Báo cáo khoa học: Complementation of coenzyme Q-deficient yeast by coenzyme Q analogues requires the isoprenoid side chain ppt

Complementation of coenzyme Q-deficient yeast by
coenzyme Q analogues requires the isoprenoid side chain
Andrew M. James
1
, Helena M. Cocheme
´
1,2
, Masatoshi Murai
3
, Hideto Miyoshi
3
and Michael P. Murphy
1
1 Medical Research Council Mitochondrial Biology Unit, Wellcome Trust ⁄ MRC Building, Cambridge, UK
2 Institute of Healthy Ageing and GEE, University College London, Darwin Building, London, UK
3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan
Introduction
CoenzymeQ (CoQ) is composed of a head group that
cycles between reduced ubiquinol and oxidized ubiqui-
none forms and a hydrophobic isoprenoid tail that
keeps the redox activity of the head group located
within the lipid bilayer. The length of the isoprenoid tail
varies between species, with Saccharomyces cerevisiae,
rats and humans predominantly synthesizing forms of
CoQ containing six (CoQ
6
), nine (CoQ
9
) and 10
(CoQ
10

) isoprenoid units, respectively. CoQ is synthe-
sized endogenously by a series of enzymes localized to
Keywords
coenzyme Q; diauxic shift; mitochondria;
ubiquinone; yeast
Correspondence
A. M. James, Medical Research Council
Mitochondrial Biology Unit, Wellcome
Trust ⁄ MRC Building, Hills Road, Cambridge
CB2 0XY, UK
Fax: +44 1223 252905
Tel: +44 1223 252903
E-mail:
Website:
(Received 18 December 2009, revised
2 February 2010, accepted 22 February
2010)
doi:10.1111/j.1742-4658.2010.07622.x
The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a
large, hydrophobic isoprenoid side chain. It functions in mitochondrial res-
piration as well as protecting membranes from oxidative damage. Yeast
that cannot synthesize CoQ (DCoQ) are viable, but cannot grow on nonfer-
mentable carbon sources, unless supplied with ubiquinone. Previously we
demonstrated that the isoprenoid side chain of the exogenous ubiquinone
was important for growth of a DCoQ strain on the nonfermentable sub-
strate glycerol [James AM et al. (2005) J Biol Chem 280, 21295–21312]. In
the present study we investigated the structural requirements of exoge-
nously supplied CoQ
2
for growth on glycerol and found that the first dou-

ble bond of the initial isoprenoid unit is essential for utilization of
respiratory substrates. As CoQ
2
analogues that did not complement growth
on glycerol supported respiration in isolated mitochondria, discrimination
does not occur via the respiratory chain complexes. The endogenous form
of CoQ in yeast (CoQ
6
) is extremely hydrophobic and transported to mito-
chondria via the endocytic pathway when supplied exogenously. We found
that CoQ
2
does not require this pathway when supplied exogenously and
the pathway is unlikely to be responsible for the structural discrimination
observed. Interestingly, decylQ, an analogue unable to support growth on
glycerol, is not toxic, but antagonizes growth of DCoQ yeast in the pres-
ence of exogenous CoQ
2
. Using a DCoQ double-knockout library we iden-
tified a number of genes that decrease the ability of yeast to grow on
exogenous CoQ. Here we suggest that CoQ or its redox state may be a sig-
nal for growth during the shift to respiration.
Abbreviations
CoQ, coenzyme Q with an isoprenoid side chain; CoQ
1–10
, coenzyme Q with a side chain of one to 10 isoprenoid units; DCoQ, yeast strains
lacking the ability to synthesize endogenous CoQ
6
; FCCP, carbonylcyanide-p-trifluoromethoxy-phenylhydrazone; I, fluorescence after the
addition of ubiquinone; I

0
, initial fluorescence; Isc1, inositol sphingolipid phospholipase C; PP2A, protein phosphatase 2A; Pyr16, 1-pyrene
hexadecanoic acid; YPD, yeast extract, peptone, glucose; YPG, yeast extract, peptone, glycerol; YPGG, yeast extract, peptone, glycerol with
glucose.
FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2067
the mitochondrial inner membrane, yet is a component
of lipid bilayers throughout the cell [1]. In addition to
its function as an electron carrier in the mitochondrial
inner membrane, the reduced ubiquinol form of CoQ
acts as a recyclable antioxidant that protects biological
membranes from oxidative damage [2]. CoQ
10
levels in
humans decrease in many pathological situations. For
this reason it has been used as a therapy in diseases
where oxidative damage is thought to be important, for
example very high doses of CoQ
10
have been used with
some beneficial effect in Parkinson’s disease [3]. As the
requirement for high doses probably results from the
extreme hydrophobicity of CoQ
10
and consequently its
low bioavailability, efforts have been made to improve
its water solubility by developing analogues with shorter
and more hydrophilic hydrocarbon tails [4,5].
In the yeast S. cerevisiae, CoQ is not essential for via-
bility, as strains lacking the ability to synthesize ubiqui-
none (DCoQ) grow by fermentation on glucose.

However, they do not grow on nonfermentable sub-
strates unless they are supplemented with exogenous
CoQ, in which case mitochondrial respiration is restored
[6]. It has generally been thought that the redox active
ubiquinone head group is the important moiety and that
the hydrophobic tail merely anchors this activity in the
membrane. Recently we have shown that when decylQ
and idebenone, two artificial analogues of CoQ contain-
ing a saturated 10-carbon alkane tail, are supplied
exogenously they are unable to restore growth on non-
fermentable substrates in DCoQ yeast [6]. As more
hydrophilic (CoQ
2
) or hydrophobic (CoQ
4
or CoQ
6
) iso-
prenoid analogues could restore growth on nonferment-
able substrates [6], it would appear that the inability of
decylQ to do this does not result from differences in pas-
sive diffusion to mitochondria in yeast. Instead, it proba-
bly arises from a selective protein interaction that can
differentiate between an isoprenoid and a saturated
alkane tail. This is important, as shorter alkyl ubiquinon-
es, such as idebenone and decylQ, are easier to synthesize
and have been used therapeutically [4,5]. Therefore, we
set out to ascertain the structural requirements for the
utilization of exogenous ubiquinone in yeast and to iden-
tify proteins that might be involved in discriminating

between alkane and isoprenoid ubiquinones.
Results
Complementation of cell growth in CoQ-deficient
yeast by short-chain CoQ analogues shows a
dramatic dependence on the isoprenoid side chain
Yeast strains that cannot synthesize CoQ endoge-
nously (DCoQ) are unable to grow on nonfermentable
substrates, such as glycerol. However, when CoQ is
supplied exogenously in the growth medium, the abil-
ity to grow on nonfermentable substrates is restored.
The utilization of glycerol as an energy source for
growth requires the presence of CoQ in the mitochon-
drial inner membrane to support respiration. That
DCoQ strains grow when supplemented with exoge-
nous CoQ indicates that some of the externally sup-
plied CoQ is reaching the mitochondrial inner
membrane within yeast.
Previous work showed that CoQ
2
and CoQ
6
sup-
ported growth when supplied exogenously to DCoQ
yeast [6]. However, not all short-chain ubiquinone ana-
logues could do this, as decylQ and idebenone, both of
which contain a 10-carbon saturated side chain, failed
to support growth on nonfermentable substrates once
glucose was depleted (Fig. 1A). This suggested that the
structure of the side chain was important for determin-
ing whether DCoQ strains were able to grow on exoge-

nously supplied ubiquinone. Each isoprenoid unit
contains a double bond and a methyl group that could
explain the differential reactivity. To identify the struc-
tural basis of this interaction we utilized a series of
ubiquinone analogues with varying similarities to
CoQ
2
(shown in Fig. 1B) [7,8]. The analogues differed
from CoQ
2
in a systematic way, with either the
removal of a double bond, the deletion of a methyl
group or the addition of a carbon atom. When we
investigated whether each analogue supported growth
in a DCoQ strain on the nonfermentable substrate
glycerol, a consensus structural pattern emerged
(Fig. 1C). Analogues in which the first (A1-Q
2
) or sec-
ond (A2-Q
2
) methyl group or second double bond
(A3-Q
2
) were removed exhibited normal growth in
glygerol-containing media, suggesting that none of
these is required for growth (Fig. 1C). This was in
stark contrast to the analogues in which the first dou-
ble bond was removed (A4-Q
2

