 |
INTRODUCTION |
Peroxisomes consist of a dense matrix and surrounding membrane.
Proteins are targeted to the peroxisomal matrix from the cytosol by
virtue of peroxisomal targeting signals
(PTSs)1 (1). Most matrix
proteins contain a PTS1 sequence at their extreme carboxyl terminus
consisting of three amino acids identical or related to the tripeptide
SKL. A few matrix proteins contain a PTS2 sequence close to the amino
terminus, which is often cleaved upon entering the peroxisomal assembly
pathway. Receptors for PTS1 and PTS2 have been identified, as have
docking proteins for the PTS receptors on the peroxisomal surface (2,
3). Receptor and cargo may translocate through the membrane before
receptors are recycled to the cytosol (4, 5).
Integral membrane proteins lack these PTS sequences and must be
targeted by alternative sequences. We have used Pmp47 from the
methylotrophic yeast Candida boidinii as a model protein to understand targeting to the peroxisome membrane. Pmp47 shares homology
with members of the mitochondrial carrier protein family (6). These
proteins span the membrane six times, and sequence similarity within
them suggests that the family was derived from an ancient gene
triplication. Pmp47 is important in the metabolism of medium-chain
fatty acids and may transport ATP (7), and it is also critical for the
import of dihydroxyacetone synthase, an abundant matrix protein (8).
Pmp47 contains two loops of amino acids on the matrix side and three on
the cytoplasmic side of the membrane. We previously identified a
targeting motif contained within the second matrix loop that we termed
the mPTS (9). We showed that the most important attribute of the
targeting loop is a basic cluster of amino acids of the sequence KIKKR.
Matrix-oriented basic clusters have since been shown to be an important
targeting element in several other peroxisomal membrane proteins
(10-13).
The Pmp47 second matrix loop fused to a carrier protein targets to
peroxisomes very weakly (9), as determined by organelle fractionation.
Similarly, a basic cluster from the cotton seed ascorbate peroxidase
(APX), fused to a synthetic membrane span and carrier protein, targets
to punctate and reticular structures, although peroxisomal localization
was not demonstrated (13). In contrast, a cytosol-oriented region of 22 amino acids in rat Pmp22 can target a different fragment of the
protein, which by itself is not sufficient for targeting, to the
peroxisomal membrane (14). Because some peroxisomal membrane proteins
integrate into peroxisomal precursor organelles before arriving at
peroxisomes (15), a more complex targeting signal may be required to
accommodate such a pathway.
We now describe experiments to indicate that Pmp47 requires discrete
regions for effective targeting. To gain evidence for the relative
contribution of each region we have established a sensitive and
quantitative in vivo fluorescence localization assay in
Saccharomyces cerevisiae. Results from the assay suggest
that charge is not the sole factor governing targeting by the basic cluster; side-chain structure and residue position may also be important. We show that targeting at low efficiency occurs even when
the basic cluster is eliminated, indicating other targeting elements
must exist within Pmp47. We have identified the other important signal
as consisting of a cytosol-oriented charged domain within cytoplasmic
loop 1 and the adjacent transmembrane domain, TMD2. TMD2 by itself is
poorly hydrophobic. Efficient targeting with high fidelity is only
achieved by combining these discrete motifs (including the matrix basic
cluster) with a strong transmembrane anchoring domain. Thus, effective
targeting of Pmp47 requires discrete regions of the protein on both
sides, and within, the membrane.
 |
EXPERIMENTAL PROCEDURES |
Strains and Culturing Conditions
Escherichia coli strain TG-1 (F' traD36 laclq
[lacZ]M15 proA+B+/supE
[hsdM-mcrB]5
[rk
mk
McrB
]
thi
(lac-proAB)) was used for plasmid
amplification. Yeast S. cerevisiae strain MMYO11
was used
in all the expression experiments (16). MMYO11
was transformed by
the lithium acetate method (17). Yeast transformants were maintained on
synthetic complete plates (2% glucose and 0.67% yeast nitrogen base
without amino acids (Difco Laboratories, Detroit, MI)) supplemented
with appropriate amino acids and bases. For glucose liquid culture,
MMYO11
was grown in synthetic dextrose medium (2% glucose and
0.67% yeast nitrogen base) supplemented with appropriate amino acids
and bases. To induce peroxisome proliferation in MMYO11
, cells were
cultured in SGd medium (3% glycerol, 0.1% glucose, 0.67% yeast
nitrogen base, supplemented with appropriate amino acids and bases) to an A600 of 1 to 2. They were then
"boosted" by adding 0.1 volume of 10% yeast extract, 20% peptone
and cultured for 4 h. Cells were harvested by centrifugation,
re-inoculated to a final A600 of 1 into oleate
medium (a semisynthetic medium (18) supplemented with 0.1% (v/v) oleic
acid, 0.2% (v/v) Tween 40 and appropriate amino acids and bases), and
cultured for 16-20 h.
