Universitas Carolina Pragensis
Facultas Mathematica Physicaque
Abstract of
Doctoral Thesis
Fakulta Matematicko-fyzikální, Univerzita Karlova Praha, Ceská Republika
&
Fyziologický ústav Akademie ved Ceské republiky Ceské Republiky
Transport zprostredkovaný odprahujícím proteinem-1 (UCP1) a jeho mutanty
Autoreferát doktorské disertacní práce
Eva Urbánková
Obor
F4 – Fyzika molekulárních a biologických struktur
Praha 2002
Faculty of Mathematics and Physics, Charles Universtiy Prague, Czech Republic
&
Institute of Physiology, Academy of Sciences of the Czech Republic
The transport mediated by wild type and mutants of uncoupling protein 1 (UCP1)
Summary of Ph.D. Thesis
Eva Urbánková
Branch
F4 – Physics of molecular and biological structures
Prague 2002
Výsledky této disertacní práce byly získány behem doktorandského studia na Matematicko-fyzikální fakulte Univerzity Karlovy v Praze v letech 1997-2002.
Uchazec: Mgr. Eva Urbánková
Fyziologický ústav AV CR
Vídenská 1083, 142 20 Praha 4-Krc
Školitel: RNDr. Petr Ježek, DrSc.
Fyziologický ústav AV CR
Vídenská 1083, 142 20 Praha 4-Krc
Konzultant:
Školící pracovište: Fyziologický ústav AV CR
Vídenská 1083, 142 20 Praha 4-Krc
Oponenti: Doc. RNDr. Dana Gášková, CSc.
Fyzikální ústav UK, Matematicko fyzikální fakulta Ke Karlovu 5, 121 16 Praha 2
RNDr. Jan Krušek, CSc Fyziologický ústav AV CR
Vídenská 1083, 142 20 Praha 4-Krc
Autoreferát byl rozeslán dne:
Obhajoba se koná dne v hod. pred komisí pro obhajoby
disertacních prací v oboru F4-Fyzika molekulárních a biologických struktur na MFF UK, Ke Karlovu 3, 120 00 Praha 2, v zasedací místnosti c. 105.
S disertacní prací se lze seznámit na Útvaru doktorandského studia MFF UK, Ke Karlovu 3, Praha 2.
Predseda oborové rady F4: Doc. RNDr. Otakar Jelínek, CSc.
INTRODUCTION... 1
UNCOUPLING OF MITOCHONDRIA... 1
MITOCHONDRIAL ANION CARRIER PROTEIN (MACP) FAMILY... 2
UNCOUPLING PROTEINS... 2
THEORETICAL PART: UNCOUPLING PROTEIN FAMILY - COMPARISON OF SEQUENCES [25]... 3
THE MATRIX UCP-SPECIFIC SEQUENCE DOES NOT APPEAR IN BMCP1. ... 3
UCP-SIGNATURES IN TRANSMEMBRANE SEGMENTS... 4
CONCLUSION... 4
EXPERIMENTAL PART... 6
UCP1 AND ITS MUTANTS... 6
Methods... 6
Reconstitution of UCP1 into proteoliposomes and measurement of proton and chloride transport ... 6
Results and discussion ... 7
The proton and chloride leaks... 7
The proton and chloride transport mediated by UCP1 mutants and its kinetics ... 7
The binding of 3H-GTP to UCP1 mutants. ... 10
Discussion of the transport properties of constructed UCP1 mutants ... 10
Flip-flop of fatty acids - theory and experimental results ... 10
MEASUREMENT OF CONDUCTIVITY OF PLANAR LIPID BILAYER MEMBRANES WITH INCORPORATED UCP1... 12
Methods... 12
Results and discussion ... 12
Conclusions... 14
EFFECTS CAUSED BY UCP2 AND UCP3 IN MITOCHONDRIA... 14
Materials and Methods ... 14
Mitochondria – measurement of the membrane potential ... 15
Measurement of nucleotide binding to mitochondria ... 15
Results... 15
The membrane potential ... 15
Binding of GTP to mitochondria... 16
Discussion ... 17
CONCLUSION ... 19
REFERENCES... 20
LIST OF PAPERS ... 22
INTRODUCTION
Mitochondria have been attracting attention of scientists for many decades. Recently, new themes have emerged - apoptosis, 'mitochondrial diseases' and their genetics, relations of mitochondria to several other diseases (e.g. diabetes mellitus), aging and regulation of body weight [45], [46]. One of the extensively developing topics is the research on mitochondrial uncoupling proteins.
Uncoupling proteins form a subfamily of mitochondrial anion carrier protein family (MACP). The best understood is the originally known, UCP1. It is expressed exclusively in the brown adipose tissue of mammals and has been proved to uncouple mitochondria by enabling proton back-flux into the mitochondrial matrix. The function of other uncoupling proteins, UCP2-5 (and PUMP, plant uncoupling mitochondrial protein) is not understood so well. Although these proteins are proposed to be involved in various physiological and patophysiological phenomena, such as thermoregulation, fever, regulation of body weight, regulation of radical oxygen species formation, apoptosis, diabetes mellitus type II etc., current knowledge about their function is limited and there is a lot of controversy in the field. The recent results suggest that most probably even UCP2-5 uncouple mitochondria in a similar way, as does UCP1.
The other question concerns the mechanism of uncoupling function of UCPs. Even for UCP1, there is still no consensus how this protein really functions. There are two incompatible hypotheses describing the uncoupling mechanism.
In my work, I have concentrated to several aspects of UCP1-mediated transport:
First, we took advantage of known sequences of UCP1-5 and searched for aminoacid residues, which are conserved among UCPs, but don't exist in other related proteins [25].
(2.) In the second step, a few of these residues were mutated and the proton and chloride transport was studied in the resulted mutants.
