(Received for publication, November 9, 1995; and in revised form, January 30, 1996)
From the
The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. Both are implicated in genetic disorders of peroxisome biogenesis and function. Proteins homologous to ALDP and the 70-kDa peroxisomal membrane protein have been discovered in other eukaryotic organisms and form a growing group of peroxisomal half ABC transporters. Amino acid sequence alignment of these and other ABC transporters reveals several protein motifs that are highly conserved both in sequence and location. Here we characterize two of these, designated the EAA-like and the loop1 motifs. We study them by introducing missense mutations in Pxa1p, a Saccharomyces cerevisiae ortholog of ALDP, and show that both motifs are important for Pxa1p function. Interestingly, missense mutations in corresponding amino acids in ALDP cause adrenoleukodystrophy in humans. We conclude that these motifs are important for ABC transporter function and that the yeast protein Pxa1p is a useful system for understanding the molecular basis of adrenoleukodystrophy.
ATP-binding cassette (ABC) ()transporters are members
of a superfamily of membrane proteins involved in the transport of a
variety of molecules across biological membranes (1, 2) . ABC transporters are comprised of two
homologous halves, each containing two parts: a transmembrane domain
(TMD) with multiple transmembrane (TM) segments and a nucleotide
binding domain (NBD) with Walker A and B consensus motifs(3) .
Mammalian ABC transporters are found either as complete transporters (e.g. the multiple drug resistance (MDR) transporter and the
cystic fibrosis transmembrane regulator proteins) or as half
transporters (e.g. the TAP1 and TAP2 proteins, which dimerize
to form the active TAP transporter) (4) .
Two half ABC
transporters have been identified in the human peroxisome membrane: the
adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane
protein (PMP70)(5, 6) . Mutations in the
adrenoleukodystrophy gene cause X-linked adrenoleukodystrophy, an
inborn error of peroxisomal -oxidation of very long chain fatty
acids(7, 8) . We identified mutations in the PMP70
genes of two patients with Zellweger syndrome(9) , an inborn
error of peroxisome biogenesis, although the role PMP70 plays in this
disease remains uncertain(10) . PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs
of ALDP and PMP70. Pxa1p, an ortholog of ALDP, is involved in
peroxisomal
-oxidation of fatty acids(11) . YKL741, an open reading frame found by the yeast genome
sequencing project(12) , also encodes a half ABC transporter
with high similarity to ALDP and PMP70. We have reported genetic
evidence suggesting that the YKL741 protein heterodimerizes
with Pxa1p (11) and, in recent work, have shown that it has the
expected peroxisome association. (
)For these reasons, we
designated the YKL741 gene as PXA2. However, the
exact function(s) and physical interaction(s) of both the human and
yeast peroxisomal ABC transporters are still unclear.
EAA motifs are
conserved sequences of approximately 30 residues between TM4 and TM5 of
prokaryotic ABC transporters(13, 14, 15) .
Alignment of prokaryotic ABC transporters reveals several residues that
are highly conserved in the core of this
motif(13, 14) , and missense mutations altering one of
these, a central glycine, result in loss of function in bacterial
transporters(14) . Sequence analysis of bacterial EAA motifs
suggested that they predict substrate specificity(13) . We
recently reported that certain eukaryotic ABC transporters possess a
15-amino acid motif resembling the central core of the prokaryotic EAA
motif that we designated an EAA-like motif(11) . Mutations in a
conserved glutamic acid residue in the EAA-like motif of the gene
encoding ALDP have been reported in four unrelated adrenoleukodystrophy
patients(8, 16, 17, 18) . A deletion
of 19 amino acids in this region of cystic fibrosis transmembrane
regulator influences the stability of Cl channel
conductance(19) .
In S. cerevisiae, fatty acid
-oxidation takes place only in the peroxisomes(20) . Yeast pxa1 mutants have impaired growth on oleic acid medium and
reduced ability to oxidize oleate(11) . Wild type growth on
oleic acid and
-oxidation of oleate can be restored by expressing
the wild type PXA1 gene in the pxa1::URA3 mutant
yeast(11) . We have used this expression system to assess the
functional consequences of missense mutations in the EAA-like motif and
in a newly characterized loop1 motif on eukaryotic ABC transporter
function.
Figure 1: Alignment of peroxisomal ABC transporters. The amino acid sequences of the human PMP70 and ALDP are aligned with a putative peroxisomal transporter encoded by a C. elegans open reading frame (C.e C44B7.8) and S. cerevisiae Pxa1p. Amino acids present in a plurality of these sequences are boxed in black. The six putative transmembrane domains (TM) segments are indicated by overlines. The loop1 and the EAA-like protein motifs as well as the Walker-A, Walker-B, and the C sequences of the nucleotide binding fold are indicated.
Figure 2: A hypothetical model of Pxa1p showing the transmembrane segments and the locations of the protein motifs. L1, loop1 motif; EAA, EAA-like motif; A, Walker A motif; B, Walker B motif; C, nucleotide binding fold conserved sequence; N-ter; N-terminal.
Figure 3: Alignment of the EAA-like motifs of eukaryotic ABC transporters and the location of mutations. Listed from top to bottom are the S. cerevisiae Pxa1p, S. cerevisiae Pxa2p, human PMP70, human ALDP, C. elegans open reading frame C44B7.8 (C.e C44B7.8), human TAP1 (hTAP1), human MDR1 (hMDR1), murine MDR2 (mMDR2), human MDR3 (hMDR3), and Drosophila MDR4 (dMDR4) proteins. Amino acids present in a plurality of these sequences are boxed in black, and conservative amino acid substitutions (27) from this plurality are boxed in gray. Mutations in ALDP that cause adrenoleukodystrophy are indicated below. Missense mutations introduced in the PXA1 gene are indicated above.
Figure 4:
Phenotype of missense mutations in the
EAA-like motif of Pxa1p. A, growth of pxa1::LEU2 knockout (ko) yeast transformed with (from left to right): wild type, E294D, Q300L, G301P, or G301A
alleles of PXA1 or vector alone. 10-fold dilutions (top to bottom) of the transformants were inoculated on medium with
0.1% oleic acid as a sole carbon source. The amino acid sequence of the
EAA-motif from Pxa1p is indicated on the bottom with conserved
residues in bold letters. B, -oxidation of
[
C]oleic acid by pxa1::LEU2 knockout
yeast expressing from left to right: E294D, G301P, or
G301A alleles of PXA1.
Figure 5: Alignment of loop1 motifs and position of mutations. Listed from top to bottom are PMP70 (hPMP70) and ALDP (hALDP), the C. elegans open reading frame C44B7.8 (C.e C44B7.8), Pxa1p, and Pxa2p. Amino acids present in a plurality of these sequences are boxed in black, and conservative amino acid substitutions (27) from this plurality are boxed in gray. Missense mutations produced in the PXA1 gene are indicated above. A mutation in ALDP that causes adrenoleukodystrophy is indicated below.
Figure 6:
Phenotype of missense mutations in the
loop1 motif. A, phenotypes of pxa1::LEU2 knockout (ko) yeast expressing (from left to right):
wild type (wt), L70G, R108L, and R108K alleles of PXA1 or
vector alone. 10-fold dilutions (top to bottom) of
the different cells were inoculated on medium containing 0.1% oleic
acid as a sole carbon source. B, -oxidation of
[
C]oleic acid by pxa1::LEU2 knockout
yeast expressing R108L or R108K alleles of
PXA1.
Figure 7: Immunoblot analysis of Pxa1p in noncomplementing mutants. The organelle pellet fraction of oleic acid-induced cells consisting primarily of peroxisomes and mitochondria was analyzed by immunoblotting using a rabbit antiserum to Pxa1p. The cells analyzed are pxa1::LEU2 knockout (KO) yeast expressing (from left to right): wild type PXA1 (WT), vector alone, or the R108L, E294D, or G301P alleles of PXA1.
Figure 8: Localization of mutant Pxa1ps to peroxisomes. pxa1::LEU2 mutant yeast expressing the wild type (WT) or the indicated PXA1 alleles (R108L, E294D, and G301P) were induced by oleic acid. The high density organelle pellets consisting primarily of peroxisomes and mitochondria were fractionated on Nycodense gradients. Gradient fractions were assayed for a peroxisome marker (Catalase) and a mitochondrial marker (succinate dehydrogenase (SDH)) activity. Only the profile of the fractionation of yeast expressing the wild type PXA1 allele is shown. All others had a similar profile with the peaks of catalase and SDH activity in the same fractions as the wild type (data not shown). Shown below are immunoblots in which equal volumes of fractions from each of the indicated gradients were probed with a rabbit antiserum to Pxa1p. The position of Pxa1p is indicated by an arrow.
ABC transporters are present in all cellular living organisms and are involved in the transport of a variety of substrates across membranes. Members of this protein superfamily are involved in many medically relevant processes including resistance to cancer chemotherapy mediated by the MDR1 transporter (28) and antigen presentation in the immune system by the TAP transporter(29) . Moreover, mutations in genes encoding ABC transporters are responsible for genetic diseases including adrenoleukodystrophy and cystic fibrosis (7, 30) . By comparing the amino acid sequence of members in one subgroup of these transporters, we identified two conserved motifs, the EAA-like motif and the loop1 motif (see Fig. 2).
We utilized site-directed mutagenesis in the gene
encoding Pxa1p, a yeast peroxisomal ABC transporter, to further analyze
these motifs. We found that as in prokaryotic ABC transporters, the
central glycine in the EAA-like motif (Gly of Pxa1p) is
important for transporter function. This glycine is 100% conserved in a
survey of 61 prokaryotic ABC transporters(13) . Conversely, the
consensus glutamic acid (Glu
in Pxa1p) is less well
conserved in the prokaryotic transporters (16 out of 61 have glutamic
acid at this position; 9 out of 61 have aspartic acid). Conservative
substitutions of this glutamic acid in the prokaryotic PstC and PstA
ABC transporters (E202Q and E185Q) had either no effect on transport or
reduced it by only 50%, respectively(31) . Despite this, we
found that a conservative substitution at this site (E294D) completely
inactivates Pxa1p. Similarly, missense mutations altering the
corresponding glutamic acid residue of ALDP (E291) have been described
in two unrelated adrenoleukodystrophy patients (E291D and
E291L)(8, 16, 17, 18) .
Several recent studies suggest that the substrate binding site of ABC transporters is located in the TMD(1, 32, 33, 34) . In contrast, Sheps et al.(33) and Beaudet et al.(30) found that missense mutations in the NBD conserved (C) sequence also influence substrate specificity. The C sequence is a 19-mer located immediately N-terminal to the Walker B motif of the NBD and is a hallmark of ABC transporters(1, 30) . Because there is interaction between the TMD and the NBD(36) , it is possible that the C sequence mediates this interaction and exerts its effect on substrate specificity in this manner. Gottesman et al.(37) have recently suggested that the C sequence of MDR1 interacts with the TMD. The exact function of the EAA motif is not known. However, based on the evolutionary conservation in prokaryotes, Kerppola and Ames suggested that the EAA motif was the segment of the TMD that interacts with the NBD(15) . Moreover, Saurin et al.(13) presented evidence that the substrate specificity of prokaryotic ABC transporters can be predicted by sequence variation in their EAA-motifs. Our results suggest that the EAA-like motif of peroxisomal ABC transporters is important for function but not for stability or targeting. Similar results have been described for prokaryotic EAA motifs(14) . Watkins et al.(8) , however, found that the E291D allele of ALDP was antigen negative by immunohistochemistry. As shown in Fig. 3, some sequence conservation in this region is specific for certain groups of eukaryotic ABC transporters. Taking all these results together, we suggest that the C region of the NBD interacts with the TMD through the EAA and EAA-like motifs.
We also recognized a
conserved sequence in the region between TM1 and TM2 that we designated
the loop1 motif. Three residues (Leu, Arg
,
and Thr
in Pxa1p) are completely conserved in our
alignment of peroxisomal ABC transporters. Changing Arg
to either leucine or lysine reduces Pxa1p function. This result
agrees with the observation that a missense mutation in the
corresponding residue in ALDP (Arg
) causes
adrenoleukodystrophy(16) . We observed a moderately reduced
amount of Pxa1p (approximately 20%) in high density organelles from
cells expressing the R108L mutation, suggesting that this mutation does
influence Pxa1p targeting or stability. A role for the loop1 motif in
targeting is also suggested by the report that an N-terminal fragment
of the ABC transporter Ste6p functions as a signal
sequence(38) . Interestingly, in addition to TM1 of Ste6p, this
fragment also contains the first loop between TM1 and TM2. Leucine and
arginine residues are positioned similarly relative to TM1 in both
Ste6p (Leu
and Arg
) and Pxa1p (Leu
and Arg
). If the loop1 motif is important for
correct cellular placement, its function may involve orientation of the
protein in the membrane rather than targeting because Ste6p is targeted
to the endoplasmic reticulum (39) rather than the peroxisome.
The importance of positive residues in membrane protein loops for
correct insertion in the membrane is well recognized (40) .
With the striking exception of the null phenotype of E294D, the
phenotypes of mutations in both the EAA-like and loop1 motifs of Pxa1p
were closely correlated with the predicted severity of the amino acid
substitution. This observation suggests that Pxa1p function is tightly
coupled to the overall peroxisomal -oxidation process in yeast.
In summary, using site-directed mutagenesis we have shown that two protein motifs in peroxisomal ABC transporters are important for transporter function. Missense mutations in these motifs cause dysfunction of Pxa1p in yeast, and mutations in corresponding residues of ALDP cause adrenoleukodystrophy in humans. This indicates that investigating the yeast protein Pxa1p is a useful system for studying the molecular basis of adrenoleukodystrophy.
Note Added in Proof-While this manuscript was in galleys, Swartzman et al. ((1996) J. Cell. Biol.132, 549) reported independent cloning of PXA1, which they termed PAL1. Their sequence agrees with that described in our earlier publication (Shani et al.(1995) Proc. Natl. Acad. Sci. U. S. A.92, 6012) except that they find an additional end terminal extension of the open reading frame that may represent an alternative form of the protein.