol whe eby i was possible eliably o sco e
(by eye) he disappea ance o nucleoid signal (see Fig 1). This
in ol ed applying he es dsRNA o 5 days in a 96-well pla e
o ma , wi h addi ion on day 3 o a dsRNA di ec ed agains
TFAM. Al hough p olonged incuba ion wi h TFAM dsRNA i sel
led o m DNA deple ion, he sho e - e m ea men con e sely
enhanced he nucleoid signal in nega i e con ol cells, whe eas i
was dec eased o e y low le els in cells ea ed wi h he posi i e
con ol dsRNA.
The p ima y sc een, conduc ed blind, was success ul in iden i y-
ing as posi i es mos o he known ac o s in ol ed in m DNA
me abolism (Table 1, ca ego y 1), gi ing con idence in i s alidi y.
In o al, 105 dsRNA a ge s we e ini ially judged as posi i e (Supple-
men a y Table S1), o which almos hal we e eco ded also as lead-
ing o cell dea h in a ac ion o he cells. Consis en wi h p e ious
s udies (Ra
¨me e al, 2002; Bou os e al, 2004), a u he 276 a ge s
(Supplemen a y Tables S1 and S2) ga e massi e cell dea h bu no
speci ic loss o nucleoid signal and we e conside ed o ep esen
essen ial genes ha could no be s udied u he . Finally, an addi-
ional 132 a ge s we e judged o gi e an abno mal ou come wi hou
comple e loss o PicoG een nucleoid signal (Supplemen a y Tables
S1 and S3), bu we e no analyzed u he , al hough many ell in o
simila ca ego ies o pa hways as hose on he posi i es lis . The
speci ici y and knockdown e iciency o his p ocedu e has p e i-
ously been documen ed (Clemens e al, 2000; Kleino e al, 2005).
Knockdown was he e e i ied a he RNA le el by qRT-PCR o 17
speci ic a ge s (see SI).
We compa ed subjec i e judgmen agains a compu a ional
me hod o measu e punc a e luo escence in ensi y (de ails o be
published elsewhe e). The la e ga e many alse posi i es due
o a iable backg ound luo escence, as well as alse nega i es due
o cell deb is. Based on esc eening, we judged he manual me hod o
be supe io (see SI o de ails), and we se c i e ia o de ining posi-
i es as desc ibed below.
Resc eening o iden i y de ini i e posi i es
Posi i es we e conside ed as con i med i h ee posi i e bu no
nega i e indings we e ob ained. Those whe e a nega i e o ambigu-
ous inding was eco ded we e e ained only whe e h ee imes as
many clea ly posi i e indings we e ob ained upon exhaus i e
esc eening (double as e isks in column H o Table 1) o he wise
hey we e conside ed as alse posi i es (double as e isks in column
G o Supplemen a y Table S4).
Fi e a ge s ha did no gi e consis en ly posi i e indings du ing
esc eening we e no ed o gi e ise o mul iple splice a ian s,
encoding a leas one polypep ide p edic ed o be mi ochond ially
localized. Fo hese, we es ed dsRNAs a ge ed speci ically on he
ele an splice a ian s, con i ming se e al addi ional posi i es
(Table 1, g een backg ound), whe eas dsRNAs a ge ed agains
o he splice a ian s o he en i e gene we e judged nega i e
(Supplemen a y Table S4, blue backg ound).
O he o iginal 105 posi i es, 83 we e e ained, 20 we e eas-
signed as nega i e, and one was eassigned as abno mal (pink
backg ound in Supplemen a y Table S3). One was disca ded
because he dsRNA de ec ed a pseudogene o a gene al eady in he
lis , ano he because a e ised gene model combined i wi h ano he
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
2
Published online: June 21, 2014
posi i e, while ano he was subsequen ly e-anno a ed as wo sepa-
a e genes (bu shown as a single en y in Table 1). Posi i e indings
we e also ob ained o speci ic splice iso o ms o h ee o he nega-
i es. Thus, he con i ma ion o 86 ou o 106 ini ial a ge s indica es
a alse-posi i e a e o 18%. One addi ional posi i e (CG5794) was
unexpec edly iden i ied by a dsRNA nominally a ge ed agains a
di e en gene. The posi i es ell in o se en dis inc classes: mi o-
chond ial DNA eplica ion o ansc ip ion, cy osolic ansla ion, he
p o easome, ATP syn hase, mi ochond ial dynamics o biogenesis,
nuclea gene exp ession, and a se en h, miscellaneous ca ego y.
Al hough he p ima y nega i es we e no esc eened sys ema i-
cally, some ha ell in o simila unc ional pa hways as de ini i e
posi i es we e e-e alua ed using he same c i e ia. Ele en we e
p omo ed o he posi i es lis (yellow backg ound in Table 1),
including, o example, mos o he subuni s o ATP syn hase. This
implies ha he ini ial sc een may ha e missed as many posi i es as
we e ac ually e ained, implying a alse-nega i e a e o up o 1%.
The o e all esul s o he sc een a e summa ized in Fig 2. The inal
numbe o de ini i e posi i es was 97, coun ing only once hose
wi h >1 posi i e splice a ian .
Copy numbe o m DNA
Fo genes on he de ini i e posi i es lis , we ca ied ou QPCR o
assess changes in m DNA copy numbe a e 5 days o dsRNA ea -
men (wi hou concomi an TFAM knockdown), no malized agains
a single-copy nuclea DNA s anda d. Based on his assay (see
Table 1, column G, aw da a in Supplemen a y Table S5), we classi-
ied he posi i es as showing subs an ial (++,≤60%) o modes (+)
m DNA deple ion, no signi ican copy numbe change (0), o an
inc ease in m DNA ().
Al hough hese classes seem a bi a y, we ound ha RNAi
knockdown o well-cha ac e ized componen s o he m DNA epli-
ca ion machine y all ga e alues in he 20-60% ange (++). Su p is-
ingly, we iden i ied only 6 new genes om he sc een whose
knockdown p oduced a compa ably se e e m DNA deple ion. Th ee
o hese encode subuni s o ATP syn hase. The o he s we e CG5794,
encoding a de-ubiqui ina ing enzyme wi h unknown subs a e(s),
TweedleY, p e iously iden i ied as a cu icula p o ein (Guan e al,
2006), and poin ed, a well-s udied E s amily ansc ip ion ac o
(Klaes e al, 1994; Mo imo o e al, 1996). The ac ha knockdown
GFP
nega i e con ol
m DNA helicase (CG5924)
posi i e (con ol)
amas (CG8987)
posi i e con ol
CG31380
nega i e
CG6413 (Dis3)
posi i e
CG2028 (CkIα
α
)
abno mal ( ewe do s)
Figu e 1. Sc eening o D osophila dsRNA lib a y by PicoG een nucleoid luo escence in S2cells.
Mic og aphs o S2cells s ained wi h PicoG een, ollowing 5days o ea men wi h he dsRNA indica ed. GFP and amas (Polg, CG8987) we e used as nega i e and
posi i e con ols, espec i ely. In esc eening, CG5924 was also used as a posi i e con ol. Bo h we e de ec ed in he blinded sc een as posi i es. O he images show a ypical
nega i e (CG31380), a ypical posi i e (CG6413) and a ypical case o a a ge classed as abno mal, in his case CG2028 (CkIa), which showed a dec eased numbe o
nucleoid signals pe cell. Images a e op imized o b igh ness and con as bu wi h no o he manipula ions. Scale ba indica es 50 lm.