Recombinant DNA Procedures and Reagents
Restriction enzymes and other DNA-modifying enzymes were
purchased from New England BioLabs (Beverly, MA). Isolation of DNA fragments and plasmids was carried out using the QIAEXII gel extraction kit and QIAprep Spin Miniprep kit from Qiagen, Inc. (Valencia, CA).
Polymerase chain reactions (PCR) were performed with a PTC-100 programmable thermal controller from MJ Research, Inc. (Watertown, MA)
using Pfu DNA polymerase. Standard recombinant DNA
techniques were performed (19).
Sources for Antibodies and Plasmids
Antibodies used in this study include anti-3-ketoacyl-CoA
thiolase (anti-thiolase) polyclonal antibodies (kind gift of Jonathan Rothblatt), and Texas Red goat anti-rabbit IgG (H+L) conjugate (Molecular Probes, Eugene, OR).
Plasmid constructs used in this study include pEMBLy3012, containing
the yeast phosphoglycerate kinase (PGK) promoter and terminator (kind
gift of Michael White, University of Texas Southwestern Medical Center,
Dallas, TX), pRS315 and pRS316 (20), and vectors pGFP-C1 and pDsRed
(CLONTECH Laboratories, Inc., Palo Alto, CA).
Construction of Plasmids
Pmp47-(1-267)-GFP--
The 1.8-kb PGK
promoter-terminator was excised from pEMBLy3012 by HindIII
and subcloned into the unique HindIII site of yeast centromeric plasmid pRS315. The resulting construct was named pRS315-PGK. A DNA fragment containing amino acids 1-267 of Pmp47 was
isolated from Candida boidinii genomic DNA by PCR using
upper primer W5U (5'-GAA GAT CTA TGT CTA CAA GAG AGT ACG
AC-3'), which introduced a BglII site at the amino terminus
(restriction sites introduced in this oligonucleotide and those
described subsequently are underlined), and lower primer W5L
(5'-AGG TAA AGA TTG AAC GCT ATC-3'). A unique endogenous
XbaI site is present at amino acid 267. The GFP coding
region was amplified from pGFP-C1 by PCR using upper primer W4U
(5'-GCT CTA GAA TGG GTA AAG GAG AAG AAC TT-3'), which
introduced an in-frame XbaI site to its amino terminus, and
lower primer W4L (5'-GAA GAT CTT TAC TTG TAT AGT TCA TCC
ATG CC-3'), which introduced a stop codon followed by a
BglII site to its carboxyl terminus. The Pmp47-(1-267)
fragment and the GFP coding region were both digested with
BglII and XbaI, and were then cloned into the
unique BglII site between the PGK promoter and terminator of
the pRS315-PGK to yield pPmp47-(1-267)-GFP.
Pmp47-GFP Mutant Constructs--
To facilitate the construction
of mutant constructs from pPmp47-(1-267)-GFP, the BglII
site between GFP and PGK terminator was disrupted by PCR mutagenesis to
produce the construct D004. D004 has a unique BglII site
preceding the start codon of the fusion protein and a unique
NcoI site within the GFP coding region (at amino acid 55 of
GFP). The DNA fragment encoding Pmp47-(1-267)-GFP (1-55) was used as
a cassette for constructing the mutant Pmp47-GFP fusion proteins. The
mutated forms of this cassette were cloned back into D004, replacing
the original sequence.
To construct the amino-terminal truncation mutants, the coding regions
for the amino-terminally truncated Pmp47-(x-267)-GFP (1-55)
fragments were isolated from D004 by PCR using specific upper primers
with a BglII site and start codon included at the 5'-end and
a common lower primer W20L (5'-ACA CCA TAA GAG AAA GTA GTG A-3') that
is located 3' of the endogenous NcoI site.
