©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteolysis Prevents in Vivo Chimeric Fusion Protein Import into Yeast Mitochondria
CYTOSOLIC CLEAVAGE AND SUBCELLULAR DISTRIBUTION (*)

Jianzhong Zhou , Yinglin Bai , Henry Weiner (§)

From the (1)Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The in vivo import of liver mitochondrial aldehyde dehydrogenase was investigated in yeast by constructing fusion proteins between its leader sequence and -galactosidase. Only 7% of the protein was imported. If 21 or 71 amino acids from the mature portion of aldehyde dehydrogenase were included in the construct, 40% was imported. The protein remaining in cytosol was sequenced. When the leader was fused directly to -galactosidase, the first 7 residues of the leader were missing. When 21 residues of mature aldehyde dehydrogenase were included, the entire leader plus 6 residues of the mature portion were missing; if 71 residues of mature aldehyde dehydrogenase were included, the first residue found corresponds to the 66th residue of the mature portion. When the leader was fused directly to -galactosidase, no processing of the imported protein occurred, and the N-terminal amino acid was blocked, presumably by acetylation. If the 21-amino acid insert was included, processing occurred. A modified leader sequence lacking the three-amino acid linker (RGP) was imported but not processed, just as we found in vitro (Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D.G.(1993) J. Biol. Chem. 268, 19906-19914). The less than 100% import of pre-aldehyde dehydrogenase was due to the action of a post-translational protease attack which prevented import by destroying the leader peptide segment.


INTRODUCTION

It has been proposed that the leader sequences which allow precursor proteins to be transported into mitochondria contain amphilpathic helices(1, 2, 3, 4, 5) . The structure of three processed and three nonprocessed leaders sequences have been determined by two-dimensional NMR(6, 7, 8, 9, 10) . All are composed of an N-terminal amphilphilic helix, supporting the early prediction. The leader sequence for rat liver mitochondrial aldehyde dehydrogenase (ALDH)()was found to be composed of three structural components(6) . These were two short segments of helices separated by a three-amino acid linker (RGP). We investigated the necessity of each region by making deletions or by constructing chimeric leader sequences with components of cytochrome oxidase, a protein whose leader sequence also was determined by two-dimensional NMR(8) . We concluded that the minimum structural requirement of the leader peptide was just a two-turn amphilpathic helix at the N termini of the leader sequence(11) . The role of the other portions of the leader was proposed to stabilize the N-terminal helix. The experiments that led to these conclusions were performed with isolated rat liver mitochondria employing proteins synthesized in rabbit reticulocytes. Data obtained from in vitro import studies are thought to represent what occurs in vivo because the cytosolic factors necessary to allow import to occur are supplied by the reticulocyte system. We felt it would be advantageous to investigate the peptides in a true in vivo system, rather than just the in vitro one employed, to verify our observations.

Investigators have often used yeast to study import(12, 13, 14, 15, 16) . Leader sequences were fused to carrier proteins in order to ascertain residues in the leader important for import. A common carrier protein employed is -galactosidase because of the ease of assaying the enzyme. Typically, mitochondria are separated from cytosol, and the amount of -galactosidase activity in each organelle is used as a measure of import. The leader sequence from rat liver aldehyde dehydrogenase and an altered sequence we employed in the in vitro studies were fused to -galactosidase on a yeast expression vector to test whether the observations made in the in vitro system were valid in vivo. It will be shown that the fusion of the leader sequence directly to -galactosidase produced a protein that was poorly imported. In contrast, if some residues of the mature portion of aldehyde dehydrogenase were included, then the protein could be imported into mitochondria, but much of the fusion protein remained in the cytoplasm.


EXPERIMENTAL PROCEDURES

Strain and Growth Condition

Saccharomyces cerevisiae strain KX 147-2C (MAT leu1 LEU4ura52(17) was kindly provided by Prof. Gunter B. Kohlhaw of this department). Yeast cells were grown at 30 °C in SD medium, which contained 0.67% yeast nitrogen base without amino acids, 2% glucose, and supplemented with 2 mM leucine. Escherichia coli strains used were TG1 and MC1000(18) . E. coli was grown at 37 °C in LB medium or 2 yeast tryptone medium. Ampicillin was added to a final concentration of 100 µg/ml. The lithium acetate method (19) was used for yeast transformation. E. coli transformation was as described by Maniatis et al.(20) .

