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 CoQ2.. Recently we have shown th
Trang 1coenzyme Q analogues requires the isoprenoid side chain Andrew M James1, Helena M Cocheme´1,2, Masatoshi Murai3, Hideto Miyoshi3
and Michael P Murphy1
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 (CoQ6), nine (CoQ9) and 10 (CoQ10) 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
CB2 0XY, UK
Fax: +44 1223 252905
Tel: +44 1223 252903
E-mail: aj@mrc-mbu.cam.ac.uk
Website: http://www.mrc-mbu.cam.ac.uk
(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 CoQ2for growth on glycerol and found that the first dou-ble bond of the initial isoprenoid unit is essential for utilization of respiratory substrates As CoQ2analogues 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 (CoQ6) is extremely hydrophobic and transported to mito-chondria via the endocytic pathway when supplied exogenously We found that CoQ2 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 CoQ2 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
hexadecanoic acid; YPD, yeast extract, peptone, glucose; YPG, yeast extract, peptone, glycerol; YPGG, yeast extract, peptone, glycerol with glucose.
Trang 2the 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] CoQ10levels 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 CoQ10have been used with
some beneficial effect in Parkinson’s disease [3] As the
requirement for high doses probably results from the
extreme hydrophobicity of CoQ10and 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 (CoQ2) or hydrophobic (CoQ4or CoQ6)
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 CoQ2 and CoQ6 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 CoQ2 (shown in Fig 1B) [7,8] The analogues differed from CoQ2 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-Q2) or sec-ond (A2-Q2) methyl group or second double bond (A3-Q2) 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-Q2 and A6-Q2), 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-Q2) 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
Trang 3side chain of CoQ Therefore, we set out to try and
understand the nature of this interaction better
All CoQ2analogues 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-Q2, A5-Q2and A6-Q2 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 O2
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 CoQ2 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 O2 This could arise via an
Time (h)
A600
0
2
4
6
8
10
12
14
Wild-type
Dcoq2 + 50 µM CoQ2
Dcoq2 + 50 µM decylQ
Dcoq2
0
2
4
6
8
10
A600
2
DecylQ A1-Q
2
A4-Q 2 A5-Q 2 A6-Q 2 A3-Q
2 A2-Q 2
O
O MeO
O
O MeO
O
O MeO
O
O MeO
O
O MeO
O
O MeO
O
O MeO
O
O MeO
O
O MeO
C
with growth above this requiring utilization of glycerol Values are the mean ± range of two independent experiments each carried out in duplicate.
Trang 4endogenous uptake pathway that recognizes the
iso-prenoid structure of CoQ2, but cannot interact with
the alkane tail of decylQ or any of the CoQ2analogues
lacking the first double bond That a CoQ transport
pathway exists for endogenous CoQ6in yeast has been
considered probable for some time, as the enzymes for
CoQ6 synthesis are located in the mitochondrial inner
membrane, but CoQ6 is found throughout the cell
[1,9] Spontaneous diffusion through the cytosol
appeared unlikely for this dispersion, as CoQ6 is very
hydrophobic with a predicted octanol⁄ water partition
coefficient in the region of 1014 [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 CoQ6 [11] To test whether the endocytic uptake pathway was also required for the uptake of short-chain ana-logues such as decylQ and CoQ2 in our experiments,
we created two double-knockout strains and tested their ability to grow on CoQ2 Both Dcoq2Dtlg2 and Dcoq2Derg2 strains grew in the presence of CoQ2 (Fig 3A) as well as the even more hydrophobic ana-logue CoQ4(data not shown) The growth characteris-tics were similar to those seen in the Dcoq2 strain
C
G3P
+
FCCP
2 min
myxo
15 10
20
–1 ·mg
–1 ·mg
0 50 100 150 200 250
0 50 100 150 200 250
0 50 100 150 200 250
0
50
100
150
200
250
0 50 100 150 200 250
0 50 100 150 200 250
0 50 100 150 200 250
0
50
100
150
200
250
indicated by the arrowheads That the oxygen consumption was mitochondrial in origin was confirmed by addition of the inhibitor
Trang 5(Fig 3D), suggesting that the endocytic pathway is not
required for the uptake of CoQ2 to the mitochondria
and, therefore, this pathway is unlikely to be
responsi-ble for discriminating between decylQ and CoQ2 That
a vesicle-based mechanism is not required for the
uptake is reasonable as the octanol⁄ PBS partition
coef-ficients of CoQ2( 104.5) and decylQ ( 105.5) are
sev-eral orders of magnitude lower than that of CoQ6
[6,10]
This suggests that CoQ2 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 CoQ2 or whether the accumulation of decylQ was
slower than for CoQ2 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 CoQ2 and decylQ it took 3–6 h for
growth to restart (Fig 3B) To determine if there were
differences in the accumulation of CoQ2 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 CoQ2 and
10 lm decylQ to a yeast suspension, the ratio of
decylQ to CoQ2 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 CoQ2and 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
CoQ2 and CoQ4, 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 CoQ2 analogues A4-Q2, A5-Q2 and A6-Q2, as well as with CoQ1, but it was not observed with 10 lm of CoQ9 or CoQ10 (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 CoQ2and CoQ4
To demonstrate that the physiochemical properties
of decylQ and CoQ2 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 I0⁄ I – 1, where I0is 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⁄ I0) 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 CoQ4, 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, CoQ2 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, CoQ9, there was no evidence of movement between the two populations during the course of the 10 min
Trang 6incubation, as relative fluorescence remained stable at
0.82 (Fig 3E) Therefore, both CoQ2 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 CoQ2 and decylQ within cells
(Fig 3C)
Finally, we sought to confirm that decylQ and CoQ2
both reach mitochondria in intact cells directly
DecylQ and CoQ2 participate effectively in respiration