(3.) In order to introduce a new method suitable for studying UCP-mediated transport, I developed a reconstitution of UCP1 into planar lipid bilayers and measured basic properties of such a system. This part of the work was done in collaboration with Dr. Elena E. Pohl from Humboldt university, Berlin and Dr. Peter Pohl, Forschungsinstitut für Molekuläre Pharmakologie, Berlin.
(4.) I have participated in the project aimed to detect UCP2 and/or UCP3 in mitochondria isolated from various tissues. The methods developed involved measurements of the mitochondrial membrane potential, mitochondrial respiration and evaluation of nucleotide binding to intact mitochondria. Some of the results are shown in this work.
Uncoupling of mitochondria
According Mitchell's chemiosmotic theory [33], respiration chain in mitochondria creates proton electrochemical potential gradient,
Eq. 1 pH
F RT
H =∆Ψ− ∆
∆ +
3 .
~ 2 µ
which is then used by F1F0ATPase to synthetize ATP and also to transport many kinds of metabolites to mitochondria (e.g. ADP/ATP antiport, phosphate import etc.).
When an uncoupler is added to mitochondria, it enables protons to get back to matrix and dissipates the proton electrochemical potential. The result is the fall of ? µH+ and the production of heat. There are synthetic uncouplers (dinitrophenol, FCCP, CCCP) as well as natural ones - uncoupling proteins (UCPs).
Fatty acids (FAs) are required for the uncoupling mediated by UCPs. Except of uncoupling proteins, for those uncoupling is their main function (this is known at least for UCP1), some other proteins also may uncouple mitochondria at certain conditions.
Mitochondrial anion carrier protein (MACP) family
Mitochondria require for their function not only proteins of the electron transport chain and the F1F0 ATP synthase, but also numerous other membrane proteins that facilitate the traffic of the substrates, ions and proteins required in the mitochondrial matrix. The inner membrane represents the only barrier for membrane-impermeable molecules. Proteins capable to translocate anions across the inner mitochondrial membrane form a gene family ("MACP family"). Uncoupling caused by fatty acids is not taking place exclusively in BAT mitochondria, where UCP1 is present. Other mitochondria are also uncoupled by FAs and it is supposed that other MACPs are also capable to exert fatty-acid induced uncouping.
Uncoupling proteins
There were five sequences of uncoupling proteins found in the human genome (all of them are found also in genomes of other mammals and some of them also in other kinds of vertebrates and invertebrates) and three in the Arabidopsis thaliana genome [17]. The first known uncoupling protein, UCP1, was found in mitochondria of brown adipose tissue (BAT). Its properties were known already from the studies on the whole mitochondria - proton back flux induced by fatty acids and binding of purine nucleotides, that inhibit the proton transport. The UCP1-enabled proton flux causes, that BAT mitochondria are uncoupled and produce heat, unless purine nucleotides are present. UCP2 is known to be expressed in all kinds of tissues studied [28], [34] and was suggested to be involved in diabetes mellitus (type II), apoptosis, fever, body weight regulation and defence against reactive oxygen species [69]. UCP3 is skeletal-muscle- specific [5] and supposed to enable muscle thermogenesis [69], [5]. Brain-specific UCP4 [32] and BMCP1 (or UCP5) [43] were discovered. An UCP4-like protein seems to be the ancestral protein to UCP subfamily [17].
There are two theories trying to explain the UCP1 function - fatty acid cycling theory [48] and fatty acid buffering theory [14]. According the first one, UCP1 has one transport pathway for various anions.
Protons are not transported by UCP1 itself, but when FAs are present in the membrane, UCP1 transports FA anions and protonated FAs return via flip-flop. When they release protons, the cycle is closed (see Figure 1 for more detailed explanation). According the FA buffering model, FAs (in an unknown stoichiometry) bind to UCP1 and provide a 'missing' carboxyl group(s) along the proton translocation path [14].
Support for FA cycling (the reasons are ordered from more abstract to more concrete):
Ø UCP1 belongs genetically to the family of anion carriers and is indeed known to transport various anions [23], [19].
Ø UCP1 is known to transport alkylsulfonates (more hydrophobic more easily), which are very similar to FAs, but their pK is much lower. Therefore they are not activating proton transport.
However, when propranolol is present (enables to the complex alkylsulfonate-H to get back), proton transport is observed [18].
Ø Alkylsulfonates competitively inhibit FA-induced proton transport. Cl- transport is competitively inhibited by alkylsulfonates and fatty acids [24], suggesting that only one transport pathway exists in UCP1 for FAs and other anions.
Ø FAs which are not able to flip-flop (acidify liposome interior) were not activating UCP1-mediated proton transport [21],[22].
Support for FA buffering
Ø Analogy with other H+ transporters, which contain an array of carboxyl groups facilitating the H+ transfer along the chain of water molecules [29]. Bound FAs could provide some of these groups.
Ø Much lower rate for Cl- transport than for H+ transport (~10x difference) [29].
Ø Existence of the mutant (E167Q), which is not able to transport Cl-, but H+ transport exerted by it is retained.[9]
Figure 1 Fatty acid cycling provided by UCP1. When FAanions are transported by UCP1 through the membrane, some of them become protonated (in order to retain the equilibrium between FA- and FA.H).
Some of FA.H then flip-flop to the other side of the membrane (in order to retain equilibrium of FA.H between the two lipid sheets of the membrane). Some of FA.H then deprotonate on the other side and
thus, protons are transported.
It seems that FA cycling gives scientifically 'smarter' explanation and is supported more strongly by experimental results, but it has to wait for its final proof.
THEORETICAL PART: UNCOUPLING PROTEIN FAMILY - COMPARISON OF SEQUENCES
We made an attempt to get as much as possible from analysis of UCP sequences. Main part of this approach laid in aligning known sequences of UCP's and their nearest relatives.