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
3
Published online: June 21, 2014
Table 1. De ini i e posi i es
Ca ego y
numbe
Ca ego y
name
CG
numbe
O icial
name
o symbol
Human
o hologue
O he o common name(s) o pu a i e
unc ion
m DNA
deple ion >3 esc eenings No e
1m DNA
eplica ion
and ansc ip ion
CG3910 m TFB2TFB2M m ansc ip ion ac o B2+*
CG4217 TFAM TFAM m TFA, m ansc ip ion ac o A ++
CG4337 m SSB SSBP1m single-s anded DNA-binding p o ein ++
CG4644 m RNApol POLRMT m RNA polyme ase ++
CG5924 CG5924 PEO1Twinkle DNA helicase ++
CG6815 belphego ATAD3A, B, C AAA domain con aining p o ein 30**
CG7175 mTe 5m ansc ip ion e mina ion ac o 5++
CG8729 nh1RNASEH1Ribonuclease H10** Weak posi i e, diminished
nucleoid signal seen in
some cells, also some cell
dea h
CG8987 amas POLG DNA polyme ase cca aly ic subuni ++
CG18124 mTTF m ansc ip ion e mina ion ac o ++
CG33650 DNApol-c35 POLG2DNA polyme ase caccesso y subuni ,
CG8969
+**
2Cy osolic
ansla ion
CG1821 RpL31 RPL31 La ge subuni –
CG3203 RpL17 RPL17 La ge subuni –
CG3751 RpS24 RPS24 Small subuni n
CG3922 RpS17 RPS17, RPS17L Small subuni –
CG3997 RpL39 RPL39, RPL39L La ge subuni 0
CG4111 RpL35 RPL35 La ge subuni n
CG4759 RpL27 RPL27 La ge subuni n
CG7283 RpL10Ab RPL10A La ge subuni n
CG7490 RpLP0RPL0La ge subuni n
CG7622 RpL36 RPL36 La ge subuni –*
CG7726 RpL11 RPL11 La ge subuni n * Cell dea h no subs an ial
in some epea s
CG8922 RpS5a RPS5Small subuni n *
CG9282 RpL24 RPL24 La ge subuni n *
CG9677 In 6EIF3E eIF3, Ini ia ion ac o 3, subuni E n * Abno mal dis ibu ion o
some esidual nucleoid
signal wi hin cells
CG11522 RpL6RPL6La ge subuni
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
4
Published online: June 21, 2014
Table 1(Con inued)
Ca ego y
numbe
Ca ego y
name
CG
numbe
O icial
name
o symbol
Human
o hologue
O he o common name(s) o pu a i e
unc ion
m DNA
deple ion >3 esc eenings No e
3P o easome CG1341 Rp 1PSMC2,5,6ATPase egula o y subuni 2(o 5o 6)+
CG5266 P os25 PSMA2Co e a- ype subuni 2+**
CG9324 Pomp POMP P o easome ma u a ion p o ein –
CG9327 P os29 PSMA4Co e a- ype subuni 4+*
CG10149 Rpn6PSMD11 Non-ATPase egula o y subuni 11 +
CG16916 Rp 3PSMC4ATPase egula o y subuni 4+
CG18174 Rpn11 PSMD14 Non-ATPase egula o y subuni 14 0 ** Se e e cell dea h: ew cells
emained a e 5days
4ATP
syn hase
CG2968 l(1)G0230 ATP5DdSubuni o F1(s alk) +
CG3321 CG3321 ATP5I Subuni e o Fo, dime iza ion/bending ++
CG4307 Oscp ATP5O OSCP subuni o F1/s a o a m ++ **
CG4412 ATPsyn-C 6ATP5J Coupling ac o 6, subuni o Fo/s a o a m +
CG6105 l(2)06225 ATP5L, ATP5L2Subuni g o Fo, dime iza ion/bending +
CG7610 ATPsyn-cATP5C1cSubuni o F1(s alk) +*
CG6030 ATPsyn-d ATP5H Subuni d o Fo, s a o a m +*
CG8189 ATPsyn-b ATP5F1Subuni b o Fo, s a o a m ++ *
CG11154 ATPsyn-bATP5B Co e bsubuni o F1+**
5Mi ochond ial
biogenesis and
dynamics
CG3114 e ec wing NRF1Nuclea espi a o y ac o 1homologue 0**
CG6338 E s97D GABPA Nuclea espi a o y ac o 2,asubuni +**
CG6512 CG6512 AFG3L2, SPG7m-AAA p o ease subuni 0*
CG8479 opa1-like OPA1Dynamin- ela ed p o ein equi ed o inne
m memb ane usion
+*
CG9809 spa gel PPARGC1A, B,
PPRC1
PPAR ccoac i a o +** Weak posi i e, diminished
numbe o nucleoid
signals emaining in
some cells
CG14981 maggie TOMM22 Tomm 22 subuni o ou e m memb ane
anslocase
+
6Nuclea gene
exp ession
CG1057 MED31 MED31 Media o complex subuni 31, anc ip ional
elonga ion
0
CG1554 RpII215 POLR2A RNA polyme ase II 215kD subuni +
CG1810 mRNA-cap RNGTT mRNA capping enzyme 0
CG1874 No 1CNOT1CCR4-NOT ansc ip ion complex subuni 10 ** Weak posi i e, diminished
numbe o nucleoid
signals emaining in
some cells, some cell
dea h also e iden
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
5
Published online: June 21, 2014

Table 1(Con inued)
Ca ego y
numbe
Ca ego y
name
CG
numbe
O icial
name
o symbol
Human
o hologue
O he o common name(s) o pu a i e
unc ion
m DNA
deple ion >3 esc eenings No e
CG2163 Pabp2PABPN1,
PABPN1L
Nuclea poly(A)-binding p o ein –
CG3162 LS2No el U2AF- ela ed egula o o di e en ial
splicing
0
CG3675 A 2PRMT6P o ein a ginine me hyl ans e ase 2+
CG6525 pps SPOCD1,
PHF3
P o ein pa ne o sn , egula o o
al e na i e splicing o Sex le hal
+* Diminished numbe o
nucleoids emained in
many cells
CG7626 Sp 5SUPT5H T ansc ip ion elonga ion ac o SPT5(DSIF
complex)
0
CG9591 omd INTS5In eg a o complex subuni 5, snRNA
p ocessing ac o
+* Ve y ew cells emained,
a e 5days o ea men
CG9748 belle DDX3X,Y,;
DDX4
RNA helicase, implica ed in X-ch omosome
dosage compensa ion
0
CG10955 R 1RTF1Componen o RNA polyme ase II-associa ed
(PAF1) complex, ansc ip ional elonga ion
0**
CG11990 hy ax CDC73 Componen o RNA polyme ase II-associa ed
(PAF1) complex, ansc ip ional elonga ion
0*
CG17183 MED30 MED30 Media o complex subuni 30, anc ip ional
elonga ion
0**
CG17358 Ta 12 TAF12 TATA box-binding p o ein-associa ed ac o 12 +
CG17603 Ta 1TAF1, TAF1L TATA box-binding p o ein-associa ed ac o 1+
7Miscellaneous
(o he o
unknown)
CG3539 SLY-1
homologue
SCFD1ER o Golgi esicle anspo 0**
CG4268 Pi sl e CDK11A Cyclin-dependen kinase supe amily,
egula o o au ophagy
–
CG5794 CG5794 USP34 Ubiqui in-speci ic pep idase
(deubiqui ina ing enzyme)
++ *
CG6413 Dis3DIS3Exosome complex exo ibonuclease –*
CG7368 CG7368 Zn inge p o ein –**
CG8021 CG8021 SLIRP RNA-binding p o ein, egula ion o m RNA
le els
0
CG9007 upSET SETD5,
MLL5
Zn inge p o ein, ela ed o i ho ax
and his one lysine me hly ans e ases
–
CG9397-H jing AEBP2Zn inge ansc ip ion ac o ,
ole in mo phogenesis
–** Was clea ly posi i e in
5ou o 7 ials
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
6
Published online: June 21, 2014
Table 1(Con inued)
Ca ego y
numbe
Ca ego y
name
CG
numbe
O icial
name
o symbol
Human
o hologue
O he o common name(s) o pu a i e
unc ion
m DNA
deple ion >3 esc eenings No e
CG9797 CG9797 Zn inge p o ein 0** Weak posi i e, diminished
numbe o nucleoid
signals emaining in
some cells
CG10042 MBD-R2Me hyl-DNA-binding p o ein 0Some small nucleoid
signals emained in some
cells. Ano he dsRNA o
his gene ga e
consis en ly nega i e
indings
CG10144 CG10144 VPS8Vesicle so ing o lysosomes +** Nucleoid signals o
diminished size o
in ensi y seen in some
cells, ew cells emained
a e 5d
CG10395 CG10395 Zn inge p o ein 0
CG10582 Sin POLR3E Sex-le hal in e ac o , possible al e na i e
splicing, p oposed subuni o RNA
polyme ase III
0*
CG10582-C Sin Pu a i e mi ochond ially a ge ed iso o m
o Sin
0** Clea posi i e in 3 ials,
bu also some
inconsis en indings
CG12242 Gs D5Glu a hione S- ans e ase supe amily (+) Deple ion jus ou side he
bo de o signi icance,
due o la ge a iance
CG13203-C CG13203 Pu a i e mi ochond ially a ge ed iso o m o
p o ein wi h unknown unc ion
–**
CG13779 Sem1Possible endopep idase 0**
CG14084 Be 1BET1ER o Golgi esicle anspo 0*
CG14247 CG14247 Unknown unc ion 0*
CG14634 CG14634 Unknown +**
CG15231 IM4Immune-induced molecule, pep ide
ho mone?
+*
CG15343 CG15343 Py idoxamine 50-phospha e oxidase-like 0*
CG15793 Dso 1MAP2K1,2,5MAP kinase kinase 0*
CG17077 poin ed ETS1,2E s ansc ip ion ac o ++ *
CG31258 Cenp-C Cen ome e-binding, kine ocho e unc ion n *
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
7
Published online: June 21, 2014
Table 1(Con inued)
Ca ego y
numbe
Ca ego y
name
CG
numbe
O icial
name
o symbol
Human
o hologue
O he o common name(s) o pu a i e
unc ion
m DNA
deple ion >3 esc eenings No e
CG31079 Unknown (gene model la e wi hd awn
om Flybase)
n P edominan ly induced cell
dea h, ew cells
emaining seemed
posi i e
CG32085 CG32085 FBXL16 Pu a i e ubiqui in ligase 0
CG32561, 2 xmas-1, xmas-2MCM3AP
(xmas-2)
Pu a i e p o ein ace yl ans e ase in ol ed
in DNA eplica ion (xmas-2)
0** Clea posi i e in 4 ials,
bu also some
inconsis en indings
CG32570 TwdlY Sugges ed cu icula p o ein ++ *
CG32652 CG32652 Unknown +*
CG34415 mu e Muscle was ed, ch oma in p o ein, his one
locus body
0** Weak posi i e, diminished
nucleoid signal seen in
some cells, cell dea h
seen only in some ials
CG33546 g z GST-con aining Zn inge p o ein, pu a i e
mi o ic checkpoin p o ein
0
CG42666-G CG42666 REXO1Exo ibonuclease, p edic ed mi ochond ially
a ge ed iso o m
n ** Weak posi i e, diminished
numbe o nucleoid
signals emained in some
cells, dsRNA o en i e
gene was an inconsis en
weak posi i e; see also
Table S1. iso o m-speci ic
p ime s also a ge Ada ,
CG12598
CG42666-D CG42666 REXO1Exo ibonuclease, p edic ed mi ochond ially
a ge ed iso o m
0* Weak posi i e, diminished
numbe o nucleoid
signals emained in some
cells, dsRNA o en i e
gene was an inconsis en
weak posi i e; see also
Table S2
CG42281 bunched Signal ansduc ion in esponse o g ow h
ac o (dpp) binding
–
=no in o iginal posi i es lis ; =posi i e iso o m wi h pu a i e m a ge ing; =gene model wi hd awn om Flybase.