The carboxyl-terminal truncations, replacements, and internal deletions
were constructed using PCR SOEing (Splicing by Overlap Extension) (21,
22). Upper primers starting at specific amino-terminal regions of Pmp47
with an upstream BglII site and a start codon were used as
outer upper primers, and W20L was used as the common outer lower
primer. A pair of inner SOEing primers, each of which contains the
mutated sequences in its 5'-overlapping region, was used for each
replacement construct. Briefly, the outer upper primer and the lower
inner primer were used to isolate the amino-terminal flanking region of
the replacement. The inner upper primer and the outer lower primer W20L
were used to generate the carboxyl-terminal flanking region. Then the
two fragments, which contained the mutated sequences in their
overlapping region, were denatured, annealed to each other, and finally
fused together by overlap extension. The carboxyl-terminal truncations
and internal deletions were generated in a similar fashion, where the
overlapping regions served solely as fusion junctions without
introducing any replacement amino acids.
Block I and III deletion constructs were made from
pPmp47-(1-243)-GGG-GFP (see Table I). The TMD4 substitution constructs were made from pPmp47-(1-267)-GFP, the TMD3 substitution construct was
made from pPmp47-(70-267)-GFP, and the TMD2 exchange constructs were
made from pPmp47-(53-267)-GFP (see Table III).
The Pmp47-derived sequences of all fusion constructs were confirmed by
DNA sequencing.
DsRed-AKL--
First, the PGK promoter-terminator from
pEMBLy3012 was subcloned into the unique HindIII site of the
yeast centromeric plasmid pRS316 to yield pRS316-PGK. The coding region
of the red fluorescence protein DsRed was amplified from the plasmid
pDsRed by two rounds of PCR. The upper primer W71U (5'-AAT GAA
GAT CTA TGA GGT CTT CCA AGA ATG TTA-3'), which incorporated an
upstream BglII site, and the lower primer W71L-1
(5'-TTT AGC ACC TCC TCC AAG GAA CAG ATG GTG GCG TC-3') were
used for the first round, and W71U and W71L-2 (5'-AAT GAA GAT
CTT TAC AAT TTA GCA CCT CCT CCA AGG-3') were used for
the second round. The two lower primers incorporated three glycines,
the peroxisomal targeting signal type I (PTS1), alanine-lysine-leucine,
and the stop codon (all shown in boldface). Another
BglII site was also added downstream of the stop codon. The
amplified fragment was then cloned into the unique BglII
site of the pRS316-PGK to yield pDsRed-AKL, which encodes a red
fluorescence protein that targets well to peroxisomes.
Indirect Immunofluorescence, Fluorescence, and Confocal
Microscopy
Immunofluorescence of S. cerevisiae was performed as
described previously (23). Fluorescence in both living and fixed cells was visualized with a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) and an MRC 600 confocal imaging system (Bio-Rad Laboratories, Hercules, CA). The GFP was excited at 488 nm,
and the DsRed and the Texas Red fluorophore were excited at 568 nm. The images shown in the figures are single representative Z-sections through cells that were fixed, converted to spheroplasts, and permeabilized (23) to eliminate vacuolar autofluorescence.
Quantification of Peroxisomal Localization
Yeast transformants
were cultured in SGd (synthetic medium containing 3% glycerol and
0.1% dextrose) and induced with oleic acid as described above. Living
cells were harvested, spread in a monolayer on microscope slides, and
observed by confocal microscopy. Five randomly chosen fields of at
least 40 cells per field were examined for each culture. For each field
a 19-section Z-series, 0.3 µm between optical slices, was recorded.
The Z-series recordings were animated by IMAGE (version 1.60, National
Institutes of Health). The percentage of the cells showing punctate
fluorescence was determined and normalized against the positive and
negative controls (Pmp47-(1-267)-GFP and untransformed MMY011
,
respectively) in each experiment. The raw percentages of cells showing
punctate fluorescence in the controls were typically 70% and 3%,
respectively. The normalized value was termed the localization score,
such that scores of 100 and 0 correspond to the positive and negative
controls. A mean and standard error were determined from the
localization scores of the five Z-series for each fusion protein. For
the cluster modifications KIKQQ, KIQQQ, QIQQQ, AAKKA, AAAKA, and AAAAA,
the data represent the average and standard error of ten Z-series from
two independent experiments.
 |
RESULTS |
A Quantitative Assay for Peroxisomal Localization--
Fig.
