[27] ATP-binding cassette transporter in Saccharomyces cerevisiae mitochondria

[27]

ABC TRANSPORTERIN S. cerevisiae MITO('HONDP,IA

389

In binding experiments with the fluorescent DANS nueleotides to UCP in mitochondria D A N S G T P is preferred because it is not degraded by mitochondrial enzymes. 4° As shown in Fig. 4A, on addition of D A N S G T P to the mitochondria, the fluorescence increases biphasically, that is, a rapid followed by a slower phase. The equilibration can take as long as 30 rain. The specific portion of DANS nucleotide binding, that is, only to UCP, is defined by the decrease of fluorescence on addition of excess ATP or GTP. The fast phase represents binding to the free UCP, whereas the slow phase is rate limited by the slow dissociation of the prebound ATP. 4° After the prebound nucleotide is removed by treatment with Dowex at pH 8.(L the specific fluorescence (/5F) increases 54%, whereas the slower phase of fluorescence increment decreases 50% (Fig. 4B). This slow phase can be restored by preincubation with 1 /zM ATP to the Dowex-treated mitochondria (Fig. 4C). Figure 5 shows a typical fluorescence titration of Dowex-treated mitochondria with D A N S G T P . Similarly the two methods were employed to evaluate the binding. The results agree well with each other. 4oS.-G. Huang and M. Klingcnberg. Eter..1. Biochetn. 229, 718 (1995).

[27] A T P - B i n d i n g

Cassette cerevisiae

Transporter in Saccharomyces Mitochondria

B y JONATHAN LEIGHTON

Introduction During the past several years, a superfamily of membrane transport proteins termed ATP-binding cassette (ABC) transporters has received much attention. 1 These transporters are characterized by an organization of four domains (composed of between one and four polypeptides): two hydrophobic domains spanning the membrane six to eight times, and two hydrophilic domains, each containing a conserved region of about 200 amino acids termed the "ATP-binding cassette." Members of the ABC family have been discovered in a wide range of organisms and in several eukaryotic organelles. These proteins mediate the cross-membrane transport of a large spectrum of molecules, although each protein usually exhibits narrow substrate specificity. The ABC family has come to particular promi1C. F. Higgins, Ann. Rev. ('ell Biol. 8, 67 (1992).

MEFH()DS

IN P ~ N Z Y M O L O G Y . V O I . 21~0

( OpVFighl C) lgc)~ by A c a d e m i c lrhcss, hlc. All rights o l leprod{lctio[1 ill tiny hilt11 reserved.

390

ION AND METABOLITE TRANSPORT SYSTEMS

[27]

nence because some ABC transporters are of direct clinical significance, the most notable cases being the resistance to cancer chemotherapy caused by overexpression of the Mdrl P-glycoprotein,2 and the development of cystic fibrosis caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. 3 With the goal of learning more about how mitochondria communicate with the rest of the cell, we searched for and found an ABC transporter residing in the mitochondrial inner membrane of the yeast Saccharomyces cerevisiae. 3a The approach we adopted involved performing the polymerase chain reaction (PCR) on yeast genomic DNA, using degenerate oligonucleotide primers designed on the basis of the homology between ABC transporters. Ten ABC-homologous gene fragments were obtained, and these were then used to try to disrupt the corresponding genes. One of the successful disruptions resulted in a dramatic inhibition of growth. When we cloned the gene and localized its protein product within the cell by immunofluorescence, we found that the protein is located in mitochondria; we therefore called the gene A TM1 (ABC transporter of mitochondria). Analysis of purified mitochondria confirmed the mitochondrial localization of Atmlp and further demonstrated that it is located in the mitochondrial inner membrane, with its hydrophilic C-terminal domain exposed to the matrix. It is likely that mitochondria contain additional ABC transporters. The approach described here might be useful in identifying them. Although the sequencing of the complete yeast genome will soon obviate the need for a PCR approach in discovering new genes in S. cerevisiae, the approach will remain highly useful for other organisms. The following is a detailed description of some of the experimental procedures used in the course of this study. Polymerase Chain Reaction PCR has been used successfully by many laboratories to identify novel homologs of known proteins, including new ABC transporters in yeast4'5 and humans. (, Many of the details have been described elsewhere] but several considerations are stressed here. z j. A. Endicott and V. Ling, Ann. Rev. Biochem. 58, 137 (1989). 3 j. R. Riordan, J. M. R o m m e n s , B. S. Kerem, N. Alon, and R. RozmaheL Science 245, 1066 (1989). x~ j. Leighton and G. Schatz, E M B O J. 14, 188 (1995). 4 K. Kuchler, H. M. G0ransson, M. N. Viswanathan, and J. Thorner, Cold Spring Harbor Syrup. Quant. Biol. 57, 579 (1992). s M. Dean. R. Allikmets, B. Gerrard, C. Stewart, A. Kistler, B. Sharer, S. Michaelis, and J. Strathcrn, Yeast 10, 377 (1994). 6 M. F. Luciani, F. Denizot, S. Savary, M. G. Mattei, and G. Chimini, Genomics 21, 150 (1994). A. F. Wilks, this series. Vol. 200, p. 533.