Posi i es also showing subs an ial cell dea h a e shown in unde line and i alic.
>3 esc eenings: Posi i es ha equi ed many ounds o esc eening o inal alida ion. * = 4 ounds; ** ≥8 ounds.
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
8
Published online: June 21, 2014
o many genes esul ed in loss o PicoG een signal wi hou majo
changes in m DNA copy numbe sugges s ha indi ec e ec s may
be common, o example, a ec ing DNA opology, nucleoid a chi-
ec u e, memb ane po en ial o cellula dye up ake.
Posi i es implica ed in m DNA me abolism
The posi i es include mos o he p o eins wi h known oles in
m DNA eplica ion o ansc ip ion, no ably he i e shown p e i-
ously o be essen ial o m DNA main enance in D osophila (Go o
e al, 2001; Maie e al, 2001; Iyenga e al, 2002; Ma sushima e al,
2004; Humph ey e al, 2012). The lis comp ises he wo subuni s o
PolG, he ca aly ic subuni o he mi ochond ial RNA polyme ase,
m SSB, he D osophila homologue o he Twinkle helicase, an-
sc ip ion ac o s TFAM and m TFB2M, mTERF amily membe s
mTTF and mTe 5 (Jo
˜e s e al, 2013), plus nh1 (RNaseH1) and
belphego (homologue o mammalian ATAD3). All ga e signi ican
m DNA deple ion excep o nh1 and belphego . DNA ligase III
(lig3, CG17227) and mTERF amily membe s mTe 3 and CG15390
(homologue o mammalian MTERF4) we e consis en ly nega i e.
DNA ligase III was p e iously epo ed as dispensable o nuclea
DNA epai bu essen ial o m DNA main enance in human cells
(Ruhanen e al, 2011) and mouse (Puebla-Oso io e al, 2006; Gao
e al, 2011). Ou da a imply ha i is edundan o a leas one o he
m DNA ligase in D osophila. Simila a gumen s may apply o he
absence o any opoisome ase, gy ase, ecombinase, esol ase o
helicase (o he han Twinkle). Ou s udy sugges s ha ew dedi-
ca ed componen s o he m DNA eplica ion appa a us emain o be
iden i ied, bu his does no exclude ac o s wi h o e lapping oles
in o he cell compa men s.
In mammalian mi ochond ia, he ansc ip ional appa a us is
conside ed essen ial o bo h leading- and lagging-s and syn hesis
(Clay on, 1982; Fus e e al, 2010). RNase H1, also equi ed o
m DNA main enance in mouse (Ce i elli e al, 2003) and human
cells (Ruhanen e al, 2011), migh be in ol ed in p ime emo al, bu
his ypically also needs o he helicases and nucleases such as Fen1
(CG8648) and Dna2 (CG2990), bo h implica ed in m DNA eplica ion
in mammalian cells (Duxin e al, 2009; Kazak e al, 2013). Thei
absence om he posi i es lis is unsu p ising, howe e , since bo h
also unc ion in he nucleus. One posi i e om he miscellaneous
ca ego y, CG8021, appea s o be a D osophila homologue o SLRP, a
mammalian p o ein in ol ed in mi ochond ial mRNA s abiliza ion
and p ocessing (Sasa man e al, 2010; Chujo e al, 2012).
The mammalian ATAD3 amily has been implica ed in nucleoid
o ganiza ion (He e al, 2007), mi ochond ial p o ein syn hesis (He
e al, 2012), egula ion o apop osis (Huang e al, 2011) and au o-
phagy (Chen e al, 2011), choles e ol a icking (Rone e al, 2012),
mi ochond ial dynamics (Gilquin e al, 2010) and s ess esis ance
(Ho mann e al, 2012). Loss o PicoG een nucleoid signal wi h only
a mino d op in m DNA copy numbe may indica e ha belphego
unc ions also in di e se pa hways and ha i s e ec s on nucleoids
and m DNA may be indi ec .
Nucleases o he han RNase H1 ha e been shown o sugges ed o
ha e oles in m DNA me abolism in a ious o ganisms, including
EXOG (Tann e al, 2011), EndoG (McDe mo -Roe e al, 2011)
and yeas Exo5 (Bu ge s e al, 2010). Exo5 has no D osophila
NEGATIVE
POSITIVE
ABNORMAL
CELL DEATH
P ima y
sc een
105
276
132
15,506
16,019
dsRNAs
Resc een
DEFINITIVE
POSITIVES
1
83 + 3 wi h splice a ian s
11
To al = 97
10
Figu e 2. O e all esul s o he sc een.
Schema ic diag am illus a ing numbe o dsRNAs analyzed, numbe s o p ima y posi i es, nega i es and o he classes, and he esul s o esc eening.
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
9
Published online: June 21, 2014
Bogenhagen, 2012). The in ol emen o cV, he p o easome, and
some key genes o mi ochond ial dynamics and quali y con ol
sugges s ha m DNA copy numbe is dependen on a balance
be ween mi ochond ial u no e and biogenesis, in which speci ic
s esses, no ably mi ochond ial ROS p oduc ion and impai ed cy o-
solic p o ein u no e , may be c ucially impo an .
ATP syn hase was in e ed o be a key playe in homeos a ic
main enance o mi ochond ia, and hus in he amoun o m DNA
and i s gene p oduc s. Mu a ions in ATP syn hase in ungi a e
al eady known o play a de e mining ole in whe he m DNA loss
can be ole a ed (Con amine & Pica d, 2000; Le eb e-Legend e
e al, 2003), o e en acili a ed (Gi aud & Velou s, 1997; Lai-Zhang
e al, 1999; Con amine & Pica d, 2000), bu wi h memb ane po en-
ial implica ed as a key pa ame e (Du ezin-Caube e al, 2006;
Wang e al, 2007). In S2 cells, excess ROS p oduc ion was be e
co ela ed wi h he s eng h and kine ics o m DNA deple ion, bu
his could be an epi-phenomenon. The ac ha memb ane po en ial
‘pe mi ochond ion’ (Supplemen a y Fig S4D) was es o ed o i s
s a ing alue sugges s ha i s dis u bance may ye p o e o be he
p ima y induce .
Excess ROS has elsewhe e been p oposed o lead o m DNA
deple ion unde pa hological condi ions (La osche e al, 2010;
Quinzii e al, 2013), and pa hological de ec s in ATP syn hase
associa ed wi h ROS o e p oduc ion (Ba acca e al, 2007) may
down egula e mi ochond ial unc ions (Wojewoda e al, 2010,
2011) and e en lead o m DNA loss (Ve gani e al, 1999; Tu ne
e al, 2005). ROS o e p oduc ion has been widely sugges ed bo h
o p o oke m DNA damage, bu also o esul om i . Howe e , a
ecen epo showed ha un epai ed damage leads o m DNA
deple ion wi hou inc eased ROS (Shokolenko e al, 2013), and
he ole o ROS in p oducing soma ic m DNA mu a ions in he
PolgA mu a o mouse is dispu ed (T i uno ic e al, 2005; Dai
e al, 2010). Thus, he inc eased mi ochond ial ROS seen when cV
is knocked down is mo e logically a cause han a consequence o
m DNA deple ion. Fu he mo e, al hough ROS may p o oke s and
b eakage, in e e ing di ec ly wi h m DNA eplica ion (Han &
Chen, 2013), ou da a ins ead sugges ha ROS ac i a es mi o-
chond ial u no e be o e widesp ead DNA damage would be
sus ained, as occu s in mammalian cells unde TNFasignaling
(Nagakawa e al, 2005; Vad o e al, 2012). Howe e , an opposing
pa hway has also been sugges ed, in which ROS o e -p oduc ion
p omo es mi ochond ial biogenesis, no u no e (Mo eno-
Loshue os e al, 2006, 2011).
A key aim o u u e esea ch will be o iden i y he senso mole-
cule(s) in eg a ing changes in mi ochond ial ROS (o memb ane
po en ial) wi h o he me abolic signals, in o de o modula e mi o-
chond ial biogenesis and u no e . One possibili y consis en wi h
ou da a is ha ATP syn hase i sel is ha senso .
Ma e ials and Me hods
Cell main enance
D osophila S2 cells (In i ogen) we e cul u ed unde s anda d condi-
ions, in Schneide 0s medium (Sigma) and dilu ed 1:6 e e y 3-4 d. In
selec ed expe imen s, a ious d ugs we e added o glucose was
eplaced wi h he same concen a ion o galac ose. S2 cells s ably
exp essing Ciona in es inalis AOX we e gene a ed by co- ans ec ion
wi h a plasmid con e ing hyg omycin esis ance.
Sc eening o D osophila dsRNA lib a y and luo escence
mic oscopy o nucleoids
S2 cells we e seeded in o 96-well pla es and ea ed o e 5 days
wi h 0.6–1.2 lg o dsRNA om he lib a y (Open BioSys ems),
alongside posi i e and nega i e con ols in each pla e, as desc ibed
p e iously (Jo
˜e s e al, 2013). Nucleoids we e isualized by luo es-
cence mic oscopy a e s aining wi h Quan -iT
TM
PicoG een dsDNA
eagen (7.5 ll/ml, In i ogen). La ge -scale dsRNA ea men s o
m DNA copy numbe e alua ion and analysis o cellula pa ame e s
we e pe o med essen ially as p e iously (Jo
˜e s e al, 2013).