In order to predict common functional domains within the UCP subfamily, we have screened accessible UCP sequences and searched for similar sequence motifs that are unique just for UCPs within the MACP gene family. If a residue is common for UCPs and is not present in other MACPs, there is a probability, that it is involved directly in the UCP function (i.e. H+ uniport induced by fatty acids, transport of various anions and inhibition of this transport by purine nucleotides).
Some other carriers of the family were also shown to provide FA-induced uncoupling (inhibited not by PN, but by their specific inhibitors), but it is never the main function of these carriers and it is probably much weaker than UCP-provided uncoupling.
The matrix UCP-specific sequence does not appear in BMCP1.
The connecting matrix segments of UCPs always begin by the major part of the MACP signature sequence [1],[49],[50],[35]. One may speculate that this sequence predetermines the termination of the odd transmembrane segments and the formation of the matrix segments in all MACPs. Searching for the UCP-specific sequence motifs, we found first such a motif, specific for UCP-subfamily except of BMCP1, in the 2nd matrix segment. This UCP-specific sequence motif, an "UCP-signature", starts with Arg152 of UCP1 and its consensus sequence can be written as follows:
[
+
]- φ-X-Gly/Ser-Thr/n-X-NH/[−
]-Ala-φwhere [+](or [−]) stands for positively (negatively) charged residues; n is a nonpolar nonaromatic residue; φ is an aromatic residue; NH represents Asn or His. The whole matrix UCP signature is not contained in BMCP1 and in any of the MACPs of mammalian or yeast origin. But, its Arg pair and two Tyr exist in the yeast dicarboxylate transporter (DTP), while the last 7-residue motif exists in the YIA6 and YEA6 yeast carriers. In the following part of the 2nd matrix segment, Ile163 and Glu167 are conserved in UCPs and some MACPs.
UCP-signatures in transmembrane segments
The 1st UCP-specific motif appears to be the 8-residue motif preceding the MACP signature in the 1st transmembrane segment. It starts with Ala23:
Ala/Ser-Cys/Thr/n-n/Phe-Ala/Gly-[
−
]-n/Phe-n/Cys-Thr-Phe/n.With alternative first Ser, second Ile and Phe after Glu, this signature is valid also for BMCP1. The 2nd transmembrane UCP signature can be described as follows:
Gly/Ala-Ile/Leu-Gln/X-[
+
]-NH-n/Cys-Ser/n-φ/X-n/Ser-OH/Gly-n-[+
]-Ile/Met-Gly/Val-n/Thr, starting at Gly80 of UCP1, where [+
] is always Arg and the alternatives such as Cys-n instead of n-Ser, and Gly-Gly-n preceding the 2nd charge are specific for PUMPs.The 4th transmembrane segment starts at the matrix interface by one free residue followed by the Leu-Trp-[+]-Gly sequence that exists as Leu/Phe-φ-[+]/Gln(Ser)-Gly in MACPs and contains well conserved Gly. The next OH (Thr-Thr or Thr-Ser) doublet in UCP1 and UCP2 is altered by Trp, Leu or Gly in other UCPs. The next 16 residues of the 4th transmembrane segment form a homologous motif that starts with conserved Pro178 and contains one conserved negative charge, plus Arg182 in all UCPs but bovine UCP1, and 5 semiconserved residues in UCP subfamily:
Pro-Asn/Thr-n-X-[
+
]-Asn/Ser/Ala-n-Ile/Leu-n-Asn/Val-Cys/n-n/Thr-[−
]-n-n/Thr/Pro-OH/Val.We can define it as a 3rd UCP signature (4th transmembrane segment). The last residue is Thr or Ser (Val in BMCP1, that contains Pro prior to it), [+] is always Arg and [−] is Glu.
Conclusion
We have found several regions in UCP sequence, which have common character in UCPs, but are not present in other MACPs. We chose several residues from the 1st transmembrane and the 2nd matrix UCP signatures for the site-directed mutagenesis and characterized properties of respective UCP1 mutants (see the next chapter).
Figure 2 Alignment of uncoupling protein sequences as compared to the yeast dicarboxylate carrier (DTP) sequence and majority sequences of ADP/ATP carrier and phosphate carrier.