DNA Manipulation

Standard DNA techniques were performed as described elsewhere(20, 21) . Site-directed mutagenesis was performed with the ``Muta-Gene'' mutagenesis kit by Bio-Rad, which is based on the procedure of Kunkel(22) . Oligonucleotides were synthesized on an Applied Biosystems model 380A synthesizer by the Laboratory for Macromolecular Structure, Purdue University. DNA sequencing was determined by the dideoxy chain-termination method(23) , using single-stranded template or alkali-denatured, double-stranded DNA and Sequenase (U. S. Biochemical Corp.). Some DNA fragments were synthesized by the polymerase chain reaction using Taq DNA polymerase performed for 30 cycles with the following program: 1 min at 94 °C, 1 min at 52 °C, and 2 min at 72 °C.

Construction of Plasmid Vector

For mutagenesis and sequencing, plasmid pYH305 and pYH305DE were constructed. For pYH305, the 2.8-kb SacI-ScaI fragment from pGEM-3Z that contained the 1.8-kb EcoRI fragment of pALDH of rat liver was isolated and ligated to Bluescript SKII(+), which was cut with SacI and ScaI. For pYH305DE, an EcoRI site in the middle of the pALDH coding region in pYH305 was removed by mutagenesis. This just changed the codon for the same amino acid.

ALDH-lacZ Gene Fusion Constructions

The rat liver ALDH cDNA has been cloned and the entire nucleotide sequence has been determined (24). A set of plasmid pGEM-3Z containing the pALDH sequence with various modified signal peptides has been constructed for in vitro import study(11) . The plasmids were cut with EcoRI and PvuII. The 308 bp of EcoRI-PvuII fragments which include 19 amino acids of the signal region and 71 amino acids of the ALDH coding region were isolated by running digested DNA on 3% NuSieve GTG gel. They were then ligated into the EcoRI and SmaI sites of plasmid pSEYC102, a yeast centromere plasmid (10.5 kb), containing the yeast URA3, CEN4-ARS1, pBR322 sequence and unique EcoRI, SmaI, and BamHI sites to the 5` side of the truncated lacZ gene. Plasmid pYB1 has a 1.7-kb BamHI fragment including 645 bp of the leu2 promoter region, 637 bp of the leu2 coding region, and 400 bp of the bacterial sequence. Using the 1.7-kb BamHI fragment as the template, a 0.64-kb EcoRI-SphI fragment of leu2 promoter was synthesized by a polymerase chain reaction, and then this fragment was inserted into the above ligated plasmids which had been digested with EcoRI and SphI. The resulting plasmids have a leu2 promoter-ALDH-lacZ fusion (Fig. 1). The plas-mid without the DNA fragment of the leader is the same as the native plasmid except the leader sequence has been removed by creating a SphI site at the 18th amino acid position of leader region by mutagenesis.


Figure 1: Plasmid construction of leu2 promoter-ALDH-lacZ fusion. The 308-bp EcoRI-PvuII fragments containing 19 amino acids of the signal peptides and 71 amino acids of mature ALDH coding region were inserted between the EcoRI and SmaI sites of plasmid pSEYC102 and proved to be in frame by sequencing. The 0.6-kb EcoRI-SphI fragment of the leu2 promoter was synthesized by a polymerase chain reaction, using pYB1 as the template, and inserted into the EcoRI and SphI sites.



To construct a fusion protein with a shorter portion of mature ALDH and to obtain high expression of the fusion protein, other ALDH-lacZ gene fusions were constructed, and a multiple copy plasmid was adopted. An oligonucleotide with sequence CGGAATTCTCGAGGAGAAC, which corresponds to nucleotides from -653 to -634 and contains an EcoRI restriction site (underlined), was used as the sense primer for the polymerase chain reaction experiments. The antisense oligonucleotide for the construction of the fusion proteins which contain 21 amino acid residues of mALDH was AATCGGATCCTGGTTGCAGAAGACCTCGGG (corresponding to amino acid residues 15-24 of mALDH) and for the construction of the fusion proteins without any portion of mALDH was GGGATCCAGCAGGCGGCTCAGGCGTGGCCC (corresponding to -8 to +2 amino acid residues), in which a BamHI restriction site (underlined) was made, respectively. The plasmid DNAs shown in Fig. 1were used as templates. All the DNA fragments amplified by polymerase chain reaction were then subcloned into vector pSEYC101 between the EcoRI and BamHI sites. These plasmids can normally be maintained in multiple copies per yeast cell due to the presence of the 2µ gene.