and A6-Q
2
), as these
failed to promote any growth on nonfermentable sub-
strates in the DCoQ strain (Fig. 1C). The position of
the double bond was also important, as inserting an
extra carbon between the head group and the first dou-
ble bond (A5-Q
2
) abolished aerobic growth (Fig. 1C).
Therefore, the presence of a double bond between C2
and C3 in the first isoprenoid unit of exogenously sup-
plied CoQ is of critical importance for restoration of
growth in DCoQ yeast strains.
In summary, the high degree of selectivity for ubiq-
uinones containing a double bond between C2 and C3
in the first isoprenoid unit suggests the presence of a
specific interaction, presumably with a protein that is
able to recognize subtle structural differences in the
Yeast growth on exogenous ubiquinones A. M. James et al.
2068 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
side chain of CoQ. Therefore, we set out to try and
understand the nature of this interaction better.
All CoQ
2
analogues restore respiration in isolated
mitochondria from DCoQ yeast
Growth on glycerol requires CoQ to pass electrons
from glycerol-3-phosphate dehydrogenase to complex
III of the mitochondrial respiratory chain. Therefore,

we first considered the possibility that the different side
chains affected the way in which the ubiquinone ana-
logues interacted with the mitochondrial respiratory
chain. To assess this, we tested their ability to restore
respiration on glycerol-3-phosphate in isolated mito-
chondria (Fig. 2). All the analogues could do this with
equal efficacy (Fig. 2), suggesting that the inability of
decylQ and the analogues A4-Q
2
, A5-Q
2
and A6-Q
2
to
support growth on YPGG (yeast extract, peptone,
glycerol with glucose; Fig. 1C) does not result from a
failure of their mitochondria to pass electrons from
glycerol-3-phosphate to O
2
.
Short-chain ubiquinone analogues do not appear
to require an intracellular transport pathway
That DCoQ strains can grow on YPG (yeast extract,
peptone, glycerol) when supplemented with exogenous
CoQ indicates that a sufficient quantity of the supplied
CoQ is reaching the mitochondrial inner membrane.
As all of the analogues tested so far probably have
hydrophobicities similar to that of CoQ
2
and support

respiration to a comparable degree in isolated mito-
chondria (Fig. 2), it is perhaps surprising that they do
not all restore growth in intact cells (Fig. 1C). One
simple explanation would be that decylQ does not
reach the mitochondrial inner membrane within cells
in sufficient quantities to facilitate the passage of elec-
trons from glycerol to O
2
. This could arise via an
Time (h)
A
600
0
2
4
6
8
10
12
14
024487296
Wild-type
Dcoq2 + 50 µ
M CoQ
2
Dcoq2 + 50 µM decylQ
Dcoq2
0
2
4

6
8
10
A
600
DCoQ
CoQ
2
DecylQ
A1-Q
2
A4-Q
2
A5-Q
2
A6-Q
2
A3-Q
2
A2-Q
2
AB
O
O
MeO
MeO
A1-Q
2
O
O

MeO
MeO
A6-Q
2
O
O
MeO
MeO
A5-Q
2
O
O
MeO
MeO
A4-Q
2
O
O
MeO
MeO
A3-Q
2
O
O
MeO
MeO
A2-Q
2
O
O

MeO
MeO
DecylQ
O
O
MeO
MeO
CoQ
2
O
O
MeO
MeO
CoQ
6
C
Fig. 1. Structural requirements for exogenously supplied CoQ. (A) Growth curve of CENDcoq2 yeast in YPGG supplemented with CoQ
2
ana-
logues. Growth to A
600
 0.6 is glucose dependent, with growth above this requiring utilization of glycerol. (B) Structure of endogenous
CoQ
6
and several hydrophilic CoQ
2
analogues. The circles highlight structural differences in relation to the parent CoQ
2
molecule. (C) Growth
of CENDcoq2 yeast after 96 h in YPGG supplemented with 50 l

M of each CoQ
2
analogue. Growth to A
600
 0.6 is glucose dependent,
with growth above this requiring utilization of glycerol. Values are the mean ± range of two independent experiments each carried out in
duplicate.
A. M. James et al. Yeast growth on exogenous ubiquinones
FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2069
endogenous uptake pathway that recognizes the iso-
prenoid structure of CoQ
2
, but cannot interact with
the alkane tail of decylQ or any of the CoQ
2
analogues
lacking the first double bond. That a CoQ transport
pathway exists for endogenous CoQ
6
in yeast has been
considered probable for some time, as the enzymes for
CoQ
6
synthesis are located in the mitochondrial inner
membrane, but CoQ
6
is found throughout the cell
[1,9]. Spontaneous diffusion through the cytosol
appeared unlikely for this dispersion, as CoQ
6

is very
hydrophobic with a predicted octanol ⁄ water partition
coefficient in the region of 10
14
[10]. Recently it has
been shown that DCoQ yeast strains require at least
four genes (tlg2, erg2, pep12 and vps45) from the endo-
cytic pathway to grow on exogenously supplied CoQ
6
[11]. To test whether the endocytic uptake pathway
was also required for the uptake of short-chain ana-
logues such as decylQ and CoQ
2
in our experiments,
we created two double-knockout strains and tested
their ability to grow on CoQ
2
. Both Dcoq2Dtlg2 and
Dcoq2Derg2 strains grew in the presence of CoQ
2
(Fig. 3A) as well as the even more hydrophobic ana-
logue CoQ
4
(data not shown). The growth characteris-
tics were similar to those seen in the Dcoq2 strain
AB
DE
F
C
G3P

+
FCCP
2 min
100 nmol O
myxo
1
5
15
10
20
[CoQ
2
] (µM)
[CoQ
2
] (µM)
[DecylQ] (µ
M)
[A1-Q
2
] (µM) [A2-Q
2
] (µM) [A3-Q
2
] (µM)
GHI
[A4-Q
2
] (µM) [A5-Q
2

] (µM) [A6-Q
2
] (µM)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
Respiration rate
(nmol O·min
–1

·mg
–1
protein)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
Respiration rate
(nmol O·min
–1
·mg
–1
protein)
0
50
100
150
200
250
0 5 10 15 20
0
50

100
150
200
250
0 5 10 15 20
0
50
100
150
200
250
0 5 10 15 20
0
50
100
150
200
250
0 5 10 15 20
0
50
100
150
200
250
0 5 10 15 20
0
50
100
150

200
250
0 5 10 15 20
0
50
100
150
200
250
0 5 10 15 20
0
50
100
150
200
250
0 5 10 15 20
Fig. 2. CoQ
2
analogues restore respiration in isolated CoQ-deficient mitochondria. (A) An example oxygen electrode trace of isolated CEN-
Dcoq2 yeast mitochondria in mannitol buffer with glycerol-3-phosphate (G3P; 5 m
M) and FCCP (1 lM). CoQ
2
was titrated successively as
indicated by the arrowheads. That the oxygen consumption was mitochondrial in origin was confirmed by addition of the inhibitor
myxothiazol (1 l
M). (B)–(I) Uncoupled rates of mitochondrial respiration as a function of CoQ
2
(B), decylQ (C), A1-Q
2