1st cytosolic segment 1st transmembrane segment 1st matrix segment 2nd transmembrane helix
Signatures / A C n A - n n T F# # # # # # # # # Q n Q + V L G T n T T n n+ - G I Q + Q n S f n S n + I G n
MACP conserved S T F G C n* * * L M n n !* * A L X H C n X S T M V T
n (for UCP1-3, PUMP) G
UCP1 hamster V N P T T S E V H P T M G V K 16I F S A G V AAC L ADI ITFP L DT AKVRLQIQ GE G Q I S S T I RY K GV L GTI T T L A K TE GL P K L Y SGL P A G I QRQ I S F A S LRI G LY95 Human M G G L T A S D V H P T L G V Q 17L F S A P I AAC L ADV ITFP L DT AKVRLQVQ GE C P T S S V I RY K GV L GTI T A V V K TE GR M K L Y SGL P A G L QRQ I S S A S LRI G LY96 Mouse V N P T T S E V Q P T M G V K 16I F S A G V SAC L ADI ITFP L DT AKVRLQIQ GE G Q A S S T I RY K GV L GTI T T L A K TE GL P K L Y SGL P A G I QRQ I S F A S LRI G LY95 Rabbit M V G T T T T D V P P T M G V K 17I F S A G V AAC L ADV ITFP L DT AKVRQQIQ GE F P I T S G I RY K GV L GTI T T L A K TE GP L K L Y SGL P A G L QRQ I S F A S LRI G LY96 Rat V S S T T S E V Q P T M G V K 16I F S A G V SAC L ADI ITFP L DT AKVRLQIQ GE G Q A S S T I RY K GV L GTI T T L A K TE GL P K L Y SGL P A G I QRQ I S F A S LRI G LY95
Bovine 1I F S A G V AAC V ADI ITFP L DT AKVRLQIQ GE C L I S S A I RY K GV L GTI I T L A K TE GP V K L Y SGL P A G L QRQ I S L A S LRI G LY80
UCP2 human M V G F K A T D V P P T A T V K 17F L G A G T AAC I ADL ITFP L DT AKVRLQIQ GE S Q G P V R A T A S A QY R GV M GTI L T M V R TE GP R S L Y NGL V A G L QRQM S F A S VRI G LY100 Mouse M V G F K A T D V P P T A T V K 17F L G A G T AAC I ADL ITFP L DT AKVRLQIQ GE S Q G L V R T A A S A QY R GV L GTI L T M V R TE GP R S L Y NGL V A G L QRQM S F A S VRI G LY100 Rat M V G F K A T D V P P T A T V K 17F L G A G T AAC I ADL ITFP L DT AKVRLQIQ GE S Q G L A R T A A S A QY R GV L GTI L T M V R TE GP R S L Y NGL V A G L QRQM S F A S VRI G LY100 UCP3 human M V G L K P S D V P P T M A V K 17F L G A G T AAC F ADL VTFP L DT AKVRLQIQ GE N Q A V Q T A R L V QY R GV L GTI L T M V R TE GP C S P Y NGL V A G L QRQM S F A S IRI G LY99 Mouse M V G L Q P S E V P P T T V V K 17F L G A G T AAC F ADL LTFP L DT AKVRLQIQ GE N P G A Q S V QY R GV L GTI L T M V R TE GP R S P Y SGL V A G L HRQM S F A S IRI G LY96 Rat M V G L Q P S E V P P T T V V K 17F L G A G T AAC F ADL LTFP L DT AKVRLQIQ GE N P G V Q S V QY R GV L GTI L T M V R TE GP R S P Y SGL V A G L HRQM S F A S IRI G LY96 Bos M V G L Q P S E R P P T T S V K 17F L A A G T AAC F ADL LTFP L DT AKVRLQIQ GE N Q A A L A A R S A QY R GV L GTI L T M V R TE GP R S L Y SGL V A G L QRQM S F A S IRI G LY99 Sus M V G L K P P E V P P T T A V K 17L L G A G T AAC F ADL LTFP L DT AKVRLQIQ GE N Q A A R S A QY R GV L GTI L T M V R NE GP R S P Y NGL V A G L QRQM S F A S IRI G LY96 StPUMP M G G G D H G G K S D I S F A G 17I F A S S A F AAC F AEA CTLP L DT AKVRLQLQ K K A V E G D G L A L P KY R GL L GTV G T I A K EE GI A S LWKGI V P G L HRQ C I Y G G LRI GMY100 AtPUMP M V A A G K S D L S L P K 14T F A C S A F AAC V GEV CTIP L DT AKVRLQLQ K S A L A G D V T L P KY R GL L GTV G T I A R EE GL R S LWKGV V P G L HRQ C L F G G LRI GMY96 UCP4 M S V P E E E E R L L P L T Q RW P R A S K 23F L L S G C AAT V AEL ATFP L DL TKTRLQMQ GE A A L A R L G D G A R E S A PY R GM V RTA L G I I E EE GF L K LWQGV T P A I YRH V V Y S G GRM V TY109 BMCP1 M G I F P G I I L I F L R V K F A T A A insert1 WK 43P F V Y G G L A S I V AEF GTFPVDL TKTRLQVQ GQ S I D A R F K E I KY R GM F H A L F R I C K EE GV L A L Y SGI A P A L LRQ A S Y G T IKI G IY125 DTP M S T N A K E S A G K N I K Y PW W 19Y G G A A G I F A T M VTHP L DL AKVRLQ A A P M P K P T L FR M L E S I L A NE GV V G L Y SGL S A A V LRQ C T Y T T VRF G AY89 AAC majority X X X X X X X X X X X X F n X D 12F L M G G n SAA V S K T A A APIER VKL L IQNQ-E M n K Q G X n n X XYXGI n-C F X R T n+-E GF n S FWRGN L A N V nRY F P T Q A n N F A F+93
main alternationsM S D A A V K N A A I A V S V H 0 A S R A I R + K V n K n P TNH n S A L Y T F
S T A
PiC majority M F S S V A H L A R A N P F N P L V H DiA E f G S G K f f n L52C n L G G n L S C G L T H T A n VP L Dn VKCRnQV D P Q KY K Gn F N G F S V T n K EE Gn R G L A KGWA P T F L G Y S n Q G L CKF G FY125
main alternations Q L V E S S K Q K R K S n S D K F G S S T T T N E n T S n T S I K K n A G K G S T T T n n G
2nd cytosolic segment 3rd transmembrane helix 2nd matrix segment 4th transmembrane helix 3th cytosolic segment
Signatures / - G Q # # # # # # # # # Q + +f X G T X N A f - LW P N n X R N n I n N C n-n n Tf- + -
MACP * A N! - S n - ! ! ! * T T S L V n T T S!
conserved not for BMCP H ! A P V
UCP1 hamsterDT V Q E Y F S S GK E T P P T L G N R I S A G L M TGG V A V F I G QP T EVV KVRLQA Q S H L H G IKP RY T G T Y N A Y R IIA T TE S F S TLWKGT TP NL LRN V I INC VEL V TY DL MKG A L V N N Q I L ADD V P212 Human DT V Q E F L T A GK E T A P S L G S K I L A G L T TGG V A V F I G QP T EVV KVRLQA Q S H L H G IKP RY T G T Y N A Y R IIA T TE G L T GLWKGT TP NL MRS V I INC TEL V TY DL MKE A F V K N N I L ADD V P213 Mouse DS V Q E Y F S S GR E T P A S L G N K I S A G L M TGG V A V F I G QP T EVV KVRMQA Q S H L H G IKP RY T G T Y N A Y R VIA T TE S L S TLWKGT TP NL MRN V I INC TEL V TY DL MKG A L V N N K I L ADD V P212 Rabbit DT V Q E F F T S GE E T P S L G S K I S A G L T TGG V A V F I G QP T EVV KVRLQA Q S H L H G LKP RY T G T Y N A Y R IIA T TE S L T SLWKGT TP NL LRN V I INC TEL V TY DL MKG A L V R N E I L ADD V P212 Rat DT V Q E Y F S S GR E T P A S L G S K I S A G L M TGG V A V F I G QP T EVV KVRMQA Q S H L H G IKP RY T G T Y N A Y R VIA T TE S L S TLWKGT TP NL MRN V I INC TEL V TY DL MKG A L V N H H I L ADD V P212 Bovine DT V Q E F F T T GK E A S L G S K I S A G L M TGG V A V F I G QP T EVV KVRLQA Q S H L H G PKP RY T G T Y N A Y R IIA T TE G L T GLWKGT SP NL T T N V I INC TEL V TY DL MKE A L V K N K L L ADD V P195 UCP2 humanDS VKQ F Y T KGS E H A S I G S R L L A G S T TGA L A V A V A QP T DVV KVRFQA Q A R A G G GR RY Q S T V N A Y K TIA R EE G F R GLWKGT SP NV ARN A I VNC AEL V TY DL IKD A L L K A N L M TDD L P215 Mouse DS VKQ F Y T KGS E H A G I G S R L L A G S T TGA L A V A V A QP T DVV KVRFQA Q A R A G G GR RY Q S T V E A Y K TIA R EE G I R GLWKGT SP NV ARN A I VNC AEL V TY DL IKD T L L K A N L M TDD L P215 Rat DS VKQ F Y T KGS E H A G I G S R L L A G S T TGA L A V A V A QP T DVV KVRFQA Q A R A G G GR RY Q S T V E A Y K TIA R EE G I R GLWKGT SP NV ARN A I VNC TEL V TY DL IKD T L L K A N L M TDD L P215 UCP3 humanDS VKQ V Y T P KGA D N S S L T T R I L A G C T TGA M A V T C A QP T DVV KVRFQA S I H L G P S R S DR KY S G T M D A Y R TIA R EE G V R GLWKGT LP NI MRN A I VNC AEV V TY DI LKE K L L D Y H L L TDN F P218 Mouse DS VKQ F Y T P KGA D H S S V A I R I L A G C T TGA M A V T C A QP T DVV KVRFQA M I R L G T G G ER KY R G T M D A Y R TIA R EE G V R GLWKGT WP NI TRN A I VNC AEM V TY DI IKE K L L E S H L F TDN F P214 Rat DS VKQ F Y T P KGT D H S S V A I R I L A G C T TGA M A V T C A QP T DVV KVRFQA M I R L G T G G ER KY R G T M D A Y R TIA R EE G V R GLWKGT WP NI TRN A I VNC AEM V TY DI IKE K L L D S H L F TDN F P214 Bos DS VKQ F Y T P KGS D H S S I I T R I L A G C T TGA M A V T C A QP T DVV KIRFQA S M H T G L G G NR KY S G T M D A Y R TIA R EE G V R GLWKGI LP NI TRN A I VNC GEM V TY DI IKE K L L D Y H L L TDN F P217 Sus DS VKQ L Y T P KGS D H S S I T T R I L A G C T TGA M A V T C A QP T DVV KVRFQA S I H A G P R S NR KY S G T M D A Y R TIA R EE G V R GLWKGI LP NI TRN A I VNC AEM V TY DV IKE K V L D Y H L L TDN L P214 StPUMP EP VKN L Y V GK D H V G D V P L S K K I L A A L T TGA L G I T I A NP T DLV KVRLQA E G K L P A G V PR RY S G A L N A Y S TIV K QE G V R ALWTGL GP NI GRN A I INA AEL A SY DQ VKE A V L R I P G F TDN V V219 AtPUMP EP VKN L Y V GK D F V G D V P L S K K I L A G L T TGA L G I M V A NP T DLV KVRLQA E G K L A A G A PR RY S G A L N A Y S TIV R QE G V R ALWT V L GP NV ARN A I INA AEL A SY DQ VKE T I L K I P G F TDN V V215 UCP4 EH LRE V V F GK S E D E H Y P L W K S V I G G M M AGV I G Q F L A NP T DLV KV Q MQM E G K R K L E GKP LRF R G V H H A F A KIL A EG G I R GLWAGWVP NI QRA A L VNM GDL T TY DT VKH Y L V L N T P L EDN I M229 BMCP1 Q S LKR L F V E R L E D E T L L I N M I C G V V SGV I S S T I A NP T DV LKIRMQA Q G S L F Q G S M I G S F I DIY Q QE G T R GLWRGV VPT A QRA A I V V G VEL P VY DI TKK H L I L S G M M GDT I L236 DTP DL LKE N V I P R E Q L T N M A Y L L P C S M F SGA I G G L A G N F ADVVN IRMQN D S A L E A A K R RN Y K N A I D G V Y K I Y R Y EG G L K TLF TGWKP NM VRG I L M T A S Q V V TY DV FKN Y L V T K L D F D A S K N207 AAC majority DK fKX n F X X X X-+R X G YW+f F n G N L A S G G n AGA T S L X F V YPLDf A R TRL A A D A+n n K n X n-RE FNHG L n D V Y X K T n K TD G n n GLY RGF X n S V Q G I I I Y R G A Y F G nY DS nKP n n L X G X n X X NHF n214
main altern. Y n L 0 0 G + P N H X f n n M G S C S N S 0 S S S K S X Q + T N C n+ n F A S G P+A Q P C n V V A L F T F n 0 F - + OHI F
T n 0 S T T R
PiC majority EV FKX n Y S N M L G E E N A Y L f R T S L f L n A S A S A E F F A D I A L APMEA AK V RnQT Q P G f A N T L R G A A P K n Y K EE G n K A F Y KGV APLWMRQ I P Y T M M K F A C FER T V E A L Y X F V V P K P R S E240
main altern. F n D N Y T T S K N A T S A T F C F T V S K Q A P P K V D C F S H L N R K E S K S S F T L C N Y F n S Q F Y K F T n E D S
5th transmembrane segment 3rd matrix segment 6th transmembrane segment PNBD 4th cytosolic segment
Signatures / C H S n G F C # # # # # # # # # - + F n PS f L + n n S W N n n M f n C f-Q n + X X n
MACP conserved T A S L F * * * !*L X AN G T A S F S L!+ Q
G n P T
UCP1 hamster 213CHL L S A F V AGF C T T F L A SPAD VVKTRF I N S L P G Q Y P S V P SCA M T M L T KE GP T A F FK GF V P S F LRL A SWN V I M F V C FEQ LKK E L S K S R Q T V D C T T306 Human 214CHL V S A L I AGF C A T A M S SPVD VVKTRF I N S P P G Q Y K S V P NCA M K V F T NE GP T A F FK GL V P S F LRL G SWN V I M F V C FEQ LKR E L S K S R Q T M D C A T307 Mouse 213CHL L S A L V AGF C T T L L A SPVD VVKTRF I N S L P G Q Y P S V P SCA M S M Y T KE GP T A F FK GF V A S F LRL G SWN V I M F V C FEQ LKK E L M K S R Q T V D C T T306 Rabbit 213CHF V S A L I AGF C T T L L S SPVD VVKTRF I N S P P G Q Y A S V P NCA M T M F T KE GP T A F FK GF V P S F LRL G SWN V I M F V C FEK LKG E L M R S R Q T V D C A T306 Rat 213CHL L S A L V AGF C T T L L A SPVD VVKTRF I N S L P G Q Y P S V P SCA M T M Y T KE GP A A F FK GF A P S F LRL G SWN V I M F V C FEQ LKK E L M K S R Q T V D C T T306 Bovine 196CHF V S A V V AGF C T T V L S SPVD VVKTRF V N S S P G Q N T S V P NCA M M M L T RE GP S A F FK GF V P S F LRL G SWN I M F V C FER LKQ E L M K C R H T M D C A T288 UCP2 human 216CHF T S A F G AGF C T T V I A SPVD VVKTRY M N S A L G Q Y S S A G HCA L T M L Q KE GP R A F YK GF M P S F LRL G SWN V V M F V T YEQ LKR A L M A A C T S R E A P F309 Mouse 216CHF T S A F G AGF C T T V I A SPVD VVKTRY M N S A L G Q Y H S A G HCA L T M L R KE GP R A F YK GF M P S F LRL G SWN V V M F V T YEQ LKR A L M A A Y Q S R E A P F309 Rat 216CHF T S A F G AGF C T T V I A SPVD VVKTRY M N S A L G Q Y H S A G HCA L T M L R KE GP R A F YK GF M P S F LRL G SWN V V M F V T YEQ LKR A L M A A Y E S R E A P F309 UCP3 human 219CHF V S A F G AGF C A T V V A SPVD VVKTRY M N S P P G Q Y F S P L DCM I K M V A QE GP T A F YK GF T P S F LRL G SWN V V M F V T YEQ LKR A L M K V Q M L R E S P F312 Mouse 215CHF V S A F G AGF C A T V V A SPVD VVKTRY M N A P L G R Y R S P L HCM L K M V A QE GP T A F YK GF V P S F LRL G AWN V MM F V T YEQ LKR A L M K V Q V L R E S P F308 Rat 215CHF V S A F G AGF C A T V V A SPVD VVKTRY M N A P P G R Y R S P L HCM L R M V A QE GP T A F YK GF M P S F LRL G SWN V MM F V T YEQ LKR A L M K V Q V L R E S P F308 Bos 218CHF V S A F G AGF C A T L V A SPVD VVKTRY M N S P P G Q Y H S P F DCM L K M V T QE GP T A F YK GF T P S F LRL G SWN V V M F V T YEQ MKR A L M K V Q M L R D S P F311 Sus 215CHF V S A F G AGF C A T V V A SPVD VVKTRY M N S P P G Q Y Q N P L DCM L K M V T QE GP T A F YK GF T P S F LRL G SWN V V M F V S YEQ LKR A L M K V Q M L R E S P F308 StPUMP 220THL I A G L G AGF F A V C I G SPVD VVKSRMM G D S A Y K N T L DCF V K T L K ND GP L A F YK GF I P N F GRL G SWN V I M F L T LEQ AKK F V K S L E S P 306 AtPUMP 216THI L S G L G AGF F A V C I G SPVD VVKSRMM G D S G A Y K G T I DCF V K T L K SD GP M A F YK GF I P N F GRL G SWN V I M F L T LEQ AKK Y V R E L D A S K R N 306 UCP4 230THG L S S L C SGL V A S I L G TPAD VIKSRI M N Q P R D K Q G R G L L Y K S S T DCL I Q A V Q GE GF M S L YK GF L P SWLRM T PWS M V FWL T YEK IR E M S G V S P F 323 BMCP1 237THF V S S F T CGL A G A L A S NPVD VVRTRMM N Q R A I V G H V D L Y K G T V D G I L K M W K HE GF F A L YK GFWP NWLRL G PWN I I F F I T YEQ LKR L Q I 325 DTP 208THL T A S L L AGL V A T T V C SPAD VMKTRI M N G S G D H Q P A L K I L A D A V R KE GP S F M FR GWL P S F TRL G P F T M L I F F A IEQ LKK H R V G M P K E D K 298 AAC majority 214A S F n n GWS n T n n A G n n S YPfDT VRRRMM M T S G+ -n K Y X G S n DCf+K I n X XE Gn K S n FK GA G A N I LR G n A G A G V L n n YD+L Q n L n F G K K f+303
main altern. W A Q T T S T C n I L Q - n R N S T F HAn S Q V F+ P X A F CW S V F A G A F I S -I I F n Q A V n
n N T L n Q E - Q
PiC majority C S K P E Q L V V T F V A G Y I AGn F C A I V S HPADS V n S V L N K E K G S S A G X n L K R LGF X G VWK GL F A R n n M I G T L T A L QW F I YDS VKV Y F R L P R P P P P E M P E S L K K K L G A T Q346
main altern. S S S T N L n S L T S n A Q N L K G Q K S D K Q S S T E S TA C T S G F S n n T G T S G S G S G n G A S Y K K
