Keywords ABC transporter; ER targeting; membrane protein trafficking transport ATPase; mitochondrial import; mitochondrial targeting sequence Correspondence R.. Abbreviations ABC, ATP-bi
Trang 1transport complex MDL1 from post-translational
mitochondrial import to endoplasmic reticulum insertion Simone Gompf1, Ariane Zutz1, Matthias Hofacker1, Winfried Haase2, Chris van der Does1
and Robert Tampe´1
1 Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany
2 Max-Planck Institute of Biophysics, Structural Biology, Frankfurt am Main, Germany
ATP-binding cassette (ABC) transporters belong to a
large family of membrane proteins found in all three
kingdoms of life The chemical energy of ATP is used
to drive uphill transport of a broad range of solutes
across membranes [1–3] ABC transporters have a
conserved domain organization consisting of two
trans-membrane domains (TMDs) and two
nucleotide-bind-ing domains (NBDs) The TMDs form a translocation
pore, whereas the NBDs catalyze ATP hydrolysis
The ABC half-transporter multidrug resistance like
protein 1 (MDL1), composed of a TMD followed by a
NBD, is located in the inner mitochondrial membrane
(IMM) of Saccharomyces cerevisiae It has been sug-gested to be involved in the export of 6-mer to 20-mer peptides, derived from proteolysis of nonassembled inner membrane proteins by the m-AAA (i.e matrix-oriented ATPase associated with a variety of cellular activities) protease [4] It has been further reported that MDL1 mediates resistance against oxidative stress and can partially complement the function of ABC transporter of mitochondria (ATM) 1 [5] Deletion of ATM1 in S cerevisiae results in a severe growth defect because ATM1 is essential for the biogenesis of cyto-solic iron-sulfur (Fe-S) proteins [6]
Keywords
ABC transporter; ER targeting; membrane
protein trafficking transport ATPase;
mitochondrial import; mitochondrial
targeting sequence
Correspondence
R Tampe´, Institute of Biochemistry,
Biocenter, Johann Wolfgang
Goethe-University, Max-von-Laue-Strasse 9,
D-60348 Frankfurt am Main, Germany
Fax: +49 (0) 69 798 29495
Tel: +49 (0) 69 798 29475
E-mail: tampe@em.uni-frankfurt.de
Website: http://www.biochem.
uni-frankfurt.de
(Received 25 May 2007, revised 5 July
2007, accepted 20 August 2007)
doi:10.1111/j.1742-4658.2007.06052.x
The ATP-binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes In the present study,
we identified an unusually long leader sequence of 59 amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotide-binding domain oriented to the matrix By contrast, MDL1 lacking this lea-der sequence is directed into the endoplasmic reticulum membrane with the nucleotide-binding domain facing the cytosol Remarkably, in both target-ing routes, the ATP-bindtarget-ing cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis The physiological con-sequences of both targeting routes were elucidated in cells lacking the mito-chondrial ATP-binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron-sulfur proteins The mitochondrial MDL1 com-plex can complement ATM1 function, whereas the endoplasmic reticulum-targeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Datm1 growth phenotype
Abbreviations
ABC, ATP-binding cassette; ATM, ABC transporter of mitochondria; ER, endoplasmic reticulum; 5-FOA, 5-fluoroorotic acid; IMM, inner mitochondrial membrane; MDL1, multidrug resistance like protein 1; MTS, mitochondrial targeting signal; NBD, nucleotide-binding domain;
SC, synthetic complete; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane; TMD, transmembrane domain.