In frame fusions between ALDH and lacZ were isolated after transformation into E. coli strain MC1000 on plates containing X-gal. The precise fusion joints were verified by DNA sequencing. All of the plasmids can be shuttled between E. coli and yeast. The -galactosidase-positive E. coli transformants were selected. The plasmid DNA isolated from them finally were transformed to yeast XK147-2C and tested for -galactosidase expression on a yeast minimal medium containing X-gal. Yeast cells expressing -galactosidase were verified by measuring enzymatic activity in cell-free extracts.

Cellular Fractionation and Enzyme Assay

Yeast cells were grown to 1.5 optical density units at 600 nm in SD medium supplemented with leucine. Cells were fractionated into cytosolic and mitochondrial fractions essentially according to the procedure of Daum et al.(25) . Spheroplasts were prepared from yeast cells in the presence of Lyticase (Sigma). Finally, mitochondria were resuspended in 20 mM HEPES, pH 7.0, 0.6 M mannitol buffer. For enzyme assay, the reaction buffer contained 1% Triton X-100. -Galactosidase activity was measured in Z buffer by the method of Miller (26) at 30 °C. The recovery of mitochondria relative to cytosol was monitored by assaying the mitochondrial marker enzyme fumarase (27) and the cytosolic marker enzyme glucose-6-phosphate dehydrogenase(28) .

Purification of -Galactosidase-Fusion Proteins

All subsequent steps were carried out at 0-4 °C in the presence of protease inhibitors (Pefabloc SC, 1 mg/ml; o-phenanthroline, 5 mM; leupeptin, 3 µg/ml; pepstatin, 15 µg/ml; phenylmethane sulfonyl fluoride, 1 mM; EDTA, 1 mM). Nucleic acids were precipitated from the post-mitochondrial supernatant fractions by adding slowly 2.5% protamine sulfate with constant stirring to give a final concentration of 0.1%. After stirring for 30 min at 4 °C, the solution was clarified by centrifugation at 10,000 g for 15 min(29) . An equal volume of 100% (of saturation) ammonium sulfate solution was added slowly with stirring. The precipitate was collected by centrifugation at 20,000 g for 10 min. The pellet was then suspended in Tris-HCl buffer (0.25 M NaCl, 10 mM Tris HCl, pH 7.6, 10 mM MgCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.1% Triton X-100) and dialyzed against 100 volumes of the same buffer for a minimum of 2 h while changing the buffer three times. The dialysate was passed through a -galactose analog affinity column(30) . The column was washed with 10 bed volumes of the buffer, followed by 5 bed volumes of the buffer without Triton X-100. The protein was eluted from the column with 0.1 M sodium borate (pH 10) and promptly precipitated with 100% saturated ammonium sulfate.

To extract the fusion protein from mitochondria, the mitochondria were diluted with an equal volume of distilled water to give a hypotonic condition and were lysed for 15-30 min by the addition of 0.1% Triton X-100 at 0-4 °C. They were then sonicated at 0 °C for 30 s two times using a Fisher-Artek model 300 sonic dismembrator at a power setting of 85% relative output. Membrane fragments and other debris were removed by centrifugation at 48,000 g for 30 min. The supernatant was dialyzed against the same buffer used for affinity chromatography and then passed through the substrate analog affinity column.

Protein Sequencing

N-terminal protein sequencing was performed by the Purdue University Biochemistry Department Protein Sequencing Facility on samples electrophoretically transferred from 8% SDS gels to a membrane as described previously(31) .