(D), A2-Q
2
(E), A3-Q
2
(F), A4-Q
2
(G), A5-Q
2
(H) and A6-Q
2
(I). Data are the mean ± standard deviation of three independent experiments.
Yeast growth on exogenous ubiquinones A. M. James et al.
2070 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
(Fig. 3D), suggesting that the endocytic pathway is not
required for the uptake of CoQ
2
to the mitochondria
and, therefore, this pathway is unlikely to be responsi-
ble for discriminating between decylQ and CoQ
2
. That
a vesicle-based mechanism is not required for the
uptake is reasonable as the octanol ⁄ PBS partition coef-
ficients of CoQ
2
( 10
4.5
) and decylQ ( 10
5.5
) are sev-

eral orders of magnitude lower than that of CoQ
6
[6,10].
This suggests that CoQ
2
is sufficiently hydrophilic
that it can passively equilibrate quite effectively within
cells over the 48 h timeframe of the growth experi-
ments. To test this we measured whether yeast sup-
plied with ubiquinone exogenously contained a
significantly lower concentration of decylQ than that
of CoQ
2
or whether the accumulation of decylQ was
slower than for CoQ
2
. To limit complications due to
growth, a DCoQ culture was maintained in YPGG for
24 h, at which point they had consumed the available
glucose and their growth had arrested. In the contin-
ued absence of exogenous CoQ, the absorbance of this
culture remained stable, with no evidence of growth
for several days (Figs 1A and 3B). Upon the addition
of a mixture of CoQ
2
and decylQ it took 3–6 h for
growth to restart (Fig. 3B). To determine if there were
differences in the accumulation of CoQ
2
and decylQ

we measured the amount of the two ubiquinone ana-
logues in the cell pellets. From 30 min up to 48 h
after the addition of a mixture of 10 lm CoQ
2
and
10 lm decylQ to a yeast suspension, the ratio of
decylQ to CoQ
2
in the pellet remained very similar
(Fig. 3C). Although a difference was observed at a
very early 2 min time point, the equilibration rate of
both was relatively rapid in the context of the 48 h
experiments where decylQ failed to restore growth.
This suggests that the association of CoQ
2
and decylQ
with yeast cells is similar, despite decylQ not being
able to complement nonfermentative growth in DCoQ
yeast.
If more subtle differences in diffusion of decylQ to
mitochondria within cells were responsible, it might be
possible to restore growth with decylQ by adding a
large excess of it to DCoQ yeast. Therefore we mea-
sured the ability of DCoQ yeast to grow on glycerol
with concentrations of decylQ up to 100 lm. No non-
fermentative growth was observed with decylQ, even
when its concentration was up to 50- and 250-fold
higher than that required to observe such growth with
CoQ
2

and CoQ
4
, respectively (Fig. 3D). However,
there was a small, but significant, increase in the
absorbance of the culture when concentrations of
decylQ as low as 2 lm were added relative to the dim-
ethylsulfoxide carrier alone, suggesting decylQ induced
some change in the culture. The small increase was
also observed with 10 lm of the ineffective CoQ
2
analogues A4-Q
2
, A5-Q
2
and A6-Q2, as well as with
CoQ
1
, but it was not observed with 10 lm of CoQ
9
or
CoQ
10
(data not shown). The lack of any concentra-
tion dependence when an excess of decylQ was sup-
plied exogenously suggests that its failure to
complement nonfermentative growth in DCoQ yeast
does not result from subtle differences in diffusion,
particularly given that the hydrophobicity of decylQ is
intermediate between CoQ
2

and CoQ
4
.
To demonstrate that the physiochemical properties
of decylQ and CoQ
2
are grossly similar, we measured
their ability to diffuse between noncontiguous mem-
branes by mixing two populations of vesicles. Both
populations of vesicles contained equal concentrations
of the very hydrophobic fluorophore, 1-pyrene hexa-
decanoic acid (Pyr16), but only the second population
contained ubiquinone. Ubiquinone collisionally
quenches pyrene fluorescence if both are present in the
same membrane system and are capable of physical
interaction [12]. There is a linear relationship between
ubiquinone concentration and I
0
⁄ I – 1, where I
0
is the
initial fluorescence and I is the fluorescence after the
addition of ubiquinone, and for a typical ubiquinone
in our hands the slope of this line is 24 mm
)1
[12].
Using this value we would expect a decrease in relative
fluorescence (I ⁄ I
0
) from 1 to 0.83 upon the addition of

ubiquinone-containing vesicles because the initial
unquenched vesicle population (2 mL) is being diluted
with a highly quenched second vesicle population con-
taining ubiquinone (500 lL). This drop does not occur
if the added vesicle population does not contain ubi-
quinone (data not shown). We would then expect a
further decrease in relative fluorescence to 0.51 if the
ubiquinone is able to equilibrate between bilayers
because 40 lm ubiquinone in 100% of vesicles will
quench total fluorescence more effectively than 200 lm
ubiquinone in 20% of vesicles. The exchange of ubi-
quinone between the two populations of vesicles can
be seen most easily using CoQ
4
, as there is a decrease
in relative fluorescence from 0.77 to 0.57 over the
course of the experiment (Fig. 3E). When the second
population of vesicles was reconstituted with the rela-
tively more hydrophilic analogues, CoQ
2
or decylQ,
quenching was largely complete within seconds and
after approximately 10 min relative fluorescence was
 0.57 for both analogues, suggesting they are free to
exchange between the phospholipid bilayers of the two
vesicle populations (Fig. 3E). If the experiment was
repeated with the very hydrophobic analogue, CoQ
9
,
there was no evidence of movement between the

two populations during the course of the 10 min
A. M. James et al. Yeast growth on exogenous ubiquinones
FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2071
incubation, as relative fluorescence remained stable at
0.82 (Fig. 3E). Therefore, both CoQ
2
and decylQ dif-
fuse rapidly between the noncontiguous membranes of
the two vesicle populations, taking only a few seconds
to equilibrate. This is consistent with the rapid equili-
bration of both CoQ
2
and decylQ within cells
(Fig. 3C).
Finally, we sought to confirm that decylQ and CoQ
2
both reach mitochondria in intact cells directly.
DecylQ and CoQ
2
participate effectively in respiration
by isolated mitochondria in the absence of endogenous
CoQ
6
(Fig. 2). Therefore, if they can migrate to mito-
chondria their redox state should be sensitive to com-
pounds that manipulate the mitochondrial respiratory
chain. To test this we incubated intact yeast cells with
either decylQ or CoQ
2
for 3 h, after which we exposed

them to the either cyanide (KCN) or carbonylcyanide-
p-trifluoromethoxy-phenylhydrazone (FCCP) and ex-
tracted the ubiquinone. KCN inhibits complex IV and
leads to a reduced ubiquinone pool as electrons can no
Ubiquinone concentration (µM)
0
1
2
3
4
5
0 1020304050
Ubiquinone concentration (µM)
0
1
2
3
4
5
012243648
Time (h)
Growth (A
600
)
0
5
10
15
20
25

0 12243648
CoQ
2
decylQ
Pellet-associated ubiquinone normalised
for growth
(% of total Q in the pellet/A
600
unit)
Time (h)
Δcoq2Δerg2
Δcoq2Δtlg2
AB
0
1
2
3
4
5
6
7
8
0 5 10 15 20 50
100
CoQ
4
CoQ
2
decylQ
CD

Non-fermentative growth (A
600
)
Non-fermentative growth (A
600
)
0
5
10
15
20
25
012 3 45 6
(h)
(% of total Q in the pellet/A
600
unit)
Ubiquinol (%)
E
CoQ
2
/decylQ
Ubiquinone-loaded
vesicles
Normalised fluorescence (I/I
0
)
0
10
20

30
40
50
60
70
80
CoQ
2
decylQ
Ubiquinone
FCCP
KCN
Time (min)
F
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
0246810
CoQ
2
CoQ
4
CoQ
9
decylQ

Yeast growth on exogenous ubiquinones A. M. James et al.
2072 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
longer flow to O
2
, whereas FCCP uncouples mitochon-
dria, increasing respiration and oxidizing the ubiqui-
none pool as electrons rapidly flow from ubiquinone
to O
2
in the absence of a membrane potential [6].
Although CoQ
2
responded as expected, surprisingly
decylQ was not significantly reduced in the presence of
KCN (Fig. 3F). In the absence of FCCP or KCN, the
ratio of reduced to oxidized ubiquinone was similar to
that in the presence of FCCP (data not shown).
In summary, the presence of a sufficient concentra-
tion of ubiquinone in the mitochondrial membrane is
required for growth on glycerol. The above results sug-
gest that CoQ
2
and decylQ are both sufficiently hydro-
philic that they can rapidly equilibrate between
noncontiguous membranes through the aqueous phase
without this movement being mediated by a hydropho-
bic phase such as a vesicle. This makes it unlikely that
the difference between CoQ
2
and decylQ is due to dif-

ferences in their ability to diffuse to mitochondria
within cells. Paradoxically, decylQ was largely insensi-
tive to a mitochondrial inhibitor that should have led
to a reduction in decylQ in the presence of a substrate.
The reason for this discrepancy remains unclear and
will be discussed later.
DecylQ is unlikely to be exported by yeast cells
Above we dealt with the possibility that CoQ
2
is selec-
tively transported to mitochondria within cells. The
converse could equally have been true and decylQ
could have been selectively excluded by yeast cells.
This is because yeast can remove foreign molecules,
primarily by using ATP binding cassette (ABC) trans-
porters, as a mechanism for protecting themselves
from toxins [13]. Selective exclusion of decylQ from
the cell appears unlikely, as the amount of decylQ
associated with the yeast cell pellet is similar to that of
CoQ
2
(Fig. 3C), the putative export machinery cannot
be overwhelmed by high concentrations of decylQ
(Fig. 3D) and the structures that fail to restore growth
are less similar than the ones that do restore growth,
implying selective recognition of CoQ
2
rather than of
decylQ (Fig. 1B).
DecylQ is not toxic, but does confound growth