Nucleic acid isola ion and QPCR
To al RNA was isola ed om S2 cells as p e iously (Jo
˜e s e al,
2013). Fo DNA isola ion, cells om a single well o a 24-well pla e
we e p ocessed by a p ocedu e de e mined o gi e consis en esul s
i espec i e o cell densi y, in ol ing SDS lysis, p o einase K diges-
ion, isop opanol p ecipi a ion and o e nigh esuspension a 55°C
(see SI). Fo la ge -scale expe imen s, DNA was p epa ed om
1.5 ×10
6
cells cul u ed in 6-well pla es, as p e iously (Jo
˜e s e al,
2013). Mi ochond ial DNA copy numbe was assessed by QPCR
using p ime s agains COXII o 16S RNA ( o m DNA) and RpL32
(nuclea DNA, single-copy, o no maliza ion). T ansc ip le els
we e es ima ed ela i e o ha o RpL32 by a simila p ocedu e, bu
using cDNA as empla e.
Measu emen s o mi ochond ial unc ion
Mi ochond ial memb ane po en ial, ROS le el, and con en pe cell
we e de e mined by low cy ome y (Cannino e al, 2012) o cells
s ained, espec i ely, wi h 200 mM e ame hyl hodamine me hyl
es e (TMRM), 2.5 lM Mi oSox
TM
(In i ogen), o ei he 200 nM
10-nonyl ac idine o ange (NAO) o 40 nM Mi oT acke G een FM
(Li e Technologies). Oxygen consump ion o li ing cells (Cannino
e al, 2012) was measu ed using a Cla k- ype elec ode (Hansa ech
Oxy e m sys em).
Analyses o lysosomal and mi ochond ial con en
Lysosome con en pe cell was measu ed by low cy ome y o cells
s ained wi h 50 nM LysoT acke Red DND-99 (Li e Technologies).
Mi oT acke G een FM (Molecula P obes) and LysoT acke Red
DND-99 we e used o li e cell imaging by con ocal mic oscopy o
mi ochond ia and lysosomes, espec i ely, wi h spo -a ea calcula-
ion (ImageJ) and image decon olu ion (SVI, Huygens so wa e).
Wes e n blo ing
Pos -nuclea ex ac s esol ed by SDS-PAGE we e elec oblo ed and
p obed using s anda d me hods (essen ially as Fe nandez-Ayala
e al, 2009; see SI). P ima y an ibodies used we e agains NDUFS3
(Abcam, mouse), 1:10,000), ATP5A (Abcam, mouse, 1:1,000), and
GAPDH (E e es Bio ech, goa , 1:2,000), wi h app op ia e ho se-
adish pe oxidase-conjuga ed seconda y an ibodies. Visualiza ion
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
16
Published online: June 21, 2014

used he ECL sys em (Ame sham Biosciences) acco ding o he
manu ac u e ’s p o ocols.
S a is ical analyses
Compa isons be ween popula ions we e pe o med using unpai ed
wo- ailed S uden ’s - es s o analyses o a iance when mo e han
wo samples we e compa ed, wi h Bon e oni-co ec ed pos hoc - es .
Fo u he de ails, see Supplemen a y Ma e ials and Me hods.
Da a a ailabili y
O iginal images om he p ima y sc een a e deposi ed a : h p://
dx.doi.o g/10.5061/d yad. 55p5.
Supplemen a y in o ma ion o his a icle is a ailable online:
h p://msb.embop ess.o g
Acknowledgemen s
This wo k was suppo ed by unding om he Academy o Finland, Tampe e
Uni e si y Hospi al Medical Resea ch Fund, and he Sig id Juselius Founda ion.
AF was suppo ed by FY 2008 Resea che Exchange P og am be ween JSPS
and Academy o Finland. We hank Tea Tuomela, Ou i Ku onen, Hanna Ojala,
and E eliina Kaulio o echnical assis ance, and Susanna Valanne, Mika
Räme , Ian Hol , Co y Dunn, B endan Ba e sby, Anu Suomalainen, and Lau ie
Kaguni o use ul discussions and ad ice.
Au ho con ibu ions
AF ini ia ed he p ojec , conduc ed he p ima y sc een, and supe ised he
implemen a ion and in e p e a ion o he seconda y sc eening. GC conduc ed
he unc ional analysis o ATP syn hase knockdown. MG pe o med he copy
numbe analysis and p o iled he exp ession o mi ochond ial p o eases and
opa1-like a he RNA le el. SB and SK assis ed echnically wi h copy numbe
analysis. EL and AR de ised he compu a ional ool o objec i ying he Pico-
G een luo escence signals (de ails o be published elsewhe e). FS gene a ed
he AOX-exp essing S2cells. ED p o ided guidance and inpu o ATP syn hase
expe imen s. HTJ de ised he p ojec , analyzed he da a, compiled he igu es
and ables, and w o e he ex .
Con lic o in e es
The au ho s decla e ha hey ha e no con lic o in e es .
Re e ences
Alexeye M, Shokolenko I, Wilson G, LeDoux S (2013) The main enance o
mi ochond ial DNA in eg i y–c i ical analysis and upda e. Cold Sp ing Ha b
Pe spec Biol 5:a012641
Ashley N, Ha is D, Poul on J (2005) De ec ion o mi ochond ial DNA
deple ion in li ing human cells using PicoG een s aining. Exp Cell Res 303:
432 –446
Bal ze C, Tie enböck SK, Ma i M, F ei C (2009) Nu i ion con ols
mi ochond ial biogenesis in he D osophila adipose issue h ough Delg
and cyclin D/Cdk4.PLoS ONE 4:e6935
Ba acca A, Sga bi G, Ma iazzi M, Casalena G, Pagno a E, Valen ino ML,
Moggio M, Lenaz G, Ca elli V, Solaini G (2007) Biochemical pheno ypes
associa ed wi h he mi ochond ial ATP6gene mu a ions a n 8993.
Biochim Biophys Ac a 1767:913 –919
Be olin G, Fe ando-Miguel R, Jacoupy M, T a e S, G enie K, G eene AW,
Dauphin A, Waha e F, Bayo A, Salame o J, Lombès A, Bul eau AL, Fon EA,
B ice A, Co i O (2013) The TOMM machine y is a molecula swi ch in
PINK1and PARK2/PARKIN-dependen mi ochond ial clea ance. Au ophagy
9:1801 –1817
Bogenhagen DF (2012) Mi ochond ial DNA nucleoid s uc u e. Biochim
Biophys Ac a 1819:914 –920
Bonnen PE, Ya ham JW, Besse A, Wu P, Faqeih EA, Al-Asma i AM, Saleh
MA, Eyaid W, Hadeel A, He L, Smi h F, Yau S, Simcox EM, Miwa S,
Don i T, Abu-Ame o KK, Wong LJ, C aigen WJ, G aham BH, Sco KL
e al (2013) Mu a ions in FBXL4cause mi ochond ial encephalopa hy and
a diso de o mi ochond ial DNA main enance. Am J Hum Gene 93:
471 –481
Bou os M, Kige AA, A mknech S, Ke K, Hild M, Koch B, Haas SA, Pa o
R, Pe imon N; Heidelbe g Fly A ay Conso ium (2004) Genome-wide
RNAi analysis o g ow h and iabili y in D osophila cells. Science 303:
832 –835
Bozza WP, Zhuang Z (2011)Biochemical cha ac e iza ion o a mul idomain
deubiqui ina ing enzyme Ubp15 and he egula o y ole o i s e minal
domains. Biochemis y 50:6423 –6432
B a ic A, La sson NG (2013) The ole o mi ochond ia in aging. J Clin In es
123:951 –957
B uni F, G amegna P, Oli ei a JM, Ligh owle s RN, Ch zanowska-Ligh owle s
ZM (2013) REXO2is an oligo ibonuclease ac i e in human mi ochond ia.
PLoS ONE 8:e64670
Bu ge s PM, S i h CM, Yode BL, Spa ks JL (2010) Yeas exonuclease 5is
essen ial o mi ochond ial genome main enance. Mol Cell Biol 30:
1457 –1466
Campbell CT, Kolesa JE, Kau man BA (2012) Mi ochond ial ansc ip ion
ac o A egula es mi ochond ial ansc ip ion ini ia ion, DNA packaging,
and genome copy numbe . Biochim Biophys Ac a 1819:921 –929
Cannino G, El-Khou y R, Pi inen M, Hu z B, Rus in P, Jacobs HT, Du ou E
(2012) Glucose modula es espi a o y complex I ac i i y in esponse o
acu e mi ochond ial dys unc ion. J Biol Chem 287:38729 –38740
Ca e R, D ouin G (2010) The inc ease in he numbe o subuni s in
euka yo ic RNA polyme ase III ela i e o RNA polyme ase II is due o he
pe manen ec ui men o gene al ansc ip ion ac o s. Mol Biol E ol 27:
1035 –1043
Cela do I, Ma ins LM, Gandhi S (2014) Un a elling mi ochond ial pa hways
o Pa kinson’s disease. B J Pha macol 171:1943 –1957
Ce i elli SM, F olo a EG, Feng C, G inbe g A, Lo e PE, C ouch RJ (2003)
Failu e o p oduce mi ochond ial DNA esul s in emb yonic le hali y in
Rnaseh1null mice. Mol Cell 11:807 –815
Chen TC, Hung YC, Lin TY, Chang HW, Chiang IP, Chen YY, Chow KC (2011)
Human papilloma i us in ec ion and exp ession o ATPase amily AAA
domain con aining 3A, a no el an i-au ophagy ac o , in u e ine ce ical
cance . In J Mol Med 28:689 –696
Chen YJ, S e ens TH (1996) The VPS8gene is equi ed o localiza ion and
a icking o he CPY so ing ecep o in Saccha omyces ce e isiae.Eu J
Cell Biol 70:289 –297
Chujo T, Ohi a T, Sakaguchi Y, Goshima N, Nomu a N, Nagao A, Suzuki T
(2012) LRPPRC/SLIRP supp esses PNPase-media ed mRNA decay and
p omo es polyadenyla ion in human mi ochond ia. Nucleic Acids Res 40:
8033 –8047
Ciesielski GL, Plo ka M, Manicki M, Schilke BA, Du kiewicz R, Sahi C,
Ma szalek J, C aig EA (2013) Nucleoid localiza ion o Hsp40 Mdj1is
impo an o i s unc ion in main enance o mi ochond ial DNA. Biochim
Biophys Ac a 1833:2233 –2243
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
17
Published online: June 21, 2014
Cla k-Walke GD, Hansb o PM, Gibson F, Chen XJ (2000) Mu an esidues
supp essing ho(0)-le hali y in Kluy e omyces lac is occu a con ac si es
be ween subuni s o F(1)-ATPase. Biochim Biophys Ac a 1478:125 –137
Clay on DA (1982) Replica ion o animal mi ochond ial DNA. Cell 28:693 –705
Clemens JC, Wo by CA, Simonson-Le N, Muda M, Maehama T, Hemmings
BA, Dixon JE (2000) Use o double-s anded RNA in e e ence in D osophila
cell lines o dissec signal ansduc ion pa hways. P oc Na l Acad Sci USA
97:6499 –6503
Cohen MM, Lebouche GP, Li na -Le anon N, Glickman MH, Weissman AM
(2008) Ubiqui in-P o easome-dependen deg ada ion o a mi o usin, a
c i ical egula o o mi ochond ial usion. Mol Biol Cell 19:2457 –2464
Con amine V, Pica d M (2000) Main enance and in eg i y o he
mi ochond ial genome: a ple ho a o nuclea genes in he budding yeas .