1A illustrates the Pmp47-GFP
fusion protein used as the basis of deletion and mutation analysis in
this study. The Pmp47 section consists of the first 267 of a total of
423 amino acids of the full-length protein. This region comprises the
amino-terminal matrix fragment, the first two cytosolic and two
matrix-oriented loops, and the first five of the six transmembrane
domains (TMDs). The sequence of matrix loop 2, containing the KIKKR
basic cluster previously shown to be important for targeting, is also
shown. Amino acids 1-267 have been shown previously to be sufficient for efficient targeting to peroxisomes in S. cerevisiae (9, 24).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Pmp47-(1-267)-GFP sorts to oleate-induced
peroxisomes in S. cerevisiae. A, a
topological map of Pmp47-(1-267)-GFP. White rectangles
denote the first five (of six) TMDs and contain the numbers of their
first and last amino acids. The amino acids of matrix loop 2 are shown,
divided into three blocks so that block II consists of the basic
cluster. The fusion junction is indicated by an arrow.
B, representative pairs of fluorescence images of single
cells that were transformed with Pmp47-(1-267)-GFP (here labeled
1-267) or doubly transformed with Pmp47-(1-267)-GFP and
DsRed-AKL and cultured in oleic acid to induce peroxisomes. Cells were
prepared for indirect immunofluorescence to detect thiolase
(first row) or permeabilized to remove vacuolar
autofluorescence (second row and subsequent figures). In
both sets GFP was detected by fluorescence. Scale bar, 2 µm.
|
|
Correct targeting of Pmp47-(1-267)-GFP to peroxisomes was evident
either with 3-ketoacyl-CoA thiolase, a PTS2 protein used in previous
studies (24), or DsRed-AKL, a PTS1-tagged red fluorescence protein
(Fig. 1B), as observed by confocal microscopy of cells grown
in oleic acid to induce peroxisomal proliferation. Peroxisomes of most
cells were clustered by Pmp47-(1-267)-GFP, as apparent in the figure.
The clustering was dependent on an intact Pmp47 amino terminus (data
not shown) and is of unknown physiological relevance.
In preliminary experiments with several Pmp47-GFP fusion proteins we
observed negligible mistargeting to organelles other than peroxisomes
in living cells. If any fluorescence was seen, it was punctate and
peroxisomal (i.e. colocalized with peroxisomal markers),
suggesting that mistargeted Pmp47 fusion proteins were unstable in
general; the few exceptions will be noted below. We also observed a
correlation between the intensity of the fluorescence pattern and the
percentage of cells displaying punctate fluorescence. Fusion proteins
with relatively intense punctate fluorescence displayed this pattern in
about 70% of cells. Weakly fluorescent punctate patterns were seen in
10-20% of cells (the remainder displayed no visible GFP
fluorescence). We therefore developed a "localization score" as an
indicator of peroxisomal targeting and retention of Pmp47-GFP fusion
proteins. The localization score is based on the percentage of the
cells displaying a peroxisome-like punctate pattern normalized to
positive and negative controls (see "Experimental Procedures" for
details). We separately verified the identity of the green fluorescent
organelles of key constructs by assaying colocalization with a
peroxisomal marker. The localization score discriminates between fusion
proteins that are efficiently sorted to and retained in peroxisomes and
those that are deficient in stability or targeting.
Basicity, Position, and Side-chain Structure Are Important Elements
of the Matrix Basic Cluster--
An initial set of GFP fusion proteins
were expressed to confirm the importance of the basic cluster contained
within matrix loop 2 and to understand the important elements of the
cluster (Table I). Both the localization
scores and results of colocalization assays with thiolase or DsRed-AKL
are shown. Removal of TMD5 had little effect on localization,
indicating that it contained no essential targeting information.
Further removal of blocks II (the basic cluster) and III of the second
matrix loop completely obliterated localization, suggesting that this
region contains important targeting information. This conclusion was
supported first by replacement of each block of the loop with glycines
or alanines. Only replacement of block II resulted in a low
localization score. The colocalization assay revealed that, although
correct targeting to peroxisomes occurred in some of the few cells that showed punctate fluorescence in the block II substitution, many of the
green fluorescent organelles were not peroxisomes (Fig. 2), yielding partial colocalization.