complementation by CoQ
2
The previous experiments suggest that decylQ can
migrate to mitochondria within cells. Therefore, it is
possible that decylQ is in some way toxic to yeast cells
and thereby prevents nonfermentative cell growth,
despite complementing respiration (Fig. 2). To deter-
mine if this was the case, we added increasing equimo-
lar amounts of CoQ
2
and decylQ to DCoQ cultures
and measured their growth in YPGG over 48 h
(Fig. 4A). The addition of equimolar decylQ to CoQ
2
had no significant detrimental effect on growth com-
pared with CoQ
2
alone. Therefore, decylQ appears to
Fig. 3. CoQ
2
does not require a transport pathway to reach mitochondria in yeast. (A) Deletion of proteins in the endocytic pathway does
not prevent exogenous CoQ
2
restoring growth. Dcoq2Dtlg2 and Dcoq2Derg2 double-knockout strains where grown in YPGG supplemented
with CoQ
2
(0–50 lM) for 48 h. Data are the mean ± range of two independent experiments. The growth of the two strains in the absence of
ubiquinone was subtracted to give the growth achieved on glycerol. (B) Time lag in the growth response of stationary phase yeast to exoge-
nously supplied CoQ
2

. The BY4743Dcoq2 strain was grown in YPGG for 18 h, at which point it had reached A
600
 0.8 and already been sta-
tionary for several hours and would remain that way for a number of days (squares). At the point indicated by the arrow, a mixture of 10 l
M
decylQ and 10 lM CoQ
2
was added and the cultures were incubated for a further 48 h with A
600
measurements taken at intervals (circles).
Data are the mean ± standard error of the mean of three independent experiments. (C) The equilibration of exogenously supplied CoQ
2
and
decylQ with yeast cells is rapid. As in (B), but samples were taken at intervals and centrifuged before CoQ
2
and decylQ were extracted with
hexane from both the supernatant and the pellet. They were separated by HPLC and quantitated using electrochemical detection. The values
are expressed as the percentage of the total CoQ
2
or decylQ in the yeast pellet normalized for the growth (A
600
) of the culture shown in (B).
Data are the mean ± standard error of the mean of three independent experiments. (D) Even a 50-fold excess of decylQ above that required
for CoQ
2
does not restore growth of the BY4743Dcoq2 strain. The BY4743Dcoq2 strain was grown in YPGG supplemented with decylQ
(0–100 l
M), CoQ
2
(0–20 lM) or CoQ

4
(0–20 lM) for 48 h. Data are the mean ± standard error of the mean of three independent experiments.
The growth of the BY4743Dcoq2 strain in the absence of ubiquinone was subtracted to give the growth achieved on glycerol. (E) DecylQ
and CoQ
2
move freely between noncontiguous phospholipid bilayers. An initial population of phosphatidylcholine vesicles containing 4 lM
Pyr16 in 2 mL KP
i
buffer was mixed with 500 lL of a second population created containing 4 lM Pyr16 and either CoQ
2
, decylQ, CoQ
4
or
CoQ
9
. A decrease in fluorescence indicated collisional quenching via a physical interaction between ubiquinone and Pyr16. The data are
expressed as the relative amount of fluorescence at a given point in time (I) to initial fluorescence just before the addition of ubiquinone-
containing vesicles (I
0
). (F) The redox state of decylQ is largely insensitive to KCN. The BY4743Dcoq2 strain was grown in YPGG for 24 h at
which point it had reached A
600
 0.8 before either 10 lM decylQ or 10 lM CoQ
2
was added and the cultures were incubated for a further
3 h. Then, either 1 l
M FCCP or 200 lM KCN was added, followed by a 2 min incubation at 30 °C. The cultures were centrifuged before
CoQ
2
and decylQ were extracted with hexane from the pellet. They were separated by HPLC and quantitated using electrochemical detec-

tion. The values are the percentage of the total CoQ
2
or decylQ found in the ubiquinol form. Data are the mean ± standard error of the mean
of three independent experiments.
A. M. James et al. Yeast growth on exogenous ubiquinones
FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2073
be nontoxic. We next reasoned that because decylQ
should diffuse to mitochondria (Fig. 3) and once there
function effectively in oxidative phosphorylation
(Fig. 1D), that decylQ might be unable to restore
growth because CoQ
2
is required in trace amounts for
some secondary function. To determine if this was the
case, we added increasing amounts of decylQ
(0–100 lm) in combination with 5 lm CoQ
2
to DCoQ
cultures in YPGG and measured their growth over
48 h. This showed that trace amounts of CoQ
2
did not
allow decylQ to complement growth (Fig. 4B). In fact,
rather surprisingly, at higher concentrations decylQ
largely inhibited the growth that would have been
observed with 5 lm CoQ
2
alone (Fig. 4B). Interest-
ingly, there was a strong correlation between the frac-
tion of the total added ubiquinone present as CoQ

2
and growth (Fig. 4C; r
2
= 0.99). Thus, it would
appear that decylQ in some way prevents CoQ
2
inter-
acting with a protein that is required for nonfermenta-
tive growth. This might occur via decylQ binding
directly to the protein and preventing CoQ
2
interacting
or growth could require an interaction with the
reduced or the oxidized form of CoQ
2
and decylQ dis-
turbs the normal CoQ
2
H
2
⁄ CoQ
2
ratio.
In summary, decylQ is not toxic, but can antagonize
the stimulation of growth by CoQ
2
, especially when its
concentration markedly exceeds that of CoQ
2
. This

suggests that there may be a protein that regulates
growth that can bind to CoQ
2
and that decylQ antago-
nizes this interaction in some way.
Screen for proteins influencing CoQ-dependent
growth in yeast
The data so far suggest that there is a protein that can
distinguish between CoQ
2
and decylQ that is essential
for growth on nonfermentable substrates. The nature
of this CoQ–protein interaction is unclear, as we have
been unable to identify an obvious secondary role for
CoQ in growth on nonfermentable substrates from the
literature. To investigate this interaction further we set
up a screen to identify potential ORFs that might be
involved in CoQ-dependent growth in yeast. The yeast
0
1
2
3
4
5
6
0 5 10 15 20
CoQ
2
CoQ
2

and
equimolar decylQ
Non-fermentative growth (A
600
)
CoQ
2
concentration (µM)
0
1
2
3
4
5
6
0 20 40 60 80 100
X µM decylQ + X µM CoQ
2
X µM decylQ + 5 µM CoQ
2
X µM decylQ
Ubiquinone concentration (X µM)
A
B
0
0.5
1
1.5
2
2.5

0 0.1 0.2 0.3 0.4 0.5
Fraction of ubiquinone that is CoQ
2
([CoQ
2
] / [CoQ
2
+decylQ])
C
Non-fermentative growth (A
600
)
Non-fermentative growth (A
600
)
Fig. 4. DecylQ is not toxic, but does interfere with growth comple-
mentation by CoQ
2
. (A) Growth on exogenous CoQ
2
is neither
stimulated nor inhibited by the presence of equimolar decylQ. The
BY4743Dcoq2 strain was grown in YPGG supplemented with either
CoQ
2
(0–20 lM) or equimolar CoQ
2
and decylQ (0–20 lM) for 48 h.
Data are the mean ± standard error of the mean of three indepen-
dent experiments. The growth of the BY4743Dcoq2 strain in the

absence of ubiquinone was subtracted to give the growth achieved
on glycerol. (B) Growth on trace exogenous CoQ
2
is inhibited by
the presence of higher concentrations of decylQ. The
BY4743Dcoq2 strain was grown in YPGG with either decylQ alone
(0–100 l
M), equimolar CoQ
2
and decylQ (0–100 lM) or CoQ
2
(5 lM)
and a range of decylQ concentrations (0–100 l
M) for 48 h. The
growth of the BY4743Dcoq2 strain in the absence of ubiquinone
was subtracted to give the growth achieved on glycerol. (C) Growth
is related to the proportion of total ubiquinone that is CoQ
2
.Asin
(B), the BY4743Dcoq2 strain was grown in YPGG with 5 l
M CoQ
2
and a range of decylQ concentrations (0–100 lM) for 48 h.
Yeast growth on exogenous ubiquinones A. M. James et al.
2074 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
S. cerevisiae contains  6000 ORFs, of which  5000
can be deleted with the strain still viable. To do this
we first created a double-knockout library unable to
synthesize CoQ by crossing a strain in which a gene
required for the endogenous synthesis of CoQ