by isolated mitochondria in the absence of endogenous CoQ6 (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 CoQ2for 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 (µ M )
0 1 2 3 4 5
Ubiquinone concentration (µ M )
0 1 2 3 4 5
Time (h)
A600
0 5 10 15 20 25
decylQ
A600
Time (h)
Δcoq2Δerg2 Δcoq2Δtlg2
0 1 2 3 4 5 6 7 8
decylQ
A600
A600
0 5 10 15 20 25
0 1 2 3 4 5 6
(h)
A600
E
CoQ2/decylQ
Ubiquinone-loaded vesicles
I/ 0
0 10 20 30 40 50 60 70 80
Ubiquinone
FCCP KCN
Time (min)
F
0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
CoQ2
CoQ4 CoQ 9
decylQ
Trang 7longer flow to O2, whereas FCCP uncouples
mitochon-dria, increasing respiration and oxidizing the
ubiqui-none pool as electrons rapidly flow from ubiquiubiqui-none
to O2 in the absence of a membrane potential [6]
Although CoQ2 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 CoQ2and 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 CoQ2 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 CoQ2is
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 CoQ2 (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 CoQ2 rather than of decylQ (Fig 1B)
DecylQ is not toxic, but does confound growth complementation by CoQ2
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 CoQ2 and decylQ to DCoQ cultures and measured their growth in YPGG over 48 h (Fig 4A) The addition of equimolar decylQ to CoQ2 had no significant detrimental effect on growth com-pared with CoQ2 alone Therefore, decylQ appears to
ubiquinone was subtracted to give the growth achieved on glycerol (B) Time lag in the growth response of stationary phase yeast to
hexane from both the supernatant and the pellet They were separated by HPLC and quantitated using electrochemical detection The values
Data are the mean ± standard error of the mean of three independent experiments (D) Even a 50-fold excess of decylQ above that required
The growth of the BY4743Dcoq2 strain in the absence of ubiquinone was subtracted to give the growth achieved on glycerol (E) DecylQ
expressed as the relative amount of fluorescence at a given point in time (I) to initial fluorescence just before the addition of
of three independent experiments.
Trang 8be 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 CoQ2 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 CoQ2 to DCoQ
cultures in YPGG and measured their growth over
48 h This showed that trace amounts of CoQ2did 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 CoQ2 alone (Fig 4B) Interest-ingly, there was a strong correlation between the frac-tion of the total added ubiquinone present as CoQ2 and growth (Fig 4C; r2= 0.99) Thus, it would appear that decylQ in some way prevents CoQ2 inter-acting with a protein that is required for nonfermenta-tive growth This might occur via decylQ binding directly to the protein and preventing CoQ2interacting
or growth could require an interaction with the reduced or the oxidized form of CoQ2 and decylQ dis-turbs the normal CoQ2H2⁄ CoQ2ratio
In summary, decylQ is not toxic, but can antagonize the stimulation of growth by CoQ2, especially when its concentration markedly exceeds that of CoQ2 This suggests that there may be a protein that regulates growth that can bind to CoQ2and 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 CoQ2 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
CoQ2 CoQ2 and equimolar decylQ
A600
0
1
2
3
4
5
6
X µ M decylQ + X µ M CoQ 2
X µ M decylQ + 5 µ M CoQ2
X µ M decylQ
Ubiquinone concentration (X µM)
A
B
0
0.5
1
1.5
2
2.5
([CoQ2] / [CoQ2+decylQ])
C
A600
A600
Fig 4 DecylQ is not toxic, but does interfere with growth
stimulated nor inhibited by the presence of equimolar decylQ The BY4743Dcoq2 strain was grown in YPGG supplemented with either
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
BY4743Dcoq2 strain was grown in YPGG with either decylQ alone
growth of the BY4743Dcoq2 strain in the absence of ubiquinone was subtracted to give the growth achieved on glycerol (C) Growth
Trang 9S 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 CoQ6 (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 CoQ2 This identified 379
double-knockout strains that could not grow on YPGG
containing CoQ2(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 CoQ4 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 Dcoq2-DORF counterparts from the original commercial library All
of these Dcoq2DORF strains grew by fermentation in YPD (yeast extract, peptone, glucose) to an A600 of
10 (data not shown) and in YPGG without CoQ4to
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
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.
Trang 10an A600 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 CoQ4, 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 CoQ4 for 48 h at
30C 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 30C 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 CoQ4 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 CoQ2 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 CoQ2 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-Q2, A5-Q2 and A6-Q2are not taken up correctly by this pathway Deletion of two endocytic proteins shown to be involved in the transport of exogenous CoQ6 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, CoQ2 and decylQ reached apparent equilibrium within minutes of being added to a culture of DCoQ yeast (Fig 3C) Even though 2 lm CoQ2 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, CoQ2 and CoQ4 indicated rapid redistribution
of decylQ and CoQ2 over a timeframe of seconds and CoQ4 within minutes (Fig 3E) CoQ2and 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 CoQ2 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 CoQ2 diffuses around blocks in the endocytic pathway used to transport CoQ6 to support growth on nonfer-mentable substrates (Fig 3A) However, even though
it appears possible for CoQ2 to diffuse to mitochon-dria, growth on glycerol with exogenously supplied CoQ2 is significantly slower than that observed on endogenous CoQ6 (Fig 1) One reason for the dimin-ished growth on exogenous CoQ2 relative to endoge-nous CoQ6 could be because the concentration of CoQ
in mitochondrial membranes appears significantly higher than that of other cellular membranes [11] Because CoQ2 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 CoQ2 cannot be accumulated and retained in a membrane system or via a decrease in