Miscellaneous Chemicals

All the restriction and modifying enzymes were purchased from New England BioLabs, Boehringer-Mannheim, Promega, or Life Technologies, Inc. A prestained molecular weight standard for SDS-polyacrylamide gel electrophoresis was from Bio-Rad. Alkaline-phosphatase conjugate anti-mouse IgG and mouse anti--galactosidase monoclonal antibody were from Promega. Rabbit anti-pALDH signal peptide antibody was previously prepared in our laboratory. Protease inhibitor set was from Boehringer Mannheim. p-Aminophenyl -D-thiogalactopyranoside-Sepharose 4B was from Sigma. Western blotting was as previously described(32) . All other reagents were commercially available analytical reagents.


RESULTS

-Galactosidase Expression in Yeast

The plasmids for the gene fusion constructs which contained the leu2 promoter/N terminal native and modified precursor (Fig. 2) ALDH were fused in frame to the 10th amino acid of lacZ (Fig. 1). -Galactosidase activity for all constructs was found in both E. coli and yeast, and no activity was expressed from the parent vector in these cells. The level of -galactosidase expressed in crude yeast extracts from all of the gene fusions was essentially identical. The reason is that these have been constructed in the plasmid pSEYC102 which contains the yeast centromere sequence CEN4 and the sequence ARS1 which allows for the maintenance of essentially one copy of the plasmid per cell.


Figure 2: The amino acid sequences of the signal peptides. Amino acids in italics are from the mature part of ALDH. Linker deletion is wild type presequence without the (RGP) linker which connects the N- and C-terminal helices.



The constructs containing the ALDH/-galactosidase fusion was also cloned into the SEYC101 vector which are maintained in multicopy in the cells. Activity assays showed that all constructs were expressed to the same extent but at a level more than 10-fold higher than with the single copy plasmid ().

Assaying for -galactosidase activity in isolated mitochondrial and cytosolic fractions allowed us to determine whether import occurred in vivo. If the leader sequence of ALDH was not included in the construct, 99% of the total -galactosidase activity was found associated with the cytosol fraction, showing that, as expected, the enzyme could not be imported into mitochondria if it did not possess a mitochondrial leader sequence.

Import of -Galactosidase Chimera Possessing a Leader Sequence Requires the Presence of a Portion of the Mature Protein

The leader sequence of aldehyde dehydrogenase was fused directly to the 10th residue of -galactosidase. It was unexpected to find that for this construct only 7% of the -galactosidase activity was found in the mitochondrial pellet with the remainder in the cytosolic fraction. The constructs containing 21 or 31 amino acids of mature ALDH between the leader sequence and -galactosidase were imported to a much greater extent (40%). The distribution of -galactosidase activity be-tween mitochondria and cytosol for the ALDH chimera is presented in .

Amino Acid Sequence of the -Galactosidase Chimera Found in Cytosol

Lack of import could have resulted from at least two unrelated events. We showed that a disruption of the N-terminal helix of the leader sequence affected its ability to function(11) . Possibly, the three-dimensional structure of the leader sequence was affected by the presence of -galactosidase being fused to it. Alternatively, if the leader sequence was modified in vivo prior to import, the precursor protein would not enter mitochondria. To test for this possibility, -galactosidase was isolated from the cytosol and subjected to amino acid sequencing. When the chimera with no intervening sequence between the leader and -galactosidase was employed, the protein was found to be lacking the N-terminal portion of the leader sequence. The first amino acid identified corresponded to the 6th residue of the leader. The proteins which were designed to contain intervening segments of mature aldehyde dehydrogenase were found to be missing all of the leader sequence plus part of the mature aldehyde dehydrogenase portion. When the construct with 71 residues of mature ALDH was analyzed, the first amino acid corresponded to the 66th residue of the mature portion of ALDH. When the construct with 21 intervening residues was analyzed, the first amino acid found corresponded to the 7th residue of the mature portion of ALDH. These sequences are shown in and illustrated in Fig. 3.


Figure 3: Cleavage site of the fusion protein isolated from cytosol. The numbers 1-19 above the filled lines indicate the leader sequence. The numbers below the single line indicate the intervening sequence of mature ALDH in the fusion protein. The position of the first amino acid found from sequencing the fusion protein is indicated by the numbers 66, 7, and 6, respectively.