6
(coq2)
was deleted with a commercial library containing
 4800 strains each harbouring a deletion in a single
nonessential gene. To generate a stable haploid
double-knockout library we utilized an approach
developed by Tong and coworkers [14]. The stability
arises because BY4743Dcoq2 contains a histidine
synthesis gene under the control of a mating-type ‘a’
promoter (MFA1pr-HIS3) inserted into the middle of
an arginine permease gene (CAN1). This allows
mating-type ‘a’ progeny to be selected without cloning.
For details on how the double-knockout library was
created, see the Materials and Methods section.
After its creation, the Dcoq2DORF strains in the
double-knockout library were screened for their ability
to grow in liquid YPGG when supplemented exoge-
nously with 100 lm CoQ
2
. This identified 379 double-
knockout strains that could not grow on YPGG
containing CoQ
2
(Fig. 5A). Of these, 324 were not con-
sidered further for three reasons. First, theDcoq2DORF
strain had grown in liquid YPGG in the absence of
CoQ2, suggesting they may not be DCoQ (Fig. 5B).
Second, the corresponding DORF strain failed to grow
on solid YPG, indicating that the ORF deleted in the
strain was a gene essential for nonfermentative growth,

e.g. a nuclear-encoded respiratory chain complex sub-
unit (Fig. 5C). Finally, they did not produce viable
progeny after mating and sporulation on the final
selective plate. Therefore, there was no Dcoq2DORF
strain to test for dependence on exogenous CoQ
(Fig. 5D). The remaining 55 strains potentially har-
boured a deletion that removes a gene that influences
growth on exogenous CoQ. Consequently, these strains
were cloned and analysed further to confirm whether
they respond abnormally to exogenous CoQ.
Confirmation of double-knockout strains with
poor growth on exogenously supplied CoQ
An inoculation from each of the 55 Dcoq2DORF
strains identified from the screen were grown in 3 mL
YPGG and 10 lm CoQ
4
. In addition, nine control
Dcoq2DORF strains were selected to control for the
mating and sporulation (see Materials and Methods).
After 48 h their growth was measured and the 24
strains with decreased growth relative to the
BY4743Dcoq2 parental strain and the nine Dcoq2-
DORF control strains were cloned, as were their DORF
counterparts from the original commercial library. All
of these Dcoq2DORF strains grew by fermentation in
YPD (yeast extract, peptone, glucose) to an A
600
of
 10 (data not shown) and in YPGG without CoQ
4

to
A
B
C
D
Fig. 5. Creation of the double-knockout
library. (A, B) Double-knockout Dcoq2DORF
strains grown in liquid glycerol media (YPG)
supplemented with canavanine, G418 and
cloneNAT with (A) or without (B) 100 l
M
CoQ
2
. (C) Parental single-deletion DORF
strains grown on solid YPG. (D) Double-dele-
tion Dcoq2DORF strains grown in solid
glucose-based synthetic media lacking histi-
dine and arginine and supplemented with
canavanine, G418 and cloneNAT. The red
squares indicate strains that failed to give
information about whether the deleted ORF
is or is not involved in CoQ-dependent
growth. Unmarked strains have exogenous
CoQ-dependent growth and are uninterest-
ing, whereas black squares indicate the
desired combination of phenotypes and
these were analysed further. The top left
black square is Srb8 and the bottom right is
Kcs1.
A. M. James et al. Yeast growth on exogenous ubiquinones

FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2075
an A
600
of  0.8 (data not shown). Therefore, their
utilization of glucose for growth was grossly normal
and they were unable to make the transition to the uti-
lization of glycerol as a carbon source in the absence
of CoQ. This can also be seen in the slightly positive
values obtained for growth in YPGG containing
10 lm CoQ
4
, as a failure to grow on glucose would
lead to negative values (Table 1).
These 24 Dcoq2DORF clones were grown in glass
tubes with 3 mL YPGG and 10 lm CoQ
4
for 48 h at
30 °C. The growth of 21 of these fell below the range
of the nine Dcoq2DORF control strains and the
BY4743Dcoq2 parental strain. To ensure the defect in
growth was related to an inability to utilize exogenous
CoQ and not related to a general defect in mitochon-
drial metabolism, the corresponding DORF strains
were grown in glass tubes with 3 mL YPGG for 48 h
at 30 °C. For five of the remaining ORFs, poor
growth of the Dcoq2DORF strain could be sufficiently
explained by similar poor growth of their DORF
strain. This left 16 ORFs where the Dcoq2DORF strain
could not grow as well when supplemented with exoge-
nously supplied CoQ

4
as any of the nine control
Dcoq2DORF strains yet contained mitochondria that
were at least partially functional in their DORF strain
(Table 1). Unlike the control Dcoq2DORF strains, for
many of the potentially interesting Dcoq2DORF strains
the counterpart DORF strain had reduced growth on
glycerol (Table 1). However, when the growth of the
Dcoq2DORF strain is expressed as a percentage of that
of its corresponding single knockout, it is apparent
that any decrease in growth on glycerol in the DORF
strain is compounded by the introduction of a Dcoq2
deletion. In summary, we have identified 16 ORFs, the
deletion of which dramatically decreased the ability of
exogenous CoQ to support growth on glycerol in the
absence of an ability to synthesize endogenous CoQ.
Discussion
Yeast lacking the ability to synthesize CoQ endoge-
nously require exogenous CoQ to grow on nonfer-
mentable substrates. Utilizing several very similar
analogues of CoQ
2
we have shown that exogenous
supplementation of decylQ and all analogues lacking
the first double bond of the side chain failed to sup-
port the growth of DCoQ yeast on glycerol. This sug-
gests that there is very selective structural recognition
of elements within the side chain, presumably by a
protein, and that this interaction is required to com-
plete the diauxic shift to nonfermentable substrates

(Fig. 1). The most obvious level at which this struc-
tural recognition could occur is mitochondrial respira-
tion. However, isolated mitochondria lacking CoQ
respired normally on glycerol-3-phosphate when sup-
plemented with all CoQ
2
analogues tested (Fig. 2).
This suggests that the selectivity does not arise from
the respiratory chain complexes.
The next possibility is that the protein of interest is
involved in the transport of exogenous CoQ from the
extracellular medium into the cell and on to mitochon-
dria, and that decylQ and the analogues A4-Q
2
, A5-Q
2
and A6-Q
2
are not taken up correctly by this pathway.
Deletion of two endocytic proteins shown to be
involved in the transport of exogenous CoQ
6
to mito-
chondria [11] failed to prevent growth of DCoQ yeast
on glycerol (Fig. 3A), suggesting that this pathway is
not an absolute requirement for the uptake of more
hydrophilic CoQ analogues. Consistent with this,
CoQ
2
and decylQ reached apparent equilibrium within

minutes of being added to a culture of DCoQ yeast
(Fig. 3C). Even though 2 lm CoQ
2
led to appreciable
growth, 100 lm decylQ did not, suggesting that uptake
of decylQ to mitochondria is not the factor limiting
growth. Furthermore, mixing populations of vesicles
containing fluorescent pyrenes with vesicles containing
decylQ, CoQ
2
and CoQ
4
indicated rapid redistribution
of decylQ and CoQ
2
over a timeframe of seconds and
CoQ
4
within minutes (Fig. 3E). CoQ
2
and decylQ were
also rapidly lost into the bulk aqueous phase during
cell subfractionation or cell pellet washing steps
(unpublished observations). Together this suggests that
even though decylQ and CoQ
2
are hydrophobic, they
are not hydrophobic enough to be retained within a
membrane system over the 48 h course of the growth
experiments and presumably equilibrate throughout all

the lipid bilayers of the cell.
The experiments outlined above suggest that enough
CoQ
2
diffuses around blocks in the endocytic pathway
used to transport CoQ
6
to support growth on nonfer-
mentable substrates (Fig. 3A). However, even though
it appears possible for CoQ
2
to diffuse to mitochon-
dria, growth on glycerol with exogenously supplied
CoQ
2
is significantly slower than that observed on
endogenous CoQ
6
(Fig. 1). One reason for the dimin-
ished growth on exogenous CoQ
2
relative to endoge-
nous CoQ
6
could be because the concentration of CoQ
in mitochondrial membranes appears significantly
higher than that of other cellular membranes [11].
Because CoQ
2
could redistribute between bilayers

(Fig. 3E) there may be no way of maintaining an ele-
vated mitochondrial CoQ concentration, thereby
resulting in suboptimal growth. Alternatively, it could
arise from a decrease in substrate concentration for an
enzyme because CoQ
2
cannot be accumulated and
retained in a membrane system or via a decrease in
Yeast growth on exogenous ubiquinones A. M. James et al.
2076 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
Table 1. Identification of 16 strains from the Dcoq2D ORF double-knockout library with a diminished response to exogenous CoQ. The growth of the Dcoq2DORF strains was measured
after 48 h in YPGG with 10 l
M CoQ
4
. Growth of the DORF strains was measured after 48 h in YPGG. The BY4743Dcoq2 strain grew to A
600
 0.8 in YPGG and this was subtracted from
the A
600
of each Dcoq2DORF and DORF strain to give the level of growth on glycerol. All Dcoq2DORF colonies could utilize glucose and grew to A
600
 0.8 in YPGG without CoQ
4
(data
not shown). The ratio between Dcoq2DORF and DORF growth was normalized against nine randomly selected Dcoq2DORF and DORF strains. The data are the mean ± standard error of
the mean of three independent experiments. Soluble NSF attachment protein receptors, SNAREs.
Control Description
Dcoq2DORF+
CoQ
4

(A
600
) DORF (A
600
)
Dcoq2DORF ⁄
DORF (%)
BY4743Dcoq2 Original parental aA2 Dcoq2 single-knockout strain used in creation of the double-knockout
library
6.59 ± 0.29 0.00 ± 0.00 n ⁄ a
Dcoq2DORF Basket (n = 9) Basket of nine strains to control for the mating and sporulation during creation of the
double-knockout library
5.84 ± 0.73 10.09 ± 0.3 100 ± 11.7
DORF (protein name) DORF description ()
Dcoq2DORF +
CoQ
4
(A
600
) DORF (A
600
)
Dcoq2DORF ⁄
DORF (%)
YMR273C (ZDS1) Protein that interacts with silencing proteins at the telomere, involved in transcriptional
silencing; has a role in localization of Bcy1p, a regulatory subunit of protein kinase A;
implicated in mRNA nuclear export
0.06 ± 0.04 10.01 ± 0.57 1.0
YOR014W (RTS1) B-type regulatory subunit of PP2A; homologue of the mammalian B’ subunit of PP2A 0.56 ± 0.04 8.35 ± 0.09 11.8
YLR384C (IKI3) Subunit of Elongator complex, which is required for modification of wobble nucleosides in

tRNA; maintains structural integrity of Elongator; homologue of human IKAP, mutations
in which cause familial dysautonomia
0.62 ± 0.03 6.95 ± 0.41 15.5
YPL101W (ELP4) Subunit of Elongator complex, which is required for modification of wobble nucleosides in
tRNA; required for Elongator structural integrity
0.75 ± 0.05 6.87 ± 0.45 18.9
YGR183C (QCR9) Subunit 9 of the ubiquinol cytochrome c reductase complex, which is a component of the
mitochondrial inner membrane electron transport chain; required for electron transfer at the
ubiquinol oxidase site of the complex
0.0 ± 0.02 3.38 ± 0.56 0.0
YLR304C (ACO1) Aconitase, required for the tricarboxylic acid cycle and also independently required for
mitochondrial genome maintenance; phosphorylated; component of the mitochondrial nucleoid;
mutation leads to glutamate auxotroph
0.45 ± 0.06 10.10 ± 0.34 7.8
YPL270W (MDL2) Mitochondrial inner membrane half-type ATP-binding cassette transporter 0.32 ± 0.05 4.69 ± 0.41 11.8
YKL212W (SAC1) Phosphatidylinositol phosphate (PtdInsP) phosphatase involved in hydrolysis of PtdIns[4]P;
transmembrane protein localizes to endoplasmic reticulum and Golgi; involved in protein
trafficking and processing, secretion, and cell wall maintenance
0.43 ± 0.05 5.45 ± 0.16 13.6
YDR126W (SWF1) Palmitoyltransferase that acts on the SNAREs Snc1p, Syn8p, Tlg1p and probably on all
SNAREs; member of a family of putative palmitoyltransferases containing an
Asp-His-His-Cys-cysteine rich (DHHC-CRD) domain; may have a role in vacuole fusion
1.86 ± 0.03 9.11 ± 0.29 35.5
YLR242C (ARV1) Protein functioning in transport of glycosylphosphatidylinositol intermediates into the
endoplasmic reticulum lumen; required for normal intracellular sterol distribution and for
sphingolipid metabolism; similar to Nup120p and Caenorhabditis elegans R05H5.5 protein
1.13 ± 0.08 8.42 ± 0.17 23.3
YCR002C (CDC10) Component of the septin ring of the mother-bud neck that is required for cytokinesis; septins
recruit proteins to the neck and can act as a barrier to diffusion at the membrane, and they
comprise the 10 nm filaments seen with electron microscopy

0.56 ± 0.02 7.82 ± 0.41 12.5
A. M. James et al. Yeast growth on exogenous ubiquinones
FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2077
binding affinity of CoQ
2
or CoQ
4
for a hydrophobic
pocket in a protein through the loss of isoprenoid
units from the side chain of CoQ
6
. To identify proteins
or pathways that might be involved in CoQ-dependent
growth on nonfermentable substrates, we created a
yeast double-knockout library (DORFDcoq2) and
screened for strains whose growth on nonfermentable
substrates was hypersensitive to supplementation with
exogenous CoQ (Table 1). We note that ORFs in
which the single-knockout DORF strain could not
grow on nonfermentable substrates when able to syn-
thesize their own CoQ
6
endogenously could not be
considered for further analysis, e.g. nuclear-encoded
respiratory complex genes. This is because even though
these ORFs may be part of a CoQ-dependent growth
pathway, they could provide no useful information in
the subsequently derived double-knockout DORFDcoq2
strain about whether CoQ was required for growth on
glycerol.

Two of the better candidate DORFDcoq2 strains
from the screen were Dcoq2Dzds1 and Dcoq2Drts1.
Although the descriptions from Table 1 do not indi-
cate a functional association, the literature is at least
suggestive, as both proteins have been reported to
interact with protein phosphatase 2A (PP2A) family
members [15]. PP2A are widely conserved protein ser-
ine ⁄ threonine phosphatases, with roles in a multitude
of cellular processes, many of which are involved in
the regulation of cell cycle progression. They act as tri-
meric complexes of a catalytic subunit (either Pph21 or
Pph22), a regulatory subunit (either Rts1, Rts3 or
Cdc55) and a scaffold subunit (Tpd3) in budding
yeast. Recently, a physical interaction has been shown
between Zds1 and a PP2A complex containing the
Rts1 homologue Cdc55 [15]. This Zds1-containing
complex forms part of the mitotic exit machinery, per-
haps suggesting that CoQ may be required at some
point during the cell cycle.
One paradoxical observation was that unlike CoQ
2
,
decylQ does not become reduced on exposure to cya-
nide in intact cells grown on glycerol (Fig. 3F). This
occurs even though isolated DCoQ mitochondria pass
electrons through a ubiquinone pool composed of
decylQ, as they can respire when supplemented with
glycerol-3-phosphate and decylQ (Fig. 2). Cyanide
should reduce the mitochondrial ubiquinone pool in the
presence of a metabolizable carbon source. Therefore,