BMCP1 insert1 21V I V S G H Q K S T T V S H E M S G L N PiC majority i 21G L X D S R S S S P n P X G P R R R N L
E n P A n D A Q H H
The absolutely conserved residues and charges in UCPs are white in black boxes, the semiconserved residues (at least in 3 UCPs) are shadowed. The MACP-signatures are marked by #, the defined UCP-signatures are written above as consensus sequences (dotted background; f stands for aromatic residue, other symbols see text). Stars depict the residues well conserved in the MACP family members (up to 10 exceptions); exclamation marks refer to the "quite conserved" residues (from 10 to 20 exceptions). Majority sequences of AAC and PiC are based on the prevailing residues (with the most frequent alternatives listed in the row below) among 19 and 6 sequences of different species of AAC and PiC, respectively. Unique AAC sequences are outlined by the dotted background. The transmembrane regions are considered according to Klingenberg [28]. The alignment was performed using the clustal method (Megalign program from the Lasergene 99 sequence analysis system, DNASTAR).
EXPERIMENTAL PART
UCP1 and its mutants
In this chapter, the analysis of transport properties of several mutants of UCP1 is shown. The transport mediated by UCP1 (mutants) was measured in proteoliposomes, using fluorescent probe, SPQ, to detect the concentration changes of the ions studied - protons and chlorides. We characterized properties of UCP1 mutants, mutated in two important regions - the first group of mutated amino acid residues belonged to the "1st UCP-specific transmembrane motif". We studied mutants D27V, T30A and the triple- mutant C24A-D27V-T30A. The second group belonged to the second matrix loop - we tested the double mutant H145L-H147L and R152L, located in the "UCP-signature".
Figure 3 Amino acid residues of UCP1 mutated in this work. The model of transmembrane spanning of UCP1 is drawn with indicated AARs mutated (small elipses). Black regions indicate the UCP- signatures. The transmembrane segments are represented by cylinders 1-6. AARs at the interface of the
membrane are indicated by their sequence position numbers.
Methods
Rat UCP1 gene placed under the control of galactose promotor in the shuttle (Sacharomyces cerevisiae/Escherichia coli) vector pCGS 110 was PCR-amplified in the elongation process starting from two antiparallel primers carrying codon for alanine in place of original threonine. Vectors were proliferated in E. coli host, plasmid DNA was isolated and sequenced.1 Selected clones were electroporated into S. cerevisiae yeast and UCP1 expression was stimulated by addition of galactose. The isolation of yeast mitochondria followed in principle the same method as [36] with minor changes.
Reconstitution of UCP1 into proteoliposomes and measurement of proton and chloride transport I followed the method, originally based upon Klingenberg's protocol [30], adopted for fluorescent probes by Dr. Ježek [23], [19],[24] and in [37]. The fluorescent probe SPQ was used for the measurement of H+ efflux and Cl- uptake to liposomes.
The fluorescence was measured on the spectrofluorophotometer Shimadzu RF-5301PC, with Xenon lamp as an excitation source. SPQ fluorescence was excited at 340 nm (10 nm band-pass) and the signal collected at 444 nm (5 nm band-pass). The proton transport was detected via changes in SPQ fluorescence after addition of valinomycin. Typically, in the 10th second, a fatty acid was added. In the 30th second, valinomycin was added (final concentration 0.1 µM), which triggered the H+ transport. The calibration of
1 The site-directed mutagenesis was done by Mgr. Petr Hanák.
fluorescence signal was done according [23], [24]. When analyzing kinetic data of the transport, I used Michaelis-Menten formalism.
For the measurement of binding of 3H-GTP to UCP1 mutants, we adopted the anion-exchange method of Klingenberg [31] and measured GTP binding to UCP1 mutants.2 The protein content was measured by the Amido Black method, described in [27].
Results and discussion
The proton and chloride leaks
The proton and chloride leaks under different conditions (± laurate, ± K+ diffusion potential) were measured. The comparison of the proton leak and an example of UCP1-induced proton flux is shown at the Figure 4.
Figure 4 Different types of H+ leak in comparison with UCP1-induced H+ efflux.
0 10 20 30 40 50 60 70
no Val, laurate 100 µM
+Val, no laurate
+ Val, laurate 100 µM
+Val, laurate 100 µM, UCP1 wt
4 µg/mg H+ efflux [nmol.min-1 .mg lipidis-1 ]
The resembling permeability coefficient for proton leak in the presence of K+ diffusion potential is 8.5 .10-4 cm.s-1, which is a bit higher, than presented in literature under similar conditions (1.4 .10-4 cm.s-1 [8]). If we calculate the chloride permeability coefficient from Cl- leak observed in the absence of K+ diffusional potential (? Cl- 215 mM), we get the value (1.63 ± 0.5).10-10 cm.s-1. The Cl- leak in the presence of K+ diffusional potential (up to 8 nmol.min-1.mg lipids-1) was higher than observed values 4 nmol.min-1.mg lipids-1 [23] or 1.2 nmol.min-1.mg lipids-1 [36], but the ? K+ used in our experiments was also higher (215 mM vs. 150 mM).
The proton and chloride transport mediated by UCP1 mutants and its kinetics
In order to exclude the influence of non-specific proton leaks, the proton transport was analyzed by the means of the dependence of H+ flux J-J0 (in nmol.min-1) on the protein concentration in liposomes. From this dependence, the protein-dependent part of H+ flux, R (in nmol.min-1mg prot.-1), and the protein- independent part of the flux, R0 (in nmol/min) were separated according to the linear regression of Eq. 2
Eq. 2 J −J0 =R.[protein](mg)+R0
Linear regression of this dependence for wild-type UCP1 yielded ~ 13.5 µmol.min-1.mg prot-1, corresponding to 15 s-1 turnover per dimer (Figure 5b).
UCP1 mutants (except of T30A) exhibited quite flat dependence on the protein amount. Namely the triple UCP1 mutant C24A-D27V-T30A showed no dependence of H+ flux on the protein content. Typical
2 The method was introduced to our laboratory by Dr. Eva Škobisová.
examples of H+ efflux for wild type UCP1 and C24A-D27V-T30A mutant are shown at Figure 5b. As the H+ efflux was independent on protein content of C24A-D27V-T30A mutant, the curves shown resemble the H+ leak caused mainly by cycling of lauric acid via laurate-valinomycin complexes.
Figure 5 a) Lauric acid- induced H+ uniport as a function of incorporated protein for various UCP1 mutants. Rates of H+ efflux induced by 100 µM lauric acid in the presence of 0.1 µM valinomycin are plotted vs. the protein-to-lipid ratio (in µg protein per mg lipid). Full diamonds: wild-type UCP1; open
diamonds: T30A mutant; open squares: R152L mutant; full triangles: D27V mutant; open rings:
H145L-H147L mutant; full rings: C24A-D27V-T30A mutant. b) Typical runs of H+ efflux in the presence of 50 µM lauric acid shown for UCP1 wild type (wt) and C24A-D27V-T30A mutant. Runs
in the presence of 2.5 mM external ATP are shown (grey lines).
a) b)
0 10 20 30 40 50 60 70
0 2 4 6 8
protein/lipid ratio (µg/mg) H+ efflux (nmol.min-1 )
-4 -2 0 2 4 6 8 10
0 25 50 75 100
time (s) [H+ ] (nmol)
wt
+ ATP
+ ATP C24A- D27V- T30A
Other characteristics of studied mutants can be provided by the H+ uniport kinetics. The kinetic data also indicated the reduced ability to mediate FA-induced H+ uniport for all studied mutants but T30A. Figure 6a shows the direct kinetic plots..
The derived Vmax values are reduced nearly to half for the D27V and R152L mutants, to ~30% for the H145L-H147L mutant and to zero for the C24A-D27V-T30A mutant (Table 1). The apparent Kms for these mutants were 2 to 4 times higher (Km for the triple mutant cannot be derived due to zero approaching fluxes). Consequently, the apparent affinity of these mutants for lauric acid is much lower.
This affinity was ~2.5 times lowered also for the T30A mutant even when the Vmax value was not reduced (Table 1).
Figure 6 a) Direct kinetic plots for lauric acid- induced H+ uniport in proteoliposomes containing various UCP1 mutants. Rates of H+ uniport (efflux) per mg protein are plotted v.s. total concentration of
lauric acid used for uniport induction in the presence of 0.1 µM valinomycin. Rates of non-protein dependent transport (“H+ leak“), taken as intercepts of protein dependencies for each FA concentration such as shown in were subtracted from all the data. Full diamonds: wild-type UCP1; open diamonds:
T30A mutant; open squares: R152L mutant; full triangles: D27V mutant; open rings: H145L-H147L mutant; full rings: C24A-D27V-T30A mutant. b) Cl- uniport in proteoliposomes containing various
UCP1 mutants. Rates of Cl- uptake induced by 1 µM valinomycin are plotted vs. external Cl- concentration for various UCP1 mutants (the same symbols as in the left panel). The scales are the same
in order to compare the transport rates for H+ and Cl-.
a) b)
Table 1 Kinetic parameters for H+ and GTP dissociation constants for various UCP1 mutants.
Standard errors refer to the linear regressions of the data. Ratios are calculated at least from three experiments for each transport mode and mutant.
Mutant H+ efflux Vmax
(µmol.min-1mg-1)
H+ efflux Km (µM)
Kd of 3H-GTP binding [µM]
Wild type 18 ± 1 43 ± 5 1.6 ± 0.1
R152L 10 ± 1 93 ± 15 1.6 ± 0.4
H145L-H147L 5.6 ±0.9 79 ± 20 1.1 ± 0.3
D27V 11± 2 162 ± 36 1.8 ± 0.7
T30A 22 ± 2 100 ± 10 1.53 ± 0.05
C24A-D27V-T30A 0.2 ± 0.3 n.d. 1.5 ± 0.2
Unlike the protonophoric function, the ability to conduct a slow Cl- uniport was preserved in all mutants studied as shown by direct kinetic plots (Figure 6, right panel). However, because of a relatively slow transport, the results had a high experimental error. There were no significant differences in the apparent