Mic obiol Mol Biol Re 64:281 –315
Copeland WC (2012) De ec s in mi ochond ial DNA eplica ion and human
disease. C i Re Biochem Mol Biol 47:64 –74
C ame P, A mache KJ, Baumli S, Benke F, B ueckne F, Buchen C, Damsma
GE, Dengl S, Geige SR, Jasiak AJ, Jawha i A, Jennebach S, Kamenski T,
Ke enbe ge H, Kuhn CD, Lehmann E, Leike K, Sydow JF, Vannini A (2008)
S uc u e o euka yo ic RNA polyme ases. Annu Re Biophys 37:337 –352
Dai DF, Chen T, Wanaga J, La lamme M, Ma cinek DJ, Emond MJ, Ngo CP,
P olla TA, Rabino i ch PS (2010) Age-dependen ca diomyopa hy in
mi ochond ial mu a o mice is a enua ed by o e exp ession o ca alase
a ge ed o mi ochond ia. Aging Cell 9:536 –544
Dasche C, Balch WE (1996) Mammalian Sly1 egula es syn axin 5 unc ion in
endoplasmic e iculum o Golgi anspo . J Biol Chem 271:15866 –15869
Dasche C, Ossig R, Gallwi z D, Schmi HD (1991) Iden i ica ion and
s uc u e o ou yeas genes (SLY) ha a e able o supp ess he
unc ional loss o YPT1, a membe o he RAS supe amily. Mol Cell Biol 11:
872 –885
Da ies KM, Anselmi C, Wi ig I, Fa aldo-Gómez JD, Kühlb and W (2012)
S uc u e o he yeas F1Fo-ATP syn hase dime and i s ole in shaping
he mi ochond ial c is ae. P oc Na l Acad Sci USA 109:13602 –13607
Debelyy MO, Pla a HW, Sa ian D, Hensel A, Thoms S, Meye HE, Wa scheid
B, Gi zalsky W, E dmann R (2011) Ubp15p, a ubiqui in hyd olase
associa ed wi h he pe oxisomal expo machine y. J Biol Chem 286:
28223 –28234
Dong Z, Bell LR (1999) SIN, a no el D osophila p o ein ha associa es wi h
he RNA binding p o ein sex-le hal. Gene 237:421 –428
Dunn CD, Jensen RE (2003) Supp ession o a de ec in mi ochond ial p o ein
impo iden i ies cy osolic p o eins equi ed o iabili y o yeas cells
lacking mi ochond ial DNA. Gene ics 165:35 –45
Du ezin-Caube S, Rak M, Le eb e-Legend e L, Te aud E, Bonne oy N, di
Rago JP (2006)A“pe i e obliga e”mu an o Saccha omyces ce e isiae:
unc ional m DNA is le hal in cells lacking he del a subuni o
mi ochond ial F1-ATPase. J Biol Chem 281:16305 –16313
Duxin JP, Dao B, Ma insson P, Rajala N, Gui a L, Campbell JL, Spelb ink JN,
S ewa SA (2009) Human Dna2is a nuclea and mi ochond ial DNA
main enance p o ein. Mol Cell Biol 29:4274 –4282
Ehses S, Raschke I, Mancuso G, Be nacchia A, Geime S, Tonde a D, Ma inou
JC, Wes e mann B, Ruga li EI, Lange T (2009) Regula ion o OPA1
p ocessing and mi ochond ial usion by m-AAA p o ease isoenzymes and
OMA1.J Cell Biol 187:1023 –1036
Elachou i G, Vidoni S, Zanna C, Pa yn A, Boukhaddaoui H, Gage K,
Yu-Wai-Man P, Gaspa e G, Sa zi E, Dele e C, Olichon A, Loiseau D,
Reynie P, Chinne y PF, Ro ig A, Ca elli V, Hamel CP, Rugolo M, Lenae s G
(2011) OPA1links human mi ochond ial genome main enance o m DNA
eplica ion and dis ibu ion. Genome Res 21:12 –20
Faulkne KM, Lioche SI, F ido ich I (1994) S able Mn(III) po phy ins mimic
supe oxide dismu ase in i o and subs i u e o i in i o.J Biol Chem 269:
23471 –23476
Fe nandez-Ayala DJ, Sanz A, Va iainen S, Kemppainen KK, Babusiak M,
Mus alah i E, Cos a R, Tuomela T, Ze iani M, Chung J, O’Dell KM, Rus in P,
Jacobs HT (2009) Exp ession o he Ciona in es inalis al e na i e oxidase
(AOX) in D osophila complemen s de ec s in mi ochond ial oxida i e
phospho yla ion. Cell Me ab 9:449 –460
F ezza C, Cipola S, Ma ins de B i o O, Mica oni M, Beznoussenko GV, Rudka
T, Ba oli D, Polishuck RS, Danial NN, De S oope B, Sco ano L (2006)
OPA1con ols apop o ic c is ae emodeling independen ly om
mi ochond ial usion. Cell 126:177 –189
Fus e JM, Wan ooij S, Jem E, G anycome CE, Clue TJ, Shi Y, A anasso a N,
Hol IJ, Gus a sson CM, Falkenbe g M (2010) Mi ochond ial RNA
polyme ase is needed o ac i a ion o he o igin o ligh -s and DNA
eplica ion. Mol Cell 37:67 –78
Gao Y, Ka yal S, Lee Y, Zhao J, Rehg JE, Russell HR, McKinnon PJ (2011) DNA
ligase III is c i ical o m DNA in eg i y bu no X cc1-media ed nuclea
DNA epai . Na u e 471:240 –244
Ga cía-Gómez S, Reyes A, Ma ínez-Jiménez MI, Choc ón ES, Mou ón S,
Te ados G, Powell C, Salido E, Méndez J, Hol IJ, Blanco L (2013) P imPol,
an a chaic p imase/polyme ase ope a ing in human cells. Mol Cell 52:
541 –553
Gilquin B, Taillebou g E, Che adi N, Hubs enbe ge A, Gay O, Me le N, Assa d
N, Fau a que MO, Tomohi o S, Kuge O, Baudie J (2010) The AAA+ATPase
ATAD3Acon ols mi ochond ial dynamics a he in e ace o he inne and
ou e memb anes. Mol Cell Biol 30:1984 –1996
Gi aud MF, Velou s J (1997) The absence o he mi ochond ial ATP syn hase
del a subuni p omo es a slow g ow h pheno ype o ho- yeas cells by a
lack o assembly o he ca aly ic sec o F1.Eu J Biochem 245:813 –818
Gomes AP, P ice NL, Ling AJ, Moslehi JJ, Mon gome y MK, Rajman L, Whi e JP,
Teodo o JS, W ann CD, Hubba d BP, Me cken EM, Palmei a CM, de Cabo
R, Rolo AP, Tu ne N, Bell EL, Sinclai DA (2013) Declining NAD(+) induces
a pseudohypoxic s a e dis up ing nuclea -mi ochond ial communica ion
du ing aging. Cell 155:1624 –1638
Go o A, Ma sushima Y, Kadowaki T, Ki agawa Y (2001) D osophila
mi ochond ial ansc ip ion ac o A (d-TFAM) is dispensable o he
ansc ip ion o mi ochond ial DNA in Kc167 cells. Biochem J 354:243 –248
Guan X, Middleb ooks BW, Alexande S, Wasse man SA (2006) Mu a ion o
TweedleD, a membe o an uncon en ional cu icle p o ein amily, al e s
body shape in D osophila.P oc Na l Acad Sci USA 103:16794 –16799
Han Y, Chen JZ (2013) Oxida i e s ess induces mi ochond ial DNA damage
and cy o oxici y h ough independen mechanisms in human cance cells.
Biomed Res In 2013:825065
Hay JC, Chao DS, Kuo CS, Schelle RH (1997) P o ein in e ac ions egula ing
esicle anspo be ween he endoplasmic e iculum and Golgi appa a us
in mammalian cells. Cell 89:149 –158
He J, Coope HM, Reyes A, Di Re M, Sembongi H, Li win TR, Gao J, Neuman
KC, Fea nley IM, Spinazzola A, Walke JE, Hol IJ (2012) Mi ochond ial
nucleoid in e ac ing p o eins suppo mi ochond ial p o ein syn hesis.
Nucleic Acids Res 40:6109 –6121
He J, Mao CC, Reyes A, Sembongi H, Di Re M, G anycome C, Clippingdale AB,
Fea nley IM, Ha bou M, Robinson AJ, Reichel S, Spelb ink JN, Walke JE,
Hol IJ (2007) The AAA+p o ein ATAD3has displacemen loop binding
p ope ies and is in ol ed in mi ochond ial nucleoid o ganiza ion. J Cell
Biol 176:141 –146
Hensen F, Cansiz S, Ge hold JM, Spelb ink JN (2014) To be o no o be a
nucleoid p o ein: a compa ison o mass-spec ome y based app oaches in
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
18
Published online: June 21, 2014
he iden i ica ion o po en ial m DNA-nucleoid associa ed p o eins.
Biochimie 100:219 –226
Heo JM, Li na -Le anon N, Taylo EB, Jones KT, Dephou e N, Ring J, Xie J,
B odsky JL, Madeo F, Gygi SP, Ash a i K, Glickman MH, Ru e J (2010)A
s ess- esponsi e sys em o mi ochond ial p o ein deg ada ion. Mol Cell
40:465 –480
He lan M, Vogel F, Bo nho d C, Neupe W, Reiche AS (2003) P ocessing o
Mgm1by he homboid- ype p o ease Pcp1is equi ed o main enance
o mi ochond ial mo phology and o mi ochond ial DNA. J Biol Chem 278:
27781 –27788
Ho mann M, Honnen S, Maya epek E, Wä jen W, Koopman WJ, Bossinge O,
Dis elmaie F (2012) MICS-1in e ac s wi h mi ochond ial ATAD-3and
modula es li espan in C. elegans.Exp Ge on ol 47:270 –275
Ho azdo sky BF, Cowles CR, Mus ol P, Holmes M, Em SD (1996) A no el
RING inge p o ein, Vps8p, unc ionally in e ac s wi h he small GTPase,
Vps21p, o acili a e soluble acuola p o ein localiza ion. J Biol Chem 271:
33607 –33615
Hou D, Ruiz M, And ulis ED (2012) The ibonuclease Dis3is an essen ial
egula o o he de elopmen al ansc ip ome. BMC Genomics 13:359
Hou koope RH, Mouchi oud L, Ryu D, Moullan N, Ka syuba E, Kno G,
Williams RW, Auwe x J (2013) Mi onuclea p o ein imbalance as a
conse ed longe i y mechanism. Na u e 497:451 –457
Hu P, Wu S, Sun Y, Yuan CC, Kobayashi R, Mye s MP, He nandez N (2002)
Cha ac e iza ion o human RNA polyme ase III iden i ies o hologues o
Saccha omyces ce e isiae RNA polyme ase III subuni s. Mol Cell Biol 22:
8044 –8055
Huang KH, Chow KC, Chang HW, Lin TY, Lee MC (2011) ATPase
amily AAA domain con aining 3Ais an an i-apop o ic ac o and
a sec e ion egula o o PSA in p os a e cance . In J Mol Med 28:
9–15
Humph ey DM, Pa sons RB, Ludlow ZN, Riemenspe ge T, Esposi o G,
Ve s eken P, Jacobs HT, Bi man S, Hi h F (2012) Al e na i e oxidase
escues mi ochond ia-media ed dopamine gic cell loss in D osophila. Hum
Mol Gene 21:2698 –2712
Hwang GW, Kimu a Y, Takahashi T, Lee JY, Naganuma A (2012) Iden i ica ion
o deubiqui ina ing enzymes in ol ed in me hylme cu y oxici y in
Saccha omyces ce e isiae.J Toxicol Sci 37:1287 –1290
I o JK, B ai hwai e DK (1991) Compila ion and alignmen o DNA polyme ase
sequences. Nucleic Acids Res 19:4045 –4057
Iyenga B, Luo N, Fa CL, Kaguni LS, Campos AR (2002) The accesso y
subuni o DNA polyme ase gamma is essen ial o mi ochond ial DNA
main enance and de elopmen in D osophila melanogas e .P oc Na l Acad
Sci USA 99:4483 –4488
Jõe s P, Lewis SC, Fukuoh A, Pa hiala M, Ellilä S, Hol IJ, Jacobs HT (2013)
Mi ochond ial ansc ip ion e mina o amily membe s mTTF and mTe 5
ha e opposing oles in coo dina ion o m DNA syn hesis. PLoS Gene 9:
e1003800
Johnson ML, Nagengas AA, Salz HK (2010) PPS, a la ge mul idomain p o ein,
unc ions wi h sex-le hal o egula e al e na i e splicing in D osophila.
PLoS Gene 6:e1000872
Jones BA, Fangman WL (1992) Mi ochond ial DNA main enance in yeas
equi es a p o ein con aining a egion ela ed o he GTP-binding domain
o dynamin. Genes De 6:380 –389
Joseph AM, Adhihe y PJ, Bu o d TW, Wohlgemu h SE, Lees HA, Nguyen LM,
A anda JM, Sandesa a BD, Paho M, Manini TM, Ma ze i E, Leeuwenbu gh
C(2012) The impac o aging on mi ochond ial unc ion and biogenesis
pa hways in skele al muscle o seden a y high- and low- unc ioning
elde ly indi iduals. Aging Cell 11:801 –809
Kaguni LS (2004) DNA polyme ase gamma, he mi ochond ial eplicase. Annu
Re Biochem 73:293 –320
Kang D, Hamasaki N (2005) Mi ochond ial ansc ip ion ac o A in he
main enance o mi ochond ial DNA: o e iew o i s mul iple oles. Ann NY
Acad Sci 1042:101 –108
Kanga S, Be na d D, Mage -Heckel AM, E papazoglou Z, Ma i oli F, Sixma TK,
Léon S, U ban-G imal D, Ta asso I, Haguenaue -Tsapis R (2012)A
deubiqui yla ing complex equi ed o neosyn hesis o a yeas
mi ochond ial ATP syn hase subuni . PLoS ONE 7:e38071
Ka niely S, Pines O (2005) Single ansla ion–dual des ina ion: mechanisms o
dual p o ein a ge ing in euka yo es. EMBO Rep 6:420 –425
Kassa e is GA, P akash P, Shim E (2010) The C53/C37 subcomplex o RNA
polyme ase III lies nea he ac i e si e and pa icipa es in p omo e
opening. J Biol Chem 285:2695 –2706
Kazak L, Reyes A, He J, Wood SR, B ea-Cal o G, Holen TT, Hol IJ (2013)A
c yp ic a ge ing signal c ea es a mi ochond ial FEN1iso o m wi h ailed
R-Loop binding p ope ies. PLoS ONE 8:e62340
Klaes A, Menne T, S ollewe k A, Scholz H, Klämb C (1994) The E s
ansc ip ion ac o s encoded by he D osophila gene poin ed di ec glial
cell di e en ia ion in he emb yonic CNS. Cell 78:149 –160
Kleino A, Valanne S, Ul ila J, Kallio J, Myllymäki H, Enwald H, S ö en S,
Poide in M, Ueda R, Hul ma k D, Lemai e B, Räme M (2005) Inhibi o o
apop osis 2and TAK1-binding p o ein a e componen s o he D osophila
Imd pa hway. EMBO J 24:3423 –3434
Koonin EV, Deu sche MP (1993) RNase T sha es conse ed sequence mo i s
wi h DNA p oo eading exonucleases. Nucleic Acids Res 21:2521 –2522
Ko nblum C, Nicholls TJ, Haack TB, Schöle S, Pee a V, Danhause K,
Hallmann K, Zsu ka G, Ro bach J, Iuso A, Wieland T, Sciacco M, Ronchi D,
Comi GP, Moggio M, Quinzii CM, DiMau o S, Cal o SE, Moo ha VK,
Klops ock T e al (2013) Loss-o - unc ion mu a ions in MGME1impai
m DNA eplica ion and cause mul isys emic mi ochond ial disease. Na
Gene 45:214 –219
Kushna e a YE, Ge encse AA, Bossy B, Ju WK, Whi e AD, Waggone J,
Ellisman MH, Pe kins G, Bossy-We zel E (2013) Loss o OPA1dis u bs
cellula calcium homeos asis and sensi izes o exci o oxici y. Cell Dea h
Di e 20:353 –365
Lai-Zhang J, Xiao Y, Muelle DM (1999) Epis a ic in e ac ions o dele ion
mu an s in he genes encoding he F1-ATPase in yeas Saccha omyces
ce e isiae.EMBO J 18:58 –64
Land ieux E, Alic N, Duc o C, Acke J, Ri a M, Ca les C (2006) A subcomplex
o RNA polyme ase III subuni s in ol ed in ansc ip ion e mina ion and
eini ia ion. EMBO J 25:118 –128
La osche I, Le é on P, Be son A, F omen y B, Huang TT, Mo eau R,
Pessay e D, Mansou i A (2010) Hepa ic mi ochond ial DNA deple ion
a e an alcohol binge in mice: p obable ole o pe oxyni i e and
modula ion by manganese supe oxide dismu ase. J Pha macol Exp The
332:886 –897
La sson NG, Wang J, Wilhelmsson H, Old o s A, Rus in P, Lewandoski M,
Ba sh GS, Clay on DA (1998) Mi ochond ial ansc ip ion ac o A is
necessa y o m DNA main enance and emb yogenesis in mice. Na Gene
18:231 –236
Le eb e-Legend e L, Balgue ie A, Du ezin-Caube S, Gi aud MF, Slonimski PP,
Di Rago JP (2003)F1-ca alysed ATP hyd olysis is equi ed o
mi ochond ial biogenesis in Saccha omyces ce e isiae g owing unde
condi ions whe e i canno espi e. Mol Mic obiol 47:1329 –1339
Lui TT, Lac oix C, Ahmed SM, Goldenbe g SJ, Leach CA, Daula AM, Ange s S
(2011) The ubiqui in-speci ic p o ease USP34 egula es axin s abili y and
Wn /b-ca enin signaling. Mol Cell Biol 31:2053 –2065
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
19
Published online: June 21, 2014
Maie D, Fa CL, Poeck B, Alaha i A, Vogel M, Fische S, Kaguni LS, Schneuwly
S(2001) Mi ochond ial single-s anded DNA-binding p o ein is equi ed
o mi ochond ial DNA eplica ion and de elopmen in D osophila
melanogas e .Mol Biol Cell 12:821 –830
Malc E, Dzie zbicki P, Kaniak A, Skoneczna A, Ciesla Z (2009) Inac i a ion o
he 20S p o easome ma u ase, Ump1p, leads o he ins abili y o m DNA
in Saccha omyces ce e isiae. Mu a Res 669:95 –103
Mamolen MC (2010)D osophila melanogas e Dis 3is dynamic endo- and
30-50exo ibonuclease. PhD hesis, Case Wes e n Rese e Uni e si y;
pe malink h p:// a e.ohiolink.edu/e dc/ iew?acc_num=case1278525341
Ma ginean u DH, Eme son CB, Diaz D, Hockenbe y DM (2007) Hsp90
inhibi ion dec eases mi ochond ial p o ein u no e . PLoS ONE 2:e1066
Ma inelli P, Ruga li EI (2007) Eme ging oles o mi ochond ial p o eases in
neu odegene a ion. Biochim Biophys Ac a 1797:1–10
Ma sushima Y, Ga esse R, Kaguni LS (2004)D osophila mi ochond ial
ansc ip ion ac o B2 egula es mi ochond ial DNA copy numbe and
ansc ip ion in Schneide cells. J Biol Chem 279:26900 –26905
Ma sushima Y, Go o Y, Kaguni LS (2010) Mi ochond ial Lon p o ease
egula es mi ochond ial DNA copy numbe and ansc ip ion by selec i e
deg ada ion o mi ochond ial ansc ip ion ac o A (TFAM). P oc Na l Acad
Sci USA 107:18410 –18415
McDe mo -Roe C, Ye J, Ahmed R, Sun XM, Se a ín A, Wa e J, Bo olo L,
Mucke P, Cañas X, Zhang J, Rowe GC, Buchan R, Lu H, B ai hwai e A,
Mancini M, Hau on D, Ma í R, Ga cía-A umí E, Hubne N, Jacob H e al
(2011) Endonuclease G is a no el de e minan o ca diac hype ophy and
mi ochond ial unc ion. Na u e 478:114 –118
McKinney EA, Oli ei a MT (2013) Replica ing animal mi ochond ial DNA.
Gene Mol Biol 36:308 –315
McLelland GL, Soubannie V, Chen CX, McB ide HM, Fon EA (2014) Pa kin and
PINK1 unc ion in a esicula a icking pa hway egula ing mi ochond ial
quali y con ol. EMBO J 33:282 –295
Meeusen S, DeVay R, Block J, Cassidy-S one A, Wayson S, McCa e y JM, Nunna i
J(2006) Mi ochond ial inne -memb ane usion and c is a main enance
equi es he dynamin- ela ed GTPase Mgm1.Cell 127:383 –395
Mo eno-Loshue os R, Acín-Pé ez R, Fe nández-Sil a P, Mo illa N,
Pé ez-Ma os A, Rod iguez de Co doba S, Galla do ME, En íquez JA (2006)
Di e ences in eac i e oxygen species p oduc ion explain he pheno ypes
associa ed wi h common mouse mi ochond ial DNA a ian s. Na Gene
38:1261 –1268
Mo eno-Loshue os R, Fe ín G, Acín-Pé ez R, Galla do ME, Viscomi C,
Pé ez-Ma os A, Ze iani M, Fe nández-Sil a P, En íquez JA (2011)
E olu ion mee s disease: pene ance and unc ional epis asis o
mi ochond ial RNA mu a ions. PLoS Gene 7:e1001379
Mo imo o AM, Jo dan KC, Tie ze K, B i on JS, O’Neill EM, Ruohola-Bake H
(1996) Poin ed, an ETS domain ansc ip ion ac o , nega i ely egula es
he EGF ecep o pa hway in D osophila oogenesis. De elopmen 122:
3745 –3754
Nagakawa Y, Williams GM, Zheng Q, Tsuchida A, Aoki T, Mon gome y RA,
Klein AS, Sun Z (2005) Oxida i e mi ochond ial DNA damage and dele ion
in hepa ocy es o ejec ing li e allog a s in a s: ole o TNF-a.
Hepa ology 42:208 –215
Na end a D, Walke JE, Youle R (2012) Mi ochond ial quali y con ol
media ed by PINK1and Pa kin: links o pa kinsonism. Cold Sp ing Ha b
Pe spec Biol 4: pii: a011338 .
Neu eld TP (2012) Au ophagy and cell g ow h – he yin and yang o nu ien
esponses. J Cell Sci 125:1–10
Newman AP, Shim J, Fe o-No ick S (1990) BET1, BOS1, and SEC22 a e
membe s o a g oup o in e ac ing yeas genes equi ed o anspo
om he endoplasmic e iculum o he Golgi complex. Mol Cell Biol 10:
3405 –3414
Oli ei a MT, Ga esse R, Kaguni LS (2010) Animal models o mi ochond ial
DNA ansac ions in disease and ageing. Exp Ge on ol 45:489 –502
Poalas K, Ha chi EM, Co dei o N, Dubois SM, Leclai HM, Le eau C, Alexia C,
Ga a d J, Vazquez A, Bidè e N (2013) Nega i e egula ion o NF-jB
signaling in T lymphocy es by he ubiqui in-speci ic p o ease USP34.Cell
Commun Signal 11:25
Puebla-Oso io N, Lacey DB, Al FW, Zhu C (2006) Ea ly emb yonic le hali y
due o a ge ed inac i a ion o DNA ligase III. Mol Cell Biol 26:3935 –3941
Quinzii CM, Ga one C, Emmanuele V, Tadesse S, K ishna S, Do ado B, Hi ano
M(2013) Tissue-speci ic oxida i e s ess and loss o mi ochond ia in
CoQ-de icien Pdss2mu an mice. FASEB J 27:612 –621
Rahman M, Kyls en P (2011) Rhomboid-7o e -exp ession esul s in Opa1-like
p ocessing and mal unc ioning mi ochond ia. Biochem Biophys Res
Commun 414:315 –320
Räme M, Man uelli P, Pea son A, Ma hey-P e o B, Ezekowi z RA (2002)
Func ional genomic analysis o phagocy osis and iden i ica ion o a
D osophila ecep o o E. coli.Na u e 416:644 –648
Ranie i M, B ajko ic S, Riboldi G, Ronchi D, Rizzo F, B esolin N, Co i S, Comi
GP (2013) Mi ochond ial usion p o eins and human diseases. Neu ol Res
In 2013:293893
Reyes A, He J, Mao CC, Bailey LJ, Di Re M, Sembongi H, Kazak L, Dzionek K,
Holmes JB, Clue TJ, Ha bou ME, Fea nley IM, C ouch RJ, Con i MA,
Adels ein RS, Walke JE, Hol IJ (2011) Ac in and myosin con ibu e o
mammalian mi ochond ial DNA main enance. Nucleic Acids Res 39:
5098 –5108
Rich e U, Lah inen T, Ma inen P, Myöhänen M, G eco D, Cannino G,
Jacobs HT, Lie zén N, Nyman TA, Ba e sby BJ (2013) A mi ochond ial
ibosomal and RNA decay pa hway blocks cell p oli e a ion. Cu Biol 23:
535 –541
Robe i M, B uni F, Polosa PL, Gadale a MN, Can a o e P (2006) The
D osophila e mina ion ac o DmTTF egula es in i o mi ochond ial
ansc ip ion. Nucleic Acids Res 34:2109 –2116
Robinson BH, Pe o a-Benedic R, Buncic JR, Wallace DC (1992) Non iabili y
o cells wi h oxida i e de ec s in galac ose medium: a sc eening es o
a ec ed pa ien ib oblas s. Biochem Med Me ab Biol 48:122 –126
Rone MB, Midzak AS, Issop L, Rammouz G, Jaganna han S, Fan J, Ye X,
Blonde J, Veens a T, Papadopoulos V (2012) Iden i ica ion o a dynamic
mi ochond ial p o ein complex d i ing choles e ol impo , a icking, and
me abolism o s e oid ho mones. Mol Endoc inol 26:1868 –1882
Rö ig A, Poul on J (2009) Gene ic causes o mi ochond ial DNA deple ion in
humans. Biochim Biophys Ac a 1792:1103 –1108
Ruhanen H, Ushako K, Yasukawa T (2011) In ol emen o DNA ligase III and
ibonuclease H1in mi ochond ial DNA eplica ion in cul u ed human
cells. Biochim Biophys Ac a 1813:2000 –2007
Saada A (2004) Deoxy ibonucleo ides and diso de s o mi ochond ial DNA
in eg i y. DNA Cell Biol 23:797 –806
Sa ig O, Goldshe D, Nousbeck J, Fuchs-Telem D, Cohen-Ka senelson K, Iancu
TC, Mano I, Saada A, Sp eche E, Mandel H (2013) In an ile mi ochond ial
hepa opa hy is a ca dinal ea u e o MEGDEL synd ome
(3-me hylglu aconic acidu ia ype IV wi h senso ineu al dea ness,
encephalopa hy and Leigh-like synd ome) caused by no el mu a ions in
SERAC1.Am J Med Gene A 161:2204 –2215
Sasa man F, B unel-Gui on C, An onicka H, Wai T, Shoub idge EA; LSFC
Conso ium (2010) LRPPRC and SLIRP in e ac in a ibonucleop o ein
complex ha egula es pos ansc ip ional gene exp ession in
mi ochond ia. Mol Biol Cell 21:1315 –1323
Molecula Sys ems Biology 10:734 |2014 ª2014 The Au ho s
Molecula Sys ems Biology m DNA main enance and ATP syn hase A sushi Fukuoh e al
20
Published online: June 21, 2014
Schmid O, Ha baue AB, Rao S, Ey ich B, Zahedi RP, S ojano ski D, Schön isch
B, Guia d B, Sickmann A, P anne N, Meisinge C (2011) Regula ion o
mi ochond ial p o ein impo by cy osolic kinases. Cell 144:227 –239
Schnau e A, Cla k-Walke GD, S einbe g AG, S ua K (2005) The F1-ATP
syn hase complex in bloods eam s age ypanosomes has an unusual
and essen ial unc ion. EMBO J 24:4029 –4040
Sesaki H, Sou ha d SM, Hobbs AE, Jensen RE (2003) Cells lacking Pcp1p/
Ugo2p, a homboid-like p o ease equi ed o Mgm1p p ocessing, lose
m DNA and mi ochond ial s uc u e in a Dnm1p-dependen manne , bu
emain compe en o mi ochond ial usion. Biochem Biophys Res Commun
308:276 –283
Shadel GS (2008) Exp ession and main enance o mi ochond ial DNA: new
insigh s in o human disease pa hology. Am J Pa hol 172:1445 –1456