Trang 2Mitochondria contain approximately 800–1500
dif-ferent proteins [7,8] Although they include mtDNA
and a transcription⁄ translation machinery, the vast
majority of mitochondrial proteins are encoded by
nuclear genes and synthesized as precursor proteins on
cytosolic ribosomes [9–13] Several pathways of
mito-chondrial protein import have been characterized:
(a) the presequence pathway for matrix proteins;
(b) sorting and assembly of anchored mitochondrial
outer membrane proteins by transmembrane b-strands,
and (c) the carrier pathway for hydrophobic inner
membrane proteins [14]
The mitochondrial targeting signal (MTS) of
pro-teins directed to the IMM is recognized by receptors
of the translocase of the outer mitochondrial
mem-brane (TOM complex) The classical targeting signal is
represented by an N-terminal leader sequence of 20–
35 amino acids [15], enriched in basic, hydrophobic
and hydroxylated residues [16] It has been suggested
that the leader peptide folds into a defined secondary
structure, which is essential for protein import, due to
the distribution of charged and apolar residues The
N-terminal part of the MTS forms a positively charged
amphiphilic a-helix or b-sheet, whereas the C-terminal
region probably serves as a recognition site for matrix
proteases [15,17] Positional amino acid preferences
have been found in the region immediately upstream
from the mature amino terminus [18] In particular,
arginine can be enriched in position -2, -3, -10, and -11
relative to the cleavage site The leader sequence
inter-acts with the TOM receptor, responsible for the
trans-location of the preproteins to the translocase of the
inner mitochondrial membrane (TIM)23 complex
located in the IMM The presequence
translocase-asso-ciated motor is directly assotranslocase-asso-ciated with TIM23 and
completes the translocation of the preprotein into the
matrix There the presequence is removed by the
mito-chondrial processing peptidase Subsequently, IMM
proteins are guided by a hydrophobic sorting sequence
that typically follows the positively charged
pre-sequence [19,20]
In the present study, we addressed the functional
role and physiological consequences of the unusual
long N-terminal leader sequence of MDL1
Full-length MDL1 is targeted to the IMM, whereas the
leaderless ABC transporter is exclusively inserted into
endoplasmic reticulum (ER) membrane Despite these
presequence-dependent trafficking routes, the
mem-brane insertion, the complex assembly, and the
ATPase function of MDL1 are preserved The
physiological consequence of these two targeting
routes is addressed by in vivo complementation in
cells lacking the mitochondrial ABC transporter
ATM1, which is essential for the assembly of cyto-solic Fe-S proteins
Results
Targeting of MDL1 to the IMM
It has been postulated that MDL1 is involved in the export of peptides generated (e.g from misassembled mitochondrially encoded respiratory chain subunits) [4] Unfortunately, the mechanism and transported substrate remain largely elusive due to the intrinsic dif-ficulties in studying mitochondrial export processes This is due to the fact that substrates are limited in the matrix and their concentrations are very difficult to control experimentally In addition, substrates are highly diluted after translocation into the external medium By contrast, many intracellular transport sys-tems have been characterized in detail by means of uptake assays; for example, the transporter associated with antigen processing (TAP) [21,22] and TAP-like (ABCB9) [23] We therefore set out to target MDL1 from mitochondrial import to insertion into the ER membrane in order to perform similar analyses
An introduced ClaI restriction site and the endo-genous BamHI site divided the MDL1 gene into three cassettes, facilitating the exchange of segments between different constructs By means of the inducible GAL1-promoter, the protein can be over-produced to a level
of approximately 1% of the total mitochondrial pro-tein This correlates with an over-expression compared
to native MDL1 of up to 100-fold To determine the localization of MDL1 in S cerevisiae, mitochondria of Dmdl1⁄ MDL1 cells were prepared by subcellular frac-tionation As shown in Fig 1A, MDL1 is found in the mitochondrial fraction even after over-expression Immunoblotting of marker proteins (TIM23 and SEC61) confirms that the mitochondrial fraction contains only traces of ER membranes In addition,
we analyzed the subcellular localization of MDL1 by immunogold labeling (Fig 1B) As expected, MDL1 was detected exclusively in cristae membranes, demon-strating that the nuclear encoded protein is post-transla-tionally targeted to mitochondria Identical results were obtained using a C-terminally His-tagged version
of MDL1 (data not shown)
Post-translational maturation of MDL1
Mitochondrial ABC transporters do not exhibit signifi-cant sequence similarities in their leader sequences In the case of MDL1, several algorithms for the predic-tion of mitochondrial targeting sequences gave rather
Trang 3conflicting results We therefore set out to examine
the post-translational modification experimentally
After purification via a C-terminal His-tag from
iso-lated mitochondria (Fig 2A), N-terminal sequence of
MDL1 was determined by Edman degradation An
unusually long presequence of 59 amino acids was
identified The N-terminus (position 2–6) of the
iso-lated, mature ABC transporter (ESDIAQ) matches
perfectly with residue S61 to Q65 (Swiss-Prot: P33310)
Surprisingly, we found that the glutamine expected at
position 60 (the newly generated N-terminus) had been
modified to a glutamate Sequencing of the expression
construct and comparison with the protein data bank
confirmed the glutamine at position 60 We can further
exclude modifications during purification because
MDL1 was prepared from isolated mitochondria
Taken together, we identified two post-translational
modifications of MDL1: first, cleavage after residue 59
in the mitochondrial matrix releasing a long
prese-quence and, second, an enzymatic deamidation of the