Amino Acid Sequences of Protein Isolated from Mitochondria after Import

The -galactosidase from two fusion proteins possessing the native leader sequence were isolated from mitochondria and subjected to amino acid sequence analysis. The first had the leader fused directly to -galactosidase; the second had the 21-amino acid insert. No sequence could be found from the protein missing the insert, suggesting that the N-terminal residue was blocked. A Western blot analysis was performed using an antibody generated to recognize only the leader region to verify that the leader sequence was still attached to -galactosidase (Fig. 4).


Figure 4: The Western blot analysis of the fusion proteins in mitochondria. Samples were run on 8% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and probed by anti-signal peptide antibody (A) and anti--galactosidase antibody (B). Mitochondrial fractions of pZ001 (no intervening sequence for mature ALDH, lane 1), pZ211 (21 amino acids from mature ALDH, lane 2), and pZ212 (linker deleted with 21 amino acids from mature ALDH, lane 3) were loaded, respectively. The band near 139 Kd is the fusion protein. The molecular weight standards were prestained by Coomassie Blue (not shown).



When the 21-amino acid portion of ALDH was included in the construct, the leader sequence was removed by the mitochondrial processing enzyme. The amino acid sequence corresponded to the first residues of the mature portion of ALDH, showing that processing occurred at the expected site located between RLL, the last 3 residues of the leader, and SAA of the mature protein (I and Fig. 2). Western blot analysis using the antibody against the leader sequence was negative, as expected, for a protein missing the leader.

We showed in the in vitro system that, if the three-amino acid linker (RGP) of the native rat liver aldehyde dehydrogenase leader sequence was missing, import occurred but not processing(7) . The chimeric protein from this construct was isolated from yeast mitochondria, but no sequence could be obtained, because the N-terminal amino acid was found to be blocked. No attempt was made to identify the blocking group, but Western blot analysis showed that the leader was still present.


DISCUSSION

Fusion proteins composed of the leader sequence of a mitochondrial precursor protein and a carrier protein have been used to investigate import of proteins into mitochondria(12, 13, 14, 15, 16) . Investigators have often deleted portions of the leader sequence in an attempt to determine the minimum number of amino acids necessary for a leader sequence to function. If a construct containing a modified leader was not imported, it was assumed that the section deleted was vital.

-Galactosidase is a commonly employed carrier protein used to study import in yeast(12, 13, 30) . There are reports in the literature showing that for some constructs -galactosidase did not function well(12) . Some investigators have suggested, although never proven, that the size (1023 amino acids) may have been responsible for its not allowing a leader sequence to function, for if the same peptide was fused to, say, dihydrofolate reductase, in vitro import would occur. We showed that the rate of import of precursors proteins could be a function of the size of the mature portion of the protein (33). Thus the large size of the -galactosidase fusion protein may have contributed to its not functioning well in some systems.

We found that, when the leader sequence from rat liver aldehyde dehydrogenase was fused directly to -galactosidase, little of the total enzyme activity was found associated with yeast mitochondria. This was an unexpected finding since the leader worked well in causing the import of mature ALDH and orithine transcarbamylase into rat liver mitochondria and into isolated yeast mitochondria.()Douglas et al. (34), however, reported that it was not possible to import F-ATPase fused directly to -galactosidase.

If proteolysis occurred prior to import it would not be possible for the precursor protein to be translocated into mitochondria. To test for this, the fusion proteins remaining in the cytosol were isolated and sequenced. The chimeric proteins isolated from yeast cytosol were missing some or all of the leader sequence. Undoubtedly, the portion of the protein was removed by the action of a protease located in the cytoplasm. We have previously shown that deletion of residues at the N-terminal end of the leader sequence prevented ALDH from being translocated into rat liver mitochondria(6) . Thus the reason for the lack of import of the fusion proteins employed in this study was the fact that the leader was destroyed or removed by the action of proteases in the cell prior to import.

It is impossible to state whether the proteolysis occurred in one step by the action of an endopeptidase, or by the sequential removal of single amino acids by the action of an N-terminal amino acid peptidase. The argument against the latter is the fact that only one peptide sequence was obtained for each protein. If the amino acids were being removed sequentially, it could be expected that a very ragged N-terminal sequence would have been found. We have no data to determine whether or not the protease was specific. As shown in , when 0, 21-, or 71-amino acid residues from the mature portion of ALDH were included, proteolysis occurred at different positions. Based on the deduced amino acid sequence (24) the cleavage occurred between an L-S, an S-A, and an A-R bond when 0, 21, and 71 residues of ALDH were included in the chimera, respectively. If there were three specific proteases involved, it could have been expected to have obtained three different amino acid sequences when the construct containing 71 amino acids was employed.

Even though proteolysis occurred which prevented some of the protein from entering the mitochondria, a portion of the newly synthesized precursor protein was found in the mitochondria. Although only 7% of the protein was imported into the mitochondria when the aldehyde dehydrogenase leader sequence was fused directly to -galactosidase, no processing occurred after import. This could have been a result of the destruction of the processing site. When the leader was fused to -galactosidase through 21 amino acids of the mature ALDH, a sequence was obtained. The amino acid sequence of the protein revealed that it was processed at the expected site. This is the first proof that the actual site of processing of the ALDH leader sequence was between the RLL and the SAA residues. It has always been assumed that this was the site based upon the amino acid sequence of the enzyme isolated from liver. The potential uncertainty comes about from the fact that human liver mitochondrial ALDH was reported to possess a ragged N-terminal sequence (35) and that rat and beef liver mitochondrial ALDH, isolated from fresh tissue, had a blocked N-terminal (acetylated) residue(36) .

The linker-deleted precursor was shown to direct the import of ALDH into isolated rat liver mitochondria, but was not removed by the action of the protease after import(7) . The corresponding -galactosidase fusion protein isolated from yeast mitochondria after import was found to be blocked and not processed. The blocking, presumed to be acetylation, could have occurred either after import or prior to import. It is not known whether or not the initiator methionine residue is removed in cytosol prior to import. Detailed structure analysis of the blocked precursor proteins by mass spectroscopic techniques could be used to address this question.

Most investigators (37, 38, 39, 40) argue that import is a post-translational event. There are some who have presented data which can be interpreted as implying that mitochondrial import is a co-translation event(15, 41) . Our finding that the fusion proteins have been subjected to the action of a protease prior to import is supportive of the notion that import is a post-translational event. If the leader was entering mitochondria as it was coming off the ribosome it would be protected from the action of the cytosolic proteases. Similar import data were obtained when the multicopy plasmid or the single-copy plasmid were employed. This finding shows that proteolysis occurred independent of the rate of synthesis of the precursor protein and was not simply a result of protein being synthesized faster than it was being imported.

The proteolysis observed in this study shows a potential difficulty in investigating import of fusion proteins in yeast. Investigators cannot really tell why import did not occur unless they were to sequence the protein remaining in the cytosol. Our study was performed only with -galactosidase as the carrier protein. It is not possible to state why the ALDH portion was so susceptible to cleavage or why the leader was cleaved when fused directly to -galactosidase. It is possible that using a protein which was imported slowly could have made it more susceptible to the action of nonspecific cytosolic proteases. A similar study will have to be undertaken using other proteins to determine if proteolysis is a problem common for all chimeric proteins expressed in yeast or is unique to the system employed in this study.

  
Table: Distribution of fusion proteins between mitochondria and cytosol

Cellular fractionations and enzyme assays were performed as described under ``Experimental Procedures.'' Imports are given as percentage of the total activity present in the combined fractions and calibrated by assaying for the marker enzymes. The numbers represent the average of at least two experiments. The experimental error was ±8% of the mean.


  
Table: 1768843040p4in Not determinable.

  
Table: N-terminal sequencing of purified fusion protein isolated from mitochondria



FOOTNOTES

*
This work was supported in part by National Institute on Alcohol Abuse and Alcoholism Grant AA05812. This is Journal Paper No. 14630 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of National Institute on Alcohol Abuse and Alcoholism Senior Scientist Award AA00028. To whom correspondence should be addressed. Tel.: 317-494-1650; Fax: 317-494-7897.

The abbreviations used are: ALDH, aldehyde dehydrogenase; pALDH, precursor aldehyde dehydrogenase; mALDH, mature aldehyde dehydrogenase; X-gal, 5-bromo-4-chloro-3-indoyl -D-galactoside; bp, base pair; kb, kilobase pair.

Y.-K. Pak and H. Weiner, unpublished data.


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