Finally, the importance of spacing of block II to the membrane was
assessed with block deletions. Removal of block I resulted in
localization score of 25. This may be an underestimation of the ability
of the basic cluster to function close to a membrane span, because the
matched control, Pmp47-(1-243-GGG)-GFP, had a localization score of
only 17. The low score may reflect destabilization of the fusion
protein by the triglycine spacer. Regardless, the basic cluster is
functional to some extent when placed directly behind TMD4 or close to
GFP (score of 56). Despite the low localization scores, these fusion proteins completely colocalize with peroxisomal markers (Table I).

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 2.
Residual peroxisomal targeting occurs even in
the absence of a basic cluster. Representative pairs of
fluorescence images from cells doubly transformed with block II
replacement mutants (in the context of 1-267) and DsRed-AKL.
Colocalization is assigned when all the GFP stained structures are also
stained with DsRed-AKL. Pmp47-(1-267)-GFP is denoted as
KIKKR here. Scale bar, 2 µm.
|
|
We then designed GFP fusion proteins to understand the critical
characteristics of the KIKKR cluster. First we altered the placement of
the isoleucine residue within the cluster (in the context of
Pmp47-(1-267)-GFP) and found no significant decrease in localization
efficiency (Table I).
We then performed two titrations of basicity. In the first we
substituted glutamines for basic residues beginning on the carboxyl side of the cluster. It can be seen that removal of two of the four
basic residues (KIKQQ) still resulted in a localization score of 70 (Table I). However, the score dropped precipitously with one (KIQQQ) or
no basic charge (QIQQQ) in the cluster, similar to the behavior of
Pex3p (10). We were surprised that the QIQQQ-containing fusion protein
still localized with a score of 33. The QIQQQ substitution completely
colocalized with DsRed-AKL (Fig. 2).
Similar data were obtained with the second basic titration in which
alanines replaced basic residues from the amino terminus of the
cluster. AAAKR targeted with a score of 68, whereas the score dropped
to 27 for AAAAR. Within the substituted clusters containing one or two
basic charges, significant differences were seen: AAAKR localized much
better than AAKKA (scores of 68 and 26, respectively), and AAAAR
localized better than AAAKA (27 and 8). These data suggest that not all
basic residues within the cluster are equivalent. Furthermore, the
difference in targeting between the two neutral substitutions QIQQQ and
AAAAA both in localization score (scores of 33 and 18, respectively)
and colocalization with DsRed-AKL (complete versus partial
colocalization) suggests that side-chain structure is important in
basic cluster targeting. More definitive conclusions must await an
in vitro targeting assay where stability can be controlled
and correct folding more easily measured.
TMD2 and Its Amino-terminal Adjacent Region Are Required for
Peroxisomal Localization--
We suspected that important targeting
information was contained in the first half of Pmp47, because targeting
was difficult to demonstrate without it (24). To localize the putative
targeting information a series of amino-terminal deletions of
Pmp47-(1-267)-GFP were constructed (Table
II). TMD1 was dispensable as well as most of the first cytosolic loop, because Pmp47-(70-267)-GFP targeted well.
Excellent colocalization of a fusion protein beginning at amino acid
75, 20 residues from TMD2, can be seen in Fig.
3. A fusion protein that lacked the first
78 amino acids did not display any peroxisomal fluorescence. However,
peroxisomal localization reappeared in a fusion protein that lacked the
first 86 amino acids, although the localization score dropped to 30 and
mistargeting to other organelles was observed (Fig. 3), suggesting that
the distal region of the first cytoplasmic loop may be important for peroxisomal localization. Targeting to peroxisomes in oleate-grown cells was never observed in fusion proteins with amino termini beyond
TMD2. Unlike most of the Pmp47-GFP proteins discussed earlier, a few of
these fusion proteins stably mislocalized to nonperoxisomal structures
of the oleate-grown cells, either punctate or reticular (Table II). Our
results suggest that targeting information is contained within TMD2 or
amino acids directly adjacent to it.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 3.
The importance of amino acids 70-110 for
localization to oleate-induced peroxisomes. Colocalization of the
75-267 GFP fusion is excellent, although its localization score is
lower. Colocalization of 87-267 is much weaker but still can be seen.
Replacement of TMD2 yields punctate patterns that do not correspond to
peroxisomes. Shown are representative pairs of fluorescence images from
cells doubly transformed with Pmp47-GFP fusions and DsRed-AKL.