newly generated N-terminal glutamine to glutamate
Such modification has been reported for cytosolic
proteins (N-end rule pathway) [24] and for at least two
mitochondrial proteins, TIM44 and COX4 [25,26] To
date, it is not clear whether this modification is an
artifact of Edman degradation or whether this
deami-dation is catalyzed by a N-terminal amidase during
mitochondrial translocation
We next examined the membrane targeting of MDL1 lacking the mitochondrial leader sequence iden-tified in the present study Thus, MDL1(60-695) was generated and expressed in S cerevisiae By contrast to the full-length protein, we found leaderless MDL1 cofractionated with the ER marker SEC61 (Fig 1A)
As a control, the mitochondrial marker TIM23 is found only in the mitochondrial fraction, whereas SEC61 is enriched in the ER fraction, but can also be found in the mitochondrial fraction In parallel, the subcellular localization of leaderless MDL1 was confirmed by immunogold labeling (Fig 1C) MDL1 lacking the MTS was detected in tubulo-vesicular membranes resembling the yeast ER membrane It is worth men-tioning that the N-terminally tagged MDL1(60-695) was also targeted to the ER, as demonstrated by sub-cellular fractionation and immunogold labeling (data not shown) This suggests that mistargeting is due to
of a lack of the leader sequence rather than the new N-terminus Collectively, these data demonstrate that leaderless MDL1 is targeted to and inserted into the
ER membrane by a cryptic default pathway
Directionality of membrane insertion
The orientation of the full-length and leaderless ABC transporter in mitochondrial and ER membranes, respectively, was examined by protease protection
A
leader-less MDL1 in S cerevisiae ER and mito-chondrial membranes were prepared from cells over-expressing wild-type MDL1 and leaderless MDL1(60-695) (A) and analyzed
by SDS ⁄ PAGE (10%) and immunoblotting using antibodies specific for MDL1, the mitochondrial maker TIM23 and the ER mar-ker SEC61 Immunogold labeling of sections through cells over-expressing wild-type MDL1 (B) and leaderless MDL1(60-695) (C) Full-length MDL1 is localized in the mito-chondrial cristae membranes, whereas lead-erless MDL1 is detected in tubulo-vesicular membranes belonging to or deriving from the endoplasmic reticulum M, mitochon-dria; N, nucleus; V, vacuole.
Trang 4assays As expected, MDL1 targeted to the IMM was
resistant to trypsin digestion because it was shielded
by the outer mitochondrial membrane (Fig 3A) To
determine the orientation of MDL1 in the IMM,
mitoplasts and inverted IMMs were prepared and
assayed for protease cleavage A factor Xa cleavage
site was engineered at the C-terminus of MDL1 before
the His8-tag Thus, if the C-terminus is accessible to
the protease, the His-tag will be cleaved off as detected
by immunoblotting with His-tag specific antibodies
This way, MDL1 in mitoplasts was shown to be
protected against factor Xa cleavage, whereas the
C-terminus of MDL1 was accessible in the inverted
IMMs, demonstrating that the ABC transporter was inserted with the NBDs oriented to the matrix (Fig 3B) By contrast to full-length MDL1 expressed
in the IMM, trypsin treatment of ER membranes con-taining leaderless MDL1 resulted in a limited digestion
of MDL1 (Fig 3C) Trypsin treatment digestion of ABC half-transporters resulted in the cleavage of the linker region between TMD and NBD [27] Even at
5 lgÆmL)1of trypsin, the NBDs of MDL1 were specif-ically cleaved, indicating that they were oriented in the cytoplasm In conclusion, the NBDs of mitochondrial MDL1 are located in the matrix, whereas the NBDs of the leaderless MDL1 targeted to the ER membrane face the cytosol
MDL1 forms homooligomeric complexes with similar activities independent of the targeting route
ABC half-transporters must assemble at least into dimeric complexes to gain function To analyze whether both targeting routes are comparable in com-plex assembly and ATPase activity, the full-length and leaderless MDL1 were purified to homogeneity via metal affinity chromatography, yielding approximately
20 lgÆg)1 wet weight of yeast in both cases (Fig 2) After isolation from different cellular compartments,
we investigated complex formation of MDL1 by gel fil-tration Each fraction was subsequently analyzed by SDS⁄ PAGE and immunoblotting (Fig 4A) The mito-chondrial as well as ER-resident MDL1 forms homo-oligomeric complexes of similar size The broad distribution is rather typical for digitonin solubilized ABC transport complexes Notably, no protein aggre-gates were detected at the exclusion volume Other detergents resulted in MDL1 complexes, which rapidly lost their ATPase activity [28] To demonstrate that the broad distribution is not due to misfolding, we per-formed an alternative approach, where we investigated the oligomeric state of MDL1 by Blue-Native electro-phoresis (Fig 4B) Full-length and leaderless MDL1 solubilized from yeast membranes migrate as defined bands at approximately 250 kDa, which corresponds
to a homodimeric complex, as resolved by single parti-cle electron microscopy analysis [28] In summary, MDL1 forms a homodimeric complex independent of its subcellular targeting
The ATPase activity of ABC half-transporters is critically dependent on the complex formation We therefore compared the ATPase activity of MDL1 tar-geted to different cellular compartments (Fig 5A,B) Mitochondrial MDL1 isolated from total membranes was active in ATP hydrolysis with a Km ATP of
A
B
Fig 2 Purification of MDL1 For purification of MDL1 (A) and
MDL1(60-695) (B) with C- or N-terminal His-tags, respectively, total
membranes were prepared from S cerevisiae over-expressing the
protein Membranes (10 mgÆmL)1) were solubilized in the presence
of 1% (w ⁄ v) digitonin The protein was purified to homogeneity by
metal affinity chromatography Pellet (P) and supernatant after
solu-bilization (S), flow-through (FT), and fractions with increasing
concentrations of imidazole were analyzed by SDS ⁄ PAGE (10%,
Coomassie Blue stained, upper panel) and immunoblotting with
anti-MDL1 serum (lower panel).