one explanation for this difference could be defective
substrate supply to mitochondria. Alternatively, it
could also be explained if the shift to growth on nonfer-
mentable substrates increases the proportion of cellular
lipid that is mitochondrial via mitochondrial biogenesis.
If decylQ did not effectively upregulate mitochondrial
Table 1. (Continued).
YDR017C (KCS1) Inositol hexakisphosphate and inositol heptakisphosphate kinase; generation of high-energy
inositol pyrophosphates by Kcs1p is required for many processes, such as vacuolar biogenesis,
stress response and telomere maintenance
1.21 ± 0.06 5.53 ± 0.27 37.8
YDR512C (EMI1) Nonessential protein of unknown function required for transcriptional induction of the early
meiotic-specific transcription factor IME1, also required for sporulation
)0.12 ± 0.04 5.49 ± 0.00 –3.8
YIL128W (MET18) DNA repair and TFIIH regulator, required for both nucleotide excision repair and RNA
polymerase II transcription; involved in telomere maintenance
0.15 ± 0.05 3.22 ± 0.22 7.8
YDL075W (RPL31A) Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl31Bp and has
similarity to rat L31 ribosomal protein; associates with the karyopherin Sxm1p
0.57 ± 0.11 6.09 ± 0.25 16.4
YDR225W (HTA1) Histone H2A, core histone protein required for chromatin assembly and chromosome function;
one of two nearly identical subtypes (see also HTA2); DNA damage-dependent phosphorylation
by Mec1p facilitates DNA repair; acetylated by Nat4p
1.35 ± 0.05 7.06 ± 0.05 33.3
Yeast growth on exogenous ubiquinones A. M. James et al.
2078 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
biogenesis during this shift, then there would be a smal-
ler proportion of decylQ being sensitive to KCN than
with CoQ
2

. There are some similarities in our observa-
tions to those seen with Isc1 (inositol sphingolipid
phospholipase C) mutants, as isolated mitochondria
lacking Isc1 also respire normally, even though Disc1
cells exhibit a growth defect. Disc1 mutants have been
shown to regulate the expression of many nuclear genes
involved in utilizing nonfermentable substrates during
the postdiauxic phase [16]. Isc1 encodes a neutral sphin-
gomyelinase that is localized to the endoplasmic reticu-
lum and migrates to mitochondria during the diauxic
shift [17]. Interestingly, the product of Isc1 is the signal-
ling molecule ceramide, which has many structural sim-
ilarities to CoQ [18]. Furthermore, CoQ has been
shown to inhibit a plasma membrane neutral sphingo-
myelinase in a redox-specific manner [19]. It would
be interesting if ceramide production by Isc1 were
regulated by the redox state of CoQ in mitochondria.
In summary, we have shown that the presence of a
double bond between C2 and C3 in the first isoprenoid
unit of exogenously supplied CoQ is essential for resto-
ration of growth on nonfermentative substrates in
DCoQ yeast strains. This is despite all the CoQ ana-
logues restoring respiration in isolated mitochondria.
This suggests the presence of a protein that is able to
recognize subtle differences in the side chain of CoQ.
This protein is not part of the endocytic pathway used
for the uptake of exogenous CoQ
6
nor does it appear
to be one of the mitochondrial respiratory complexes.

Growth is slowed by decylQ even though it appears
unlikely that the protein recognizes decylQ itself. One
possibility is that during the diauxic shift, the ratio of
CoQH
2
⁄ CoQ is an important growth signal and an
excess of decylQ interferes with this redox couple. The
exact nature of the interaction remains unclear and
will be the subject of future work.
Materials and methods
Chemicals
CoQ
4
, CoQ
2
and decylQ were sourced from Sigma (St Louis,
MO, USA). Other CoQ
2
analogues were prepared as
described previously [7,8].
Yeast strains
Two Dcoq2 strains were used for these experiments, CEN-
Dcoq2 (MATaDhis3 Dleu2 Dtrp1 Dura3 Dcoq2::HIS3
+
[20])
and BY4743Dcoq2 (MATa his3D1 Dleu20 Dlys2 Dura3
Dcan1::LEU2
+
-MFA1pr-HIS3 Dcoq2::natR). BY4743Dcoq2
was derived from a commercially available heterozygote

strain (MATa ⁄ aDhis3 Dleu2 Dlys2 ⁄ LYS
+
Dmet15 ⁄ MET15
+
Dura3 Dcan1::LEU2
+
-MFA1pr-HIS3 ⁄ CAN1
+
Dcoq2::kanMX ⁄
COQ2
+
; Open Biosystems, Huntsville, AL, USA). Briefly,
the heterozygote strain was grown in YPD [1% (w ⁄ v) yeast
extract, 2% (w ⁄ v) peptone, 2% (w ⁄ v) glucose] containing
200 mgÆmL
)1
G418 for 24 h before the overnight culture
was pelleted and washed twice with water. This was resus-
pended in sporulation medium (10 gÆL
)1
K
+
acetate, 1 gÆL
)1
yeast extract, 0.5 gÆL
)1
glucose supplemented with 40 mgÆL
)1
uracil and 20 mgÆL
)1

histidine) and incubated at 24 °C for
4 days. The resulting spores were transferred to YPD for
1 h and then streaked on to SD) Arg+Can+Kan haploid
selection plates (21 gÆL
)1
agar, 20 gÆL
)1
glucose, 1.7 gÆL
)1
yeast nitrogen base without ammonium sulphate or amino
acids, 1 gÆL
)1
monosodium glutamic acid, 2 gÆL
)1
amino
acid dropout lacking arginine, 70 mgÆL
)1
canavanine and
200 mgÆL
)1
G418) for 4 days at 30 °C. Thirty-two colo-
nies were replica plated on to SD ) Arg+Can+Kan,
SD ) Arg ) His+Can+Kan and YPG plates [21 gÆL
)1
agar, 1% (w ⁄ v) yeast extract, 2% (w ⁄ v) peptone, 3% (v ⁄ v)
glycerol], of which five grew on SD-Arg+Can+Kan
plates, but not SD ) Arg)His+Can+Kan and YPG plates,
indicating they had the desired MATaDcoq2 genotype, of
which one was characterized further. Genetic complementa-
tion occurred when the putative MATaDcoq2::kanMX

strain was crossed with a MATa Dcoq3::kanMX strain in
liquid YPG. In contrast, complementation did not occur
when it was crossed with a MATa Dcoq2::kanMX strain or
when putative MATa Dcoq2::kanMX colonies were crossed
with the MATa Dcoq3::kanMX strain.
As the MATa knockout library to which it would be
mated carries a kanMX resistance marker, the strain had its
kanMX marker switched to a nourseothricin (CloneNat;
Werner Bioagents, Jena, Germany) resistance marker (natR)
by transforming with the plasmid pAG25 (Euroscarf,
Frankfurt, Germany) as described previously [21]. After
transformation the cells were pelleted and resuspended in
1 mL YPD and incubated at 30 °C for 3 h to allow expres-
sion of the natR marker. The culture was then plated on to
SD ) Arg+Can+Nat plates (21 gÆL
)1
agar, 20 gÆL
)1
glucose,
1.7 gÆL
)1
YNB without ammonium sulphate without amino
acids, 1 gÆL
)1
monosodium glutamic acid, 2 gÆL
)1
amino
acid dropout lacking arginine, 70 mgÆL
)1
canavanine and

100 mgÆL
)1
CloneNat). Colonies were then replica replated
on to SD ) Arg+Can+Kan plates to ensure they had lost
their kanMX marker. One colony, which is referred to as
BY4743Dcoq2, was picked and used to mate with the MATa
knockout library and as a control. Yeast were stored as
stocks at )80 °C in 50% (v ⁄ v) YPD and 50% (v ⁄ v) glycerol.
Growth on CoQ
2
analogues
From these stocks, precultures were made by inoculating
3 mL YPD in a 50 mL Falcon tube and growing at 30 °C
A. M. James et al. Yeast growth on exogenous ubiquinones
FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS 2079
with mechanical shaking for 2 days, after which time they
had reached A
600
 10. Sufficient preculture of the yeast
strain CENDcoq2 was inoculated into 5 mL YPG contain-
ing 0.05% (w ⁄ v) glucose (YPGG) to give A
600
 0.1. The
trace glucose was added to give a reproducible transition to
a respiratory phenotype. CoQ
2
analogues were added in
dimethylsulfoxide from 5 mm stock solutions. Cultures were
grown at 30 °C with mechanical shaking for 96 h, after
which their absorbance was measured at 600 nm. The