Shio a T, Mabuchi H, Tanaka-Yamano S, Yamano K, Endo T (2011)In i o
p o ein-in e ac ion mapping o a mi ochond ial ansloca o p o ein
Tom22 a wo k. P oc Na l Acad Sci USA 108:15179 –15183
Shokolenko IN, Wilson GL, Alexeye MF (2013) Pe sis en damage induces
mi ochond ial DNA deg ada ion. DNA Repai (Ams ) 12:488 –499
Sogaa d M, Tani K, Ruby YR, Ge omanos S, Temps P, Ki chhausen T,
Ro hman JE, Sollne T (1994) A ab p o ein is equi ed o he assembly o
SNARE complexes in he docking o anspo esicles. Cell 78:937 –946
Song Z, Chen H, Fike M, Alexande C, Chan DC (2007) OPA1p ocessing
con ols mi ochond ial usion and is egula ed by mRNA splicing,
memb ane po en ial, and Yme1L. J Cell Biol 178:749 –755
Spelb ink JN, Li FY, Ti an i V, Nikali K, Yuan QP, Ta iq M, Wan ooij S, Ga ido
N, Comi G, Mo andi L, San o o L, Toscano A, Fab izi GM, Some H, C oxen
R, Beeson D, Poul on J, Suomalainen A, Jacobs HT, Ze iani M e al (2001)
Human mi ochond ial DNA dele ions associa ed wi h mu a ions in he
gene encoding Twinkle, a phage T7gene 4-like p o ein localized in
mi ochond ia. Na Gene 28:223 –231
Spelb ink JN (2010) Func ional o ganiza ion o mammalian mi ochond ial
DNA in nucleoids: his o y, ecen de elopmen s, and u u e challenges.
IUBMB Li e 62:19 –32
Spinazzola A, Viscomi C, Fe nandez-Viza a E, Ca a a F, D’Adamo P, Cal o S,
Ma sano RM, Donnini C, Weihe H, S isciuglio P, Pa ini R, Sa zi E, Chan A,
DiMau o S, Rö ig A, Gaspa ini P, Fe e o I, Moo ha VK, Ti an i V, Ze iani M
(2006) MPV17 encodes an inne mi ochond ial memb ane p o ein and is
mu a ed in in an ile hepa ic mi ochond ial DNA deple ion. Na Gene 38:
570 –575
S o ie B, A a di G (1972) Exp ession o he mi ochond ial genome in HeLa
cells. 13. E ec o selec i e inhibi ion o cy oplasmic o mi ochond ial
p o ein syn hesis on mi ochond ial nucleic acid syn hesis. J Mol Biol 71:
177 –199
Suzuki Y, Holmes JB, Ce i elli SM, Sakhuja K, Minczuk M, Hol IJ, C ouch RJ
(2010) An ups eam open eading ame and he con ex o he wo AUG
codons a ec he abundance o mi ochond ial and nuclea RNase H1.Mol
Cell Biol 30:5123 –5134
Sy SM, Jiang J, O WS, Deng Y, Huen MS (2013) The ubiqui in speci ic p o ease
USP34 p omo es ubiqui in signaling a DNA double-s and b eaks. Nucleic
Acids Res 41:8572 –8580
Syme sky J, Osowski D, Wal e s DE, Muelle DM (2008) Oligomycin ames a
common d ug-binding si e in he ATP syn hase. P oc Na l Acad Sci USA
109:13961 –13965
Tamu a K, Miya a K, Sugaha a K, Onishi S, Shuin T, Aso T (2003) Iden i ica ion
o EloA-BP1, a no el elongin A binding p o ein wi h an exonuclease
homology domain. Biochem Biophys Res Commun 309:189 –195
Tann AW, Boldogh I, Meiss G, Qian W, Van Hou en B, Mi a S, Szczesny B
(2011) Apop osis induced by pe sis en single-s and b eaks in
mi ochond ial genome: c i ical ole o EXOG (50-EXO/endonuclease) in
hei epai . J Biol Chem 286:31975 –31983
Tomecki R, K is iansen MS, Lykke-Abde sen S, Chlebowski A, La ssen KM,
Szczesny RJ, D azkowska K, Pas ula A, Ande sen JS, S epien PP,
Dziembowski A, Jensen TH (2010) The human co e exosome in e ac s wi h
di e en ially localized p ocessi e RNases: hDIS3and hDIS3L. EMBO J 29:
2342 –2357
T i uno ic A, Hansson A, W edenbe g A, Ro io AT, Du ou E, Kh o os o I,
Spelb ink JN, Wibom R, Jacobs HT, La sson NG (2005) Soma ic m DNA
mu a ions cause aging pheno ypes wi hou a ec ing eac i e oxygen
species p oduc ion. P oc Na l Acad Sci USA 102:17993 –17998
Tu ne CJ, G anycome C, Hu s R, Pohle E, Juhola MK, Juhola MI, Jacobs HT,
Su he land L, Hol IJ (2005) Sys ema ic seg ega ion o mu an
mi ochond ial DNA and accompanying loss o mi ochond ial DNA in
human NT2 e a oca cinoma cyb ids. Gene ics 170:1879 –1885
Vad o N, Ghanem S, B au F, Ga ilescu L, Pila d N, Mansou i A, Mo eau R,
Reyl-Desma s F (2012) Mi ochond ial DNA main enance is egula ed in
human hepa oma cells by glycogen syn hase kinase 3band p53 in
esponse o umo nec osis ac o a.PLoS ONE 7:e40879
Ve gani L, Rossi R, B ie ley CH, Hanna M, Hol IJ (1999) In oduc ion o
he e oplasmic mi ochond ial DNA (m DNA) om a pa ien wi h NARP in o
wo human ho deg ees cell lines is associa ed ei he wi h selec ion and
main enance o NARP mu an m DNA o ailu e o main ain m DNA. Hum
Mol Gene 8:1751 –1755
Vielhabe S, Debska-Vielhabe G, Pee a V, Schoele S, Kudin AP, Minin I,
Sch eibe S, Dengle R, Kollewe K, Zusch a e W, Ko nblum C, Zsu ka G,
Kunz WS (2013) Mi o usin 2mu a ions a ec mi ochond ial unc ion by
mi ochond ial DNA deple ion. Ac a Neu opa hol 125:245 –256
Wang Y, Singh U, Muelle DM (2007) Mi ochond ial genome in eg i y
mu a ions uncouple he yeas Saccha omyces ce e isiae ATP syn hase. J
Biol Chem 282:8228 –8236
Wilkinson S, C o DR, O’P ey J, Meedendo p A, O’P ey M, Du ès C, Ryan KM
(2011) The cyclin-dependen kinase PITSLRE/CDK11 is equi ed o
success ul au ophagy. Au ophagy 7:1295 –1301
Wojewoda M, Duszy
nski J, Szczepanowska J (2010) An ioxidan de ence
sys ems and gene a ion o eac i e oxygen species in os eosa coma cells
wi h de ec i e mi ochond ia: e ec o selenium. Biochim Biophys Ac a
1797:890 –896
Wojewoda M, Duszy
nski J, Szczepanowska J (2011) NARP mu a ion and
m DNA deple ion igge mi ochond ial biogenesis which can be
modula ed by seleni e supplemen a ion. In J Biochem Cell Biol 43:
1178 –1186
Wong ED, Wagne JA, Go sich SW, McCa e y JM, Shaw JM, Nunna i J (2000)
The dynamin- ela ed GTPase, Mgm1p, is an in e memb ane space p o ein
equi ed o main enance o usion compe en mi ochond ia. J Cell Biol
151:341 –352
Ylikallio E, Suomalainen A (2012) Mechanisms o mi ochond ial diseases. Ann
Med 44:41 –59
Yonashi o R, Ishido S, Kyo S, Fukuda T, Go o E, Ma suki Y, Ohmu a-Hoshino
M, Sada K, Ho a H, Yamamu a H, Ina ome R, Yanagi S (2006) A no el
mi ochond ial ubiqui in ligase plays a c i ical ole in mi ochond ial
dynamics. EMBO J 25:3618 –3626
License: This is an open access a icle unde he
e ms o he C ea i e Commons A ibu ion 4.0
License, which pe mi s use, dis ibu ion and ep oduc-
ion in any medium, p o ided he o iginal wo k is
p ope ly ci ed.
ª2014 The Au ho s Molecula Sys ems Biology 10:734 |2014
A sushi Fukuoh e al m DNA main enance and ATP syn hase Molecula Sys ems Biology
21
Published online: June 21, 2014