Trang 5120 ± 6 lm and a turnover rate kcat of 74 ± 1 ATPÆ
min)1 (per monomer) In comparison, leaderless
MDL1 purified from microsomes showed a Km ATP of
200 ± 1 lm and a kcat of 77 ± 1 ATPÆmin)1 (per
monomer) To exclude the possibility that the activity
is caused by contaminating ATPases, we expressed and
purified two MDL1 variants (E599Q and H631A),
each of which has a disrupted catalytic dyad
MDL1(E599Q) and MDL1(H631A) show no ATPase
activity above background but are active in ATP
bind-ing [28] We further examined whether MDL1 show
similar sensitivity towards vanadate inhibition in both targeting routes As shown in Fig 5C,D, the ATPase activity of MDL1 purified from mitochondria or ER membranes was inhibited in a dose-dependent manner
by ortho-vanadate Comparable to other ABC trans-porters [29–31], the IC50 values of 0.86 mm and 1.1 mm were determined for the mitochondrial and ER-resident MDL1, respectively Taken together, full-length and leaderless MDL1 are comparable in respect
to assembly of homooligomeric complexes, ATPase activity, and vanadate inhibition
A
C
B
Fig 3 Membrane orientation of mitochon-drial and ER-resident MDL1 Mitochonmitochon-drial (A) and ER fractions (C) (30 lg each) con-taining wild-type MDL1 and leaderless MDL1(60-695), respectively, were incubated with increasing concentrations of trypsin (0–0.1 mgÆmL)1) and analyzed by SDS ⁄ PAGE (10%) followed by immunoblot-ting (B) Mitoplasts and inverted IMMs (IMV) (30 lg each) containing MDL1 were incubated with factor Xa (0.5 lg) and analyzed by SDS ⁄ PAGE (10%) and immuno-blotting In this case, MDL1 contains a C-terminal His-tag separated by a factor Xa cleavage site.
A
B
Fig 4 Formation of homooligomeric com-plexes of mitochondrial and ER-resident MDL1 Purified MDL1 (upper panel) and leaderless MDL1(60-695) (lower panel) were analyzed by gel filtration on a Superdex 200
PC 3.2 in the presence of 0.1% (w ⁄ v) digitonin Every second fraction (30 lL) was analyzed by immunoblotting using an MDL1-specific antibody (A) Total membranes (10 mgÆmL)1) of cells expressing MDL1 or MDL1(60-695) were solubilized
in presence of 1% (w ⁄ v) digitonin.
(B) Digitonin-solubilized proteins were analyzed by Blue-Native electrophoresis and immunoblotting using anti-MDL1 serum Apoferritin (443 kDa), b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), and albumin (66 kDa) were used as markers.
Trang 6Uptake assays with isolated microsomes
containing MDL1
Leaderless MDL1 is targeted to ER membranes, where
the NBDs of the homooligomeric complex are oriented
to the cytosol In this orientation, the ATP level and
substrates can effectively be controlled To identify the
MDL1 substrate, we screened combinatorial peptide
libraries of different length Xn (n¼ 5–8, 11, 17, and
23, where X represents an equimolar distribution of all
19 amino acids except cysteine) These libraries have
been instrumental in deciphering the substrate
specific-ity of several eukaryotic and prokaryotic ABC
trans-porters [21,23,32,33] In addition, we analyzed a set of
defined peptides expected to be a putative substrate of
MDL1 These include, for example, N-formylated
pep-tides or fragments of mitochondrially encoded gene
products, which have been identified as minor antigens
[34] Systematic uptake assays with these peptidic
sub-strates, however, showed no MDL1-specific transport
activity, suggesting that MDL1 may be not a general
peptide transporter such as TAP or TAP-like, but
most likely transports a very specific or even modified
peptide
Physiological function of MDL1 targeted to
different membranes
As shown in Fig 6, MDL1 complements the severe
growth defect of Datm1 cells, indicating that the ABC
transporter can at least partially restore the assembly
of essential cytosolic Fe-S proteins We next generated
a set of mutants defective in ATP binding and hydro-lysis Mutation of the conserved lysine in the
Walk-er A motif (K473A) is known to inhibit ATP binding, whereas substitutions in the catalytic dyad (E599Q or H631A) inhibit ATP hydrolysis [35–37] Importantly, these three mutants did not complement the Datm1 phenotype Together with in vitro experiments these data demonstrate for the first time that ATP binding and hydrolysis are required for MDL1 function
It has very recently been shown that the ATPase activity of ATM1 is stimulated by cysteine-containing peptides [38] We therefore generated a cysteine-less MDL1 and examined its function by in vivo comple-mentation Datm1⁄ MDL1(Cys-less) cells found to be viable, demonstrating that cysteine residues are not essential for MDL1 function By contrast to wild-type MDL1, the leaderless protein did not restore ATM1 function Taken together, the function of MDL1 in rescuing the cytosolic Fe-S cluster assembly machinery requires ATP binding and hydrolysis and is strictly coupled to its post-translational targeting to the mito-chondrial membrane
Discussion
Most mitochondrial proteins are synthesized by free ribosomes in the cytosol Once released into the cyto-plasm with an N-terminal MTS, these preproteins are imported into the mitochondria post-translationally [39] MTS usually consists of 20–35 residues and is highly degenerated in primary sequence, but is rich in basic, hydrophobic and hydroxylated residues and
Fig 5 ATPase activity and vanadate
inhibi-tion of purified MDL1 ATPase activities
were measured as a function of ATP
concentration for 10 min at 30 C with
0.5 l M of purified protein MDL1 (A) and
MDL1(60-695) (B) showed Michaelis–
Menten kinetics with a K m ATP of
120 ± 6 l M and 200 ± 1 l M and a kcatof
74 ± 1 ATPÆmin)1and 77 ± 1 ATPÆmin)1
(per MDL1 monomer), respectively.
Inhibition of ATPase activity of MDL1 (C)
and MDL1(60-695) (D) by different
concentrations of ortho-vanadate (given in
l M ) Based on the curve fit half-maximal
inhibitory concentrations (IC50) of 860 l M
and 1.1 m M were determined All values are
derived from triplicate measurements.
Trang 7generally lacks acidic amino acids [16] For
post-trans-lational targeting of MDL1 to the IMM, a 59 amino
acid long mitochondrial leader sequence was identified,
which is cleaved in the matrix Subsequently, the
resulting N-terminal glutamine is converted to a
gluta-mate Limited protease protection assays confirmed
that MDL1, even after over-expression, is efficiently
imported into mitochondria and properly inserted into
as well as assembled in the IMM with the NBDs
facing the mitochondrial matrix Based on sequence
comparison, MDL1 should function as an exporter of
solutes to the intermembrane space It is worth noting
that murine ABCB10, the closest homolog of MDL1,
also possesses an exceptionally long presequence of
105 amino acids [40] Membrane topology algorithms predict either five or six transmembrane helices for MDL1 Protease accessibility assays and post-transla-tional modifications revealed that the NBD and the highly positively charged N-terminus of mature MDL1 are located in the mitochondrial matrix Based on these data, we propose that MDL1 comprises six transmembrane helices
In the present study, we addressed the functional role of the unusually long leader sequence of MDL1 in its subcellular targeting and physiological conse-quences By contrast to the full-length protein, which
is efficiently imported into mitochondria, leaderless MDL1 is exclusively targeted to ER membranes
Fig 6 Physiological function of MDL1 vari-ants analyzed by in vivo complementation Datm1 ⁄ ATM1 + MDL1 cells were plated on SCD without uracil and tryptophan and used for replica plating Selection plates contain-ing 5-FOA were incubated at 30 C for
7 days MDL1 can complement the severe growth defect of Datm1 cells, whereas mutants K473A, E599Q, H631A, inactive in ATP binding or hydrolysis, as well as MDL1(60-695) do not show complementa-tion of ATM1 MDL1(Cys-less) is able to take over the function of ATM1 and do not affect growth.
Trang 8Protease accessibility assays demonstrated that the
NBDs of the ER-resident transporter are oriented to
the cytosol The localization is not influenced by
addi-tional C- or N-terminal His-tags either for full-length
or leaderless MDL1
To exclude that full-length and leaderless MDL1
have different activities, the proteins were purified to
homogeneity (Fig 2) Remarkably, full-length and
leaderless MDL1 form homooligomeric complexes of
the same size and similar ATPase activities, Km ATP
values of 120 lm and 200 lm and kcat values of
74 ATPÆmin)1and 77 ATPÆmin)1(per MDL1 subunit),
respectively The ATPase activity of the transport
complex is in very good agreement with data of the
mitochondrial ABC transporter ATM1 [38] and the
purified NBD of MDL1 [36] Both full-length and
leaderless MDL1 show sensitivity to ortho-vanadate,
similar to other ABC transporters [29–31]
MDL1 over-expressed in microsomes provides an
optimal setting to study the substrate specificity and
function of this sparingly characterized ABC
trans-porter Based on a rather indirect assay, it has
previ-ously been concluded that MDL1 exports peptides of
6–20 amino acids in length [4] To our surprise, no
transport activity was observed for microsomal MDL1
by screening combinatorial peptide libraries of
differ-ent lengths (Xn, n¼ 5–23 amino acids) Notably, this
approach has been crucial in the identification of the
substrate specificity of other peptide transporters
[21,23,32,33,41] In addition, defined peptides favored
by the homologous TAP complex, such as the peptide
RRYQKSTEL, are not transported by MDL1
Recently, a peptidic fragment, named COXI, of a
mitochondrially encoded subunit of the cytochrome
oxidase was identified to be presented on MHC class I
molecules of murine cells [42] It was suggested that
COXI is transported from the matrix to the cytosol,
where the peptide is funneled into the pathway of
MHC class I antigen processing [34,43] Thus,
N-terminal 7-, 9- and 12-mer fragments of COXI were
analyzed for an MDL1-dependent transport activity
However, no uptake was detected Taken together,
these findings suggest that MDL1, if indeed a peptide
transporter, is highly specific for a small set of peptides
or even modified peptides largely under-represented in
the peptide libraries These systematic studies point to
an intriguing possibility that MDL1 may require
addi-tional factors for substrate transfer Such factors may
be absent in uptake studies or in the libraries used
Similar ATPase activities prove that the NBDs of both
MDL1 variants are correctly folded, although it
can-not be excluded that their TMDs are influenced by the
lipid compositions of the corresponding membranes
Based on the important role of ATM1 in the biogen-esis of cytosolic Fe-S proteins, Datm1 cells show a severe growth phenotype When Datm1⁄ ATM1 cells are forced to loose the plasmid-encoded ATM1 (URA3 marker) by growth on 5-fluoroorotic acid (5-FOA), Datm1 cells are almost nonviable Multicopy expres-sion of MDL1 (Datm1⁄ MDL1) can rescue this pheno-type and cells are viable on fermentable carbon sources [5] This implies that ATM1 and MDL1 have
an overlapping function by which the growth pheno-type of Datm1 cells is abrogated However, by contrast
to ATM1 [38], no stimulation of the ATPase activity
of MDL1 was observed with thiol-containing peptides
of 10–15 residues in length (data not shown) A recent report suggested that thiol-containing molecules are first translocated by ATM1 and afterwards oxidized by ERV1 These events are necessary for the maturation
of cytosolic and nuclear Fe-S proteins [38] However, a functional overlap between ATM1 and MDL1 with regard to the translocation of thiol-containing peptides appears to be very unlikely
By analyzing several mutants, we demonstrated for the first time that ATP binding and ATP hydrolysis are required for the export function of MDL1 These results are supported by data obtained in vitro showing that the mutants K473A, E599Q and H631A are inac-tive in ATP hydrolysis [28] Notably, cysteine-less MDL1 rescues ATM1 function, demonstrating that cysteines are not essential for substrate translocation across the IMM by MDL1 The conclusion that cyste-ines are not involved in substrate translocation is also
in line with the observation that the ATPase activity
of MDL1 is not stimulated by cysteine-containing pep-tides (see above) Leaderless MDL1, although correctly assembled and fully active in ATP hydrolysis, does not complement the growth phenotype of Datm1 cells This finding attests that the physiological function of the ABC transporter MDL1 is intimately linked to its correct targeting to the IMM
Experimental procedures
Materials
A rabbit polyclonal antibody was generated against the C-terminal 15 amino acids (KGGVIDLDNSVAREV) of MDL1 from S cerevisiae
Cloning and expression of MDL1
The MDL1 gene from S cerevisiae was divided into three cassettes, separated by a newly generated silent ClaI restric-tion site at S221 and the endogenous BamHI site at K422
Trang 9[28] Cassette I includes the N-terminal part of the TMD
(M1 to A220), cassette II the C-terminal part of the TMD
(S221 to K422), and cassette III the NBD of MDL1 (D423
to V695) Furthermore, leaderless MDL1 (cassette IB, Q60
to A220) was generated The different cassettes of MDL1
were amplified from genomic DNA (for sequences of the
primers, see Table 1) The corresponding PCR fragments
were cloned downstream of the GAL1-promoter in the
pYES2.1⁄ V5-His-TOPO expression vector (Invitrogen,
Carlsbad, CA, USA) resulting in plasmids pMDL1 and
pMDL1(60-695) Using primers p1C(f) and p3(r), a similar
approach was applied to insert an N-terminal His10-tag
fol-lowed by leaderless MDL1(60-695) resulting in
pMDL1(60-695,His) pMDL1(His), comprising four glycines, a
factor Xa cleavage site, and a His8-tag downstream of
MDL1, was generated with primers p1(f) and p3B(r)
Plasmids were transformed into Dmdl1 strain Y24137
(BY4743; Mat a⁄ a; his3D1 ⁄ his3D1; leu2D0 ⁄ leu2D0; lys2D0 ⁄
LYS2; MET15⁄ met15D0; ura3D0⁄ ura3D0; YLR188w::
kanMX4⁄ YLR188w) [44] Transformed cells were cultured
at 30C in synthetic complete (SC) medium in the presence
of 2% (w⁄ v) glucose without uracil [45] Cultures were
diluted to an A600 nm of 0.4 in SC medium containing 2%
(w⁄ v) galactose and growth was continued for 12 h Cells
were harvested by centrifugation and immediately used for
the isolation of total membranes [46] or mitochondria
[28,47] Microsomes were separated from mitochondria by centrifugation of the resulting supernatant at 100 000 g for
45 min at 4C (Ti45, Beckman Coulter, Fullerton, CA, USA) Mitoplasts and inverted inner mitochondrial vesicles are prepared as described [48,49] The proteins were analyzed
by SDS⁄ PAGE and immunodetection using the MDL1-specific antibody Protein concentrations were determined using the Bradford assay (Pierce, Rockford, IL, USA)
Immunogold labeling
S cerevisiae expressing full-length MDL1 or leaderless MDL1(60-695), with and without the corresponding His-tags, were fixed with 4% paraformaldehyde in 0.1 m sodium cacodylate buffer (pH 7.2) supplemented with 0.8 m sorbitol, 1 mm MgCl2and 1 mm CaCl2with or without 1% glutardialdehyde After 2 h, the fixative was exchanged for cacodylate buffer containing decreasing concentrations of sorbitol (0.5, 0.25, 0 m; three times 10-min incubation for each concentration) Cells were treated with 1% sodium meta-periodate, washed in water, and incubated in 0.05 m
NH4Cl After 12 h, cells were washed again and enclosed in agar-agar, which then was cut into small slices and passed through increasing concentrations of ethanol for dehydra-tion Samples were stepwise infiltrated with LR White resin
Table 1 Primers used for generating MDL1 constructs f, forward primer; r, reverse primer; mut, mutagenesis primer (exchanged bases underlined).
GATACGTCTTTGTAAAGG
GAAGTCC
CACCATCAATCAGACATTGCGCAAGGAAAGAAGTCC
GTCC
ATGCCGCCCTTCGATGCCGCCGCCGCCTACTTCCCGG GCAACACTATTGTCC
a Restriction endonuclease site introduced by primer b The BamHI site is found up- or downstream of the primer.
Trang 10(London Resin Company Ltd, Reading, UK) and
polymer-ized for 30 h at 55C Thin sections were cut from the
resin bloc and transferred onto formvar-coated nickel grids
For immunogold labeling, grids were placed on drops of
the respective solutions in the following order: saturated
sodium meta-periodate; water; NaCl⁄ Pi containing 2%
glycine; NaCl⁄ Pi; NaCl ⁄ Pi containing 1% BSA, 0.1%
Tween 20, NaCl⁄ Pi, 0.1% BSA, 0.05% Tween 20 Sections
were incubated with the anti-MDL1 serum After removal
of unbound antibodies, sections were incubated with
sec-ondary goat anti-rabbit serum coupled to gold particles
(diameter of 10 nm) Carefully washed slices were briefly
treated with 1% glutardialdehyde in NaCl⁄ Pi and, after
contrasting with uranyl acetate and lead citrate,
prepara-tions were analyzed by electron microscopy (EM 208S, FEI
Company, Eindhoven, the Netherlands)
Blue-Native PAGE
Total membranes (10 mgÆmL)1) were solubilized in
digito-nin buffer [20 mm Tris⁄ HCl pH 7.4, 50 mm NaCl, 10%
(v⁄ v) glycerol, 1 mm EDTA, 1 mm phenylmethanesulfonyl
fluoride, 1% (w⁄ v) digitonin (Calbiochem, Darmstadt,
Ger-many)] for 1 h at 4C under gentle rotation Loading dye
(10 mm Bis-Tris pH 7, 50 mm e-amino-n-caproic acid, 5%
(w⁄ v) Coomassie Blue (G) was added to solubilized
mate-rial after ultracentrifugation (100 000 g, 30 min, 4C) [28]
Blue-Native electrophoresis (gradient 6.0–16.5%) was
per-formed as previously described [50] Apoferritin (443 kDa),
b-amylase (200 kDa), alcohol dehydrogenase (150 kDa),
and albumin (66 kDa) were used as markers
Limited trypsin digestion and factor Xa cleavage
To determine the membrane orientation of MDL1 in
iso-lated organelles, 15 lg of organelles were incubated for
15 min on ice with increasing concentrations of trypsin (up
to 0.1 mgÆmL)1) Proteolysis was stopped by addition of
tri-chloroacetic acid to a final concentration of 7.5% (v⁄ v)
After subsequent centrifugation, pellets were washed with
ice-cold acetone and resuspended in sample buffer [51] For
factor Xa cleavage, mitoplasts and inverted IMMs (30 lg)
were incubated with 0.01 mgÆmL)1 factor Xa in 20 mm
Tris⁄ HCl pH 8.0, 100 mm NaCl, and 2 mm CaCl2 for
30 min at 25C The reaction was stopped by addition of
sample buffer and incubation for 10 min at 65C The
accessibility to trypsin and factor Xa was determined by
immunodetection with MDL1-specific and anti-His-tag
(Novagen, San Diego, CA, USA) sera
Purification of MDL1
Total membranes (10 mgÆmL)1) were solubilized in
buf-fer A (20 mm Tris⁄ HCl pH 8.0, 150 mm NaCl, 15% (v ⁄ v)
glycerol, EDTA-free complete protease inhibitor cocktail (final concentration according to manufacturer, Roche, Mannheim, Germany), 1% (w⁄ v) digitonin (Calbiochem)) for 1 h at 4C under gentle rotation Nonsolubilized mate-rial was removed by ultracentrifugation (100 000 g, 30 min,
4C; Ti80, Beckman Coulter) and the soluble fraction was loaded onto a 1 mL Ni2+-High-Trap Chelating column (GE Healthcare, Piscataway, NJ, USA) equilibrated with buffer B (20 mm Tris⁄ HCl pH 8.0, 150 mm NaCl, 15% (v⁄ v) glycerol, 2 mm imidazole, 0.1% (w ⁄ v) digitonin) After washing with buffer B containing 80 and 160 mm imidazole, the protein was eluted in buffer B containing
400 mm imidazole
Gel filtration
Full-length and leaderless MDL1 were analyzed by gel fil-tration on a Superdex 200 PC 3.2 (GE Healthcare) equili-brated with SEC buffer (20 mm Tris⁄ HCl pH 8.0, 150 mm NaCl and 0.1% (w⁄ v) digitonin); 60 lg of protein was loaded at a flow rate of 50 lLÆmin)1 30 lL fractions were collected and analyzed by SDS⁄ PAGE and immunoblotting using anti-MDL1 serum Ferritin (443 kDa), b-amylase (200 kDa), and BSA (70 kDa) in SEC buffer without deter-gent were used for calibration
ATPase assays
The ATPase activity was essentially determined as described [31] 20 mm dithiothreitol was added to 1 lm purified MDL1 The reaction was started by addition of ATP con-taining buffer (20 mm Tris⁄ HCl pH 8.0, 150 mm NaCl,
20 mm MgCl2, 0.1% (w⁄ v) digitonin, 10 mm ATP traced (370 000 : 1) with [c-32P]ATP (specific activity 110 TBqÆ mmol)1; Hartmann Analytic, Braunschweig, Germany) in a
1 : 1 ratio at 30C The reaction was stopped after 10 min
by adding 1 mL of 10 mm ammonium molybdate in 1 m HCl Subsequently, 15 lL of 20 mm H3PO4and 2 mL of a butanol⁄ cyclohexane ⁄ acetone (5 : 5 : 1) mixture were added After rigorous vortexing, the organic phase was extracted and the radioactivity was quantified by liquid scintillation b-counting (Beckman LS6500 Liquid Scintillation Counter; Beckman Coulter Inc., Fullerton, CA, USA) Km ATPvalues were derived by fitting the data to the Michaelis–Menten equation Specific inhibition of the ATPase activity was ana-lyzed at various concentrations of vanadate, using the char-coal adsorption method in combination with [c-32P]ATP [52] 0.5 lm of purified MDL1 was incubated with increasing concentrations of ortho-vanadate By addition of buffer supplemented with ATP, the reaction was initiated and incu-bated for 15 min at 30C 750 lL of ice-cold 10% charcoal
in 10 mm EDTA were added to terminate the reaction After rigorous agitation, reactions were incubated for 3 h on ice to allow maximal binding of free ATP to the charcoal After