[27]

ABC TRANSPOR'FERIN S. cerevisiae MITOCHONDRIA

391

We begin by comparing the amino acid sequences of the ATP-binding cassette of several ABC transporters t and determining which regions show sufficient conservation for use in designing degenerate oligonucleotide primers. Two conserved regions with sequences typified by SGSGKST and D E A T S A L D are selected. These sequences lie at opposite ends of the ATP-binding cassette and thus allow the rapid identification of new ABC genes by sequencing of the cloned PCR fragments (Fig. 1). Serine and leucine residues complicate primer design because each can be', encoded by six different codons, and it is therefore best to choose sequences with a minimal number of these residues, when possible. To reduce the chance of missing new genes while still maintaining primer specificity, we design separate primers to encompass the two classes of serine codons ( T C T / C / A / G and AGT/C) for one serine in each sequence. Mixes of two nucleotides are used at several positions, as appropriate, and inosine (I) is used at positions where three or four nucleotides could be present, because it is neutral in terms of binding affinity and does not increase the degeneracy of the oligonucleotides, which is kept in the range of 8- Io 32-fold. To facilitate the cloning of the PCR fragments, we incorporate B a m H I and E c o R I restriction sites into the 5' ends of the upstream and downstream primers, respectively. We also include additional nucleotides before the restriction sites to allow reasonable cutting efficiencies. The use of restriction sites has the advantage of a potentially high cloning efficiency and of directionality of cloning into the vector. A disadvantage is the potential for PCR fragments to be cleaved internally by the restriction enzymes, thus reducing the size

Primer

1

B~X

(Forward) :

S

GGC G G A T C C TCI G G C G G A T C C TCI

G

A/S/C

GGI GGI

T/GC/GI T/GC/GI

G

K

GGI GGI

AAA/G AAA/G

S

T

AGC/T TCI

AC AC

Primer 2 (Reverse): EcoRI D CGG CGG CGG CGG

GAATTC GAATTC GAATTC GAATTC

TC TC TC TC

L

A

IAG/A IAG/A IAG/A IAG/A

IGC IGC IGC IGC

S A/GCT AJGCT IGA IGA

V/T A/P IAC IGT IAC IGT

IGC/G IGC/G IGC/G IGC/G

E T/CTC T/CTC T/CTC T/CTC

D A/GTC A/GTC A/GTC A/GTC

FJ~;. 1. Design of degenerate oligonucleotide primers. A m i n o acids encoded by the various codons arc given in single-letter code. The third nucleotide of the codon encoding the last amino acid from each region was left out of each primer.

392

lON AND METABOLITE TRANSPORT SYSTEMS

[27]

of the cloned fragments: in one case this limited our ability to make further use of a PCR fragment. Alternatives that have been used successfully are to fill in the ends of the PCR fragments and then blunt-end clone, or to use one of the commercially available T-tailed vectors intended for cloning PCR fragments (PT7BIue, Novagen; T A Cloning System, Invitrogen). PCR reactions are performed in a volume of 100/xl, containing 10 ng of yeast genomic DNA, ~ 200/xM of each dNTP, 10/xl of a standard 10× PCR buffer (100 mM Tris-HC1, 15 mM MgCI2, 500 mM KC1, 1 mg/ml gelatin, pH 8.3), and pooled upstream and downstream oligonucleotide primers at concentrations such that 5 pmol of each oligonucleotide (representing one unique sequence) is present. The more degenerate oligonucleotides are thus added in proportionately higher amounts. The reaction mixtures are overlaid with paraffin oil and heated at 94 ° for 2 rain; this "hot start" is aimed at reducing background. Taq polymerase (2.5 units) is then added and a program performed of 30 cycles of 1 rain at 94 °, 2 min at 45 °, and 3 min at 72 °, followed by a final 7 rain at 72 °. The wide range of Tm's among the oligonucleotide pool makes it impossible to choose an ideal annealing temperature, but 45 ° is below the Tm of every oligonucleotide used and proved to be optimal. Among the 10 ABC-homologous PCR fragments that were finally identified, four correspond to genes that have since been sequenced in their entirety by us or others. A comparison of the oligonucleotide primers with the corresponding sequences of the four genes reveals that single-nucleotide mismatches in one or both primers are tolerated under our amplification conditions. The PCR products are electrophoresed in 2% agarose gels with wide wells and containing ethidium bromide. The bands are visualized with ultraviolet light, and those within the expected size range are excised as separately as possible. The DNA is then purified using either electroelution onto D E A E paper (Schleicher and Schtill) or the Gene Clean kit (Bio 101, CA). Restriction digestion is performed either before or after running the PCR products on a gel. The DNA is then cloned into the pUC18 vector. ~ Bacterial transformations are spread onto plates containing X-Gal and IPTG (isopropyl-/3-D-thiogalactopyranoside), and white colonies are picked. "J Clones are analyzed by restriction digestion with B a m H I and EcoRI, to confirm the presence and size of inserts, and by sequencing with the pUC reverse sequencing primer, using the United States Biochemical D N A sequencing kit. s H. Riezman, T. H a s t , A. P. G. M. van L o o n , L. A. Grivell, K. Suda, and G. Schatz, E M B O

,l. 2, 2161 (1983). '~C. Yanisch-Perron,J. Vieira. and J. Messing, Gene 33, 103 (1985). "~J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning. A Laboratory Manual." Cold Spring Harbor Laboratory Press, New York. 1989.

[271

ABC TRANSPORTERIN S. cerevisiae MITOCHONDRIA

393

Gene D i s r u p t i o n s u s i n g PCR F r a g m e n t s The 10 cloned PCR fragments with homology to the ABC family allow us to probe the role of the corresponding proteins via gene disruptions, without having to clone the complete genes. All but one of the cloned fragments are longer than 300 base pairs, and are thus potentially long enough to obtain disruptions in the corresponding genes at reasonable frequencies. Two approaches are used: In one approach, the PCR fragment is subcloned via the B a m H I and E c o R l sites into the yeast integrating vector YIp5, ~ containing the U R A 3 marker. This construct is cut at a unique restriction site as close as possible to the middle of the PCR fragment, to ensure a sufficiently long stretch of genomic D N A on either side. The DNA is then used to transform wild-type yeast, and cells are selected that had integrated the construct into the genome and thus become Ura ~. In the other approach, a selectable marker such as U R A 3 or L E U 2 is cloned into a unique restriction site of the PCR fragment in pUC18, again choosing a site lying as close as possible to the middle of the PCR fragment. The PCR fragment bearing the marker is excised from the vector using B a m H I and E c o R I and used to transform yeast. With both approaches, transformants are tested for disruption of the gene of interest by Southern blotting, using the original PCR fragment to prepare the probe [ECL (enhanced chemiluminescence), Amersham]. Hybridization is performed in a solution of 10% dextran sulfate, 1% SDS, and 1 M NaCI overnight at 68 °. Two low-stringency washes are performed of 15 min in 2× SSC, 1% SDS, and 15 min in 2 x SSC, 0.1% SDS, both at room temperature, before a high-stringency wash of 1 hr at 65 ° in 0.5x SSC, 0.1% SDS (20× SSC is 3 M NaCI, 0.3 M trisodium citrate.) Despite the sequence homology among the fragments, the hybridization and washing conditions used are sufficiently stringent that, in most cases, only the gene of interest is detected. We were successful with five of the eight genes we attempted to disrupt. Transformants from the remaining three attempted disruptions yielded ambiguous Southern blot patterns. Some of the PCR fragments thus probably inserted elsewhere within the genome at a high frequency. To determine unambiguously the null mutant phenotype of a gene, it is necessary to dissect tetrads derived from a diploid bearing a disruption in one copy of the gene. Because our goal is to identify genes with an interesting null mutant phenotype, we initially analyzed haploid disruptants for an obvious phenotype on plates containing glucose oi" nonfermentable carbon sources. (Growth on a nonfermentable carbon source requires re11K. Slruhl. D. T. Stinchcomb, S. Schcrcr, and R. W. Davis, Proc. Natl. Acad, Sci. USA 76, 1035 (1979).

394

lON AND METABOLITK TRANSPORT SYSTEMS

[271

spiring mitochondria.) None of the haploid disruptants showed a growth defect on either carbon source. However, tetrad dissection of a diploid disrupted in one copy of a gene in which we had not obtained any haploid disruptants showed a profound reduction in growth in the spores containing the disruption. On the basis of this phenotype, we cloned the gene and sequenced it, and then proceeded to localize the protein product within the cell. Epitope Tagging To localize Atmlp within the cell by immunofluorescence and biochemical fractionation, we place a c-rnyc epitope tag at its C terminus. The epitope tagging approach allows the use of a commercially available, highly specific monoclonal antibody to detect the tagged protein, eliminating problems of cross-reactivity as well as the delay in obtaining a clean rabbit antiserum. One can also easily perform double-label immunofluorescence, using a rabbit antiserum against another marker. Disadvantages are the lower avidity of a monoclonal antibody and the fact that it is not always as effective as a polyclonal antiserum for every purpose (e.g., immunofluorescence, immunoblotting, and immunoprecipitation). Furthermore, the tagged protein must be shown to be functional, which could be difficult if disruption of the gene produces no obvious phenotype. To epitope-tag A TM1, we use PCR to amplify a short 3'-terminal region of the gene, using a downstream primer that encodes the six C-terminal amino acids of Atmlp, the 10 amino acids EQKLISEEDL from c-rnyc, which are recognized by monoclonal antibody Mycl-9El0,12 and a stop codon. The 3'-terminal region of the gene, which had been cloned into both single- and multicopy yeast vectors, is excised and replaced with the tagged version, using appropriate restriction sites that had been incorporated into both primers. Alternative approaches to epitope tagging include a more general fourprimer PCR method used for performing site-directed mutagenesis, 13 as well as specialized vectors into which the gene to be tagged can be directly cloned. 14 We placed the epitope tag at the C terminus of Atmlp because others have tagged ABC transporters at the C terminus without abolishing function 15'1~', we subsequently showed that tagged Atmlp is indeed funct ' G . 1. Evan, G. K. Lewis, G. Ramsay. and J. M. Bishop, Mo/. Cell. Biol. 5, 361/) (1985). l~S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, and I,. R. Pease, Gene 77, 51 (1989). J4 p. Reisdorf, A. C. Maarse, and 13. Daignan-Fornier. Curr. Genet. 23, 181 (1993). ts p. Juranka, F. Zhang, J. Kulpa, J. Endicott. M. Blight, I. B. Holland. and V. Ling, J. Biol. Chem. 267, 3764 (1992). l,, K. Kuchler, H. G. D o h h n a n . and J. Thorner, J. Cell Biol. 120, 1203 (1993).

[27]

ABC TRANSPORTERIN S. cerevisiae MITOCHONDRIA

395

tional in rescuing the slow-growth phenotype of the null mutant. However, other proteins may need to be tagged elsewhere to avoid loss of function. This is particularly important if a C-terminal targeting or retention signal is present. Intramitochondrial Localization lmmunofluorescence ~7 as well as immunoblotting of Nycodenz-purified mitochondria (this volume, [14]) from cells expressing c-myc-tagged Arm 1p demonstrate a mitochondrial localization. To try to determine the intramitochondrial localization of Atmlp, we determine the protease accessibility of the tagged protein in mitoplasts (mitochondria subjected to osmotic shock to disrupt the outer membrane), in intact mitochondria, and in mitochondria treated with detergent to disrupt both membranes. A procedure for determining the submitochondrial location of a protein is given elsewhere in this volume in [15]. The following protocol has been adopted for the purpose of localizing c-myc-tagged Atmlp. Crude mitochondria (250 /~g; for determining the submitochondrial location of Atmlp, purification of mitochondria on a Nycodenz gradient is unnecessary), stored frozen with 10 mg/ml fatty acid-free BSA (bovine serum albumin), are washed in mitochondrial breaking buffer (0.6 M sorbitol, 20 mM K+-HEPES, pH 7.4) and resuspended in 450/,1 import buffer (this volume, [15]), and four 10(I-/zl aliquots are transferred to new microcentrifuge tubes, to be treated as follows: Sample 1, untreated: .,sample 2, protease treated: sample 3, subjected to osmotic shock in the presence of protease: and sample 4, subjected to detergent and protease. Sample 3 is washed again with breaking buffer to remove the BSA and is resuspended in 100/,l breaking buffer. Sample 1 is left untreated; sample 2 receives 1 /21 of 10 mg/ml proteinase K: sample 3 receives 100/,1 of 2% octylglucoside and 2 /,1 of proteinase K: and sample 4 receives 700 /,l of mitoplasting buffer (20 mM K--HEPES, pH 7.4) containing 8/~1 of proteinase K. Samples are incubated for 30 rain on ice. To inactivate the proteinase K, samples 2, 3, and 4 receive 1 mM PMSF (phenylmethylsulfonyl fluoride.) from a freshly prepared 200 mM stock solution in ethanol. Samples 1, 2, and 4 are spun for 5 rain, the supernatants are aspirated, and the pellets are resuspended in 180 p,1 breaking buffer containing 0.2 mM PMSF. All four samples are transferred to new tubes, and TCA (trichloroacetic acid) is added to 5% (w/v) from a 50% stock solution. The tubes are heated for 5 min at 60 °, left 5 min on ice, and then spun for 10 min. The supernatants are completely aspirated to remove the TCA, and the pellets are resuspended in /7 M. N. Hall, C. Craik, and Y. Hiraoka. Proc. Natl. Acad. Sci. USA 87, 6954 (1990).

396

tON AND METABOLITE TRANSPORT SYSTEMS

[27]

40 /xl of SDS-containing sample loading buffer containing 1 mM PMSF and 50 mM Na ~-PIPES, pH 7.5. Samples are heated for 5 min at 55 ° (to avoid possible aggregation of A t m l p at higher temperatures) and then subjected to 10% SDS-PAGE. Gels are blotted and probed with the cm y c antibody, as well as with antisera against the following proteins: porin, an outer membrane protein, which is inherently protease resistant, even in the presence of detergent, and which serves as a control for the amount of protein loaded; cytochrome t)2, an intermembrane space protein, which is released by mitoplasting; and o~-ketoglutarate dehydrogenase, a matrix protein that is highly protease sensitive and serves as a control for the intactness of the inner membrane. In mitoplasts treated with protease, tagged A t m l p remains intact and is still recognized by the c-myc antibody, while the tag is fully digested in the presence of the detergent. This result indicates that the C terminus bearing the epitope tag is located in the matrix, and that the protein must therefore be situated in the inner membrane. This procedure allows both an unambiguous localization of Atm lp and a determination of its membrane orientation by taking advantage of the specific detection of the C terminus of the protein. However, if the c-myc epitope were protease digested in mitoplasts as well, this result would indicate only that the C terminus faces the intermembrane space, and would necessitate the preparation of inner and outer membrane vesicles ([16] this volume) to determine the membrane localization. Acknowledgments 1 would like to thank Jeff Schatz for continual support, and Ben (Hick and Carolyn Suzuki for critically evaluating lhc manuscript.