Dcoq2 strains grew on YPGG to A
600
 0.6–0.8, with
the exact value extremely consistent, but dependent on the
media batch. Growth above this value in Dcoq2 strains
required utilization of exogenous ubiquinone for the meta-
bolism of glycerol.
Mitochondrial respiration on CoQ
2
analogues
Mitochondria were prepared by digesting the cell wall with
lyticase followed by homogenization and differential centri-
fugation, as described previously [6]. Respiration was mea-
sured with a Clark-type oxygen electrode (Rank Brothers,
Cambridge, UK), as described previously [6].
Creation of the double-knockout library
A genetically stable double-knockout Dcoq2DORF MATa
library was created using the approach of Tong and
coworkers [14]. We utilized the commercially available dip-
loid yeast strain MATa ⁄ aDhis3 Dleu2 Dlys2 ⁄ LYS
+
Dmet15 ⁄
MET15
+
Dura3 Dcan1::LEU2
+
-MFA1pr-HIS3 ⁄ CAN1
+
Dcoq2::kanMX ⁄ COQ2
+

(Open Biosystems) to create the
BY4743Dcoq2 MATa haploid strain, as described above.
BY4743Dcoq2 was mated with the commercially available
MATa single-knockout DORF library (Open Biosystems)
after it had been transformed to a 384 pin format. Library
creation involved pinning the MATa single-knockout
DORF library sequentially through a series of plates that
first mates the two haploids before sporulation and selec-
tion for only MATa Dcoq2::natR DORF::kanMX haploid
progeny.
Screen of the double-knockout library
Of the original 4728 single-knockout DORF strains,  8%
could not grow aerobically on YPG before crossing with
BY4743Dcoq2, indicating that they were unlikely to contain
a functioning mitochondrial respiratory chain. Although
the ORFs deleted in these strains could be involved in CoQ
uptake, they were not considered for further analysis, as
they would not be responsive to exogenous CoQ in the final
double-knockout library. After creation of the double-
knockout Dcoq2DORF library,  4% of strains were not
viable on the final selective plate, SD)Arg)His+Can+
Kan+Nat (21 gÆL
)1
agar, 20 gÆ L
)1
glucose, 1.7 gÆL
)1
YNB
without ammonium sulphate without amino acids, 1 gÆL
)1

monosodium glutamic acid, 2 gÆL
)1
amino acid dropout
lacking arginine and histidine, 70 mgÆL
)1
canavanine,
200 mgÆL
)1
G418 and 100 mgÆmL
)1
CloneNat). Of the 446
blank control wells that did not contain a single-knockout
DORF library strain, but did have the BY4743Dcoq2 strain
pinned into them, five grew, suggesting a cross-contamina-
tion rate of  1%. The screen itself was conducted in 384
well plates containing liquid YPG+Can+Kan+Nat with
or without 100 lm CoQ
2
. After 96 h, photographs were
taken. Strains with potentially interesting ORF deletions
were cloned from the double-knockout Dcoq2DORF library,
grown in YPD and stored frozen at )80 °C. These were
then grown in YPD for 48 h before inoculation at A
600
 0.1 into YPGG supplemented with 10 lm CoQ
4
. CoQ
4
was added in dimethylsulfoxide from a 5 mm stock solu-
tion. Cultures were grown at 30 °C with mechanical shak-

ing for 48 h, after which their absorbance was measured at
600 nm. Single-knockout DORF strains were also cloned
and grown under the same conditions in the absence of
exogenous CoQ
4
for comparison. In addition, systemati-
cally selected Dcoq2DORF strains were cloned from the
library and used as controls for mating and sporulation.
These were the strains at an identical, but randomly chosen,
well position on each of the fourteen 384 well library plates.
They were the ORFs YAL051W, YMR265C, YPL205C,
YPR011C, YLR174W, YJL206C-A, YOL110W, YLR436C
and YCR067C. Strains from five of the plates could not be
used for the various reasons outlined in Fig. 5.
Measurement of CoQ
2
and decylQ
Sufficient preculture of the yeast strain BY4743Dcoq2 was
inoculated into 3 mL YPGG to give A
600
 0.1. Cultures
were grown at 30 °C with mechanical shaking for 18 h,
after which 10 lm CoQ
2
and 10 lm decylQ were added.
After periods of time (2 min to 48 h) 1 mL culture was
removed to an Eppendorf tube and centrifuged for 30 s at
10 000 g. The supernatant was removed to another Eppen-
dorf tube and snap frozen. The residual supernatant was
aspirated from the pellet and the Eppendorf tube was

dried with tissue paper before the pellet was also snap fro-
zen. The pellet was not washed due to significant loss of
decylQ and particularly CoQ
2
into the washing medium
over and above that which could be explained by the
residual water space of the pellet. Supernatants and pellets
were extracted three times with 4 mL 2 : 1 diethyl
ether ⁄ hexane in a glass test tube. The combined hexane
volume was transferred to a fresh glass test tube and
evaporated to dryness under a stream of nitrogen. The
CoQ
2
and decylQ residue was resuspended in 500 lL 50%
(v ⁄ v) methanol. The sample was resolved using a C18 col-
umn (Jupiter 250 · 4.6 mm 5 lm; Phenomenex, Torrance,
CA, USA) with an isocratic 80% methanol ⁄ 20% water
Yeast growth on exogenous ubiquinones A. M. James et al.
2080 FEBS Journal 277 (2010) 2067–2082 ª 2010 The Authors Journal compilation ª 2010 FEBS
mobile phase containing 10 mm lithium perchlorate. CoQ
2
and decylQ were detected using a CoulochemIII electro-
chemical detector with the guard cell voltage set to
+650 mV and the analytical cell voltages set to E1
)500 mV, E2 + 350 mV. The order and times of elution
were approximately CoQ
2
(9 min), CoQ
2
H

2
(11 min),
decylQ (17 min) and decylQ
2
(22 min). The area under
each peak was integrated and compared with standards
containing known amounts (0–200 pmol) of CoQ
2
and
decylQ. CoQ
2
H
2
and decylQH
2
were quantitated by
assuming a similar peak intensity to their oxidized coun-
terparts and rerunning of samples after slow oxidation
under air suggested this was a valid assumption as loss of
area from the CoQ
2
H
2
and decylQH
2
peaks was compara-
ble with the gain in size of the CoQ
2
and decylQ peaks.
Determination of the fraction of CoQ

2
and decylQ that
could be reduced by mitochondria was as above with some
modifications. The BY4743Dcoq2 strain was grown in
YPGG for 24 h, at which point it had reached A
600
 0.8
and become stationary. As we were interested in their redox
state, either 10 lm decylQ or 10 lm CoQ
2
was added sepa-
rately so they could not equilibrate. The cultures were incu-
bated for a further 3 h before a sample was taken and
incubated for a further 2 min with either 1 lm FCCP or
200 lm KCN. The sample was centrifuged and the superna-
tant removed before the pellet was dried and snap frozen.
CoQ
2
or decylQ was extracted from the pellet and quanti-
tated as above.
Ubiquinone movement in mixed vesicle
populations
Vesicles were prepared largely as described previously
[12]. Briefly, a bulk population of vesicles was created
from 25 mgÆmL
)1
turkey egg yolk phosphatidylcholine
(Type XII-E, Sigma) in chloroform, supplemented with
100 lm Pyr16. This mixture was evaporated to dryness
under a stream of nitrogen in a 15 mL glass Kimax tube.

Residual chloroform was removed under vacuum over-
night before the addition of sufficient KP
i
buffer [50 mm
KP
i
-KOH (pH 7.8), 100 lm EDTA] to give a final phos-
phatidylcholine concentration of 1 mgÆmL
)1
. This was
incubated for 1 h at room temperature, then vortexed
vigorously and placed in a Decon F5 Minor sonicating
water bath for 30 min at room temperature. The second
added population was created as above, but 5 mm of
either CoQ
2
, decylQ, CoQ
4
or CoQ
9
was added to the
chloroform. Pyrene quenching was assayed by adding
2 mL of the first bulk vesicle suspension (1 mgÆmL
)1
)
containing only Pyr16 in a stirred cuvette with a Shima-
dzu RF 5301-PC fluorimeter (k
ex
346 and k
em

377) at
30 °C. One 500 lL addition of the second vesicle popula-
tion containing either CoQ
2
, decylQ, CoQ
4
or CoQ
9
was
made after 90 s, such that the final ubiquinone concentra-
tion was 40 lm. The data are expressed as the ratio of
fluorescence at a given point in time (I) to initial fluores-
cence just before this addition (I
0
).
Acknowledgement
This work was supported by the Medical Research
Council (UK).
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