75-267, Pmp47-(75-267)-GFP; 87-267,
Pmp47-(87-267)-GFP; TMD2(Aac2p) and TMD2(Mpcpp),
Pmp47-(53-267)-GFP with the TMD2 replaced with the TMD2s from
ScAac2p and ScMpcpp, respectively (see Table
III). Scale bar, 2 µm.
|
|
To further test whether TMD2 or the other remaining membrane-spanning
domains have essential targeting information, we substituted these TMDs
with spans from membrane proteins of other organelles. Membrane spans
from Sec61p (a membrane protein of the endoplasmic reticulum) or a
mitochondrial solute transporter were able to substitute for TMD4 or
TMD3, respectively, indicating that neither Pmp47 span contained
essential targeting information (Table
III). However, we were unable to
demonstrate peroxisomal targeting when Pmp47 TMD2 was substituted with
the homologous TMDs from two different mitochondrial solute
transporters; they have similar hydrophobicity to the Pmp47 TMD2. The
substituted fusion proteins targeted instead to an unknown punctate
organelle (Fig. 3). These experiments support a role of TMD2 in
peroxisomal targeting.
Three Small Regions within Pmp47 Are Sufficient for Effective
Targeting--
Experiments described so far suggest that important
peroxisomal membrane targeting information is contained in the basic
cluster within matrix loop 2 as well as in TMD2 and an adjacent region of cytosolic loop 1. To determine whether these domains are sufficient for peroxisomal targeting, amino acids 70-110 (carboxyl end of cytoplasmic loop 1 and TMD2) were fused to matrix loop 2. Hydrophobicity analysis indicates that TMD2 is a weak transmembrane
span, and previous data show that TMD2 is not sufficient to anchor the
protein onto the membrane bilayer (24); therefore, we added TMD5, a strongly hydrophobic domain without essential targeting information, to
allow membrane integration of the fusion protein. This fusion protein
targeted to peroxisomes with a localization score of 48 and yielded a
perfect colocalization pattern with DsRed-AKL, indicating that these
small regions of Pmp47 were sufficient for targeting (Fig.
4).

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 4.
Amino acids 70-110, a matrix-facing basic
cluster, and an anchoring TMD are sufficient for peroxisomal
localization. Top, diagram of the three minimal
Pmp47-GFP fusion proteins and their localization scores;
bottom, representative pairs of fluorescence images from
cells doubly transformed with DsRed-AKL and each of the three Pmp47-GFP
fusion constructs. Scale bar, 2 µm.
|
|
The first matrix loop contains three pairs of basic residues at amino
acids 123-124, 134-135, and 139-140. Because two basic residues
within matrix loop 2 could replace the basic cluster, we asked whether
the first matrix loop could substitute for the second one. The data in
Fig. 4 show that both fusion proteins had similar localization scores
and both colocalized well with DsRed-AKL. Therefore, these loops have
redundant targeting information. However, TMD2 appears to be essential;
substitution of TMD4 for TMD2 resulted in loss of fluorescence,
suggesting an inability to target to peroxisomes (Fig. 4).
In addition to the demonstrated importance of matrix-oriented basic
clusters in targeting to the peroxisomal membrane, a short cytosol-oriented region adjacent to TMD1 of rat Pmp22 has been shown to
contain a peroxisomal targeting signal (14). Similarly, data suggest
that a cytosolic region adjacent to the membrane span of Pex15p is
required for peroxisomal localization (11). We asked whether either
region could substitute for amino acids 70-94 (the region in
cytoplasmic loop 1 adjacent to TMD2) of Pmp47. No peroxisomal targeting
was seen with either substitution in yeast grown on oleic acid.
However, we found that the Pex15p substitution yielded strong
fluorescence and excellent colocalization in cells grown on glucose, a
substrate that represses peroxisomal proliferation, although a few
small peroxisomes remain (Fig. 5).
Colocalization was also seen with the Pmp22 substitution under the same
growth conditions, although fluorescence was weaker. In contrast, a
cytosol-oriented region adjacent to the TMD1 of Sec61p yielded no
fluorescence in cells grown in either medium. Although the effect of
carbon source on peroxisomal membrane targeting is unclear at present, these data suggest that the cytoplasmic regions in Pmp47, Pex15p, and
Pmp22 share a common targeting element. Comparison of the sequences
(Fig. 5) failed to identify apparent sequence similarity, although all
three sequences have amphipathic characteristics and net positive
charge.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 5.
Amino acids 70-94 contain peroxisomal
targeting information. A, the sequence of Pmp47
residues 70-94 and those from cytoplasmic regions of two peroxisomal
membrane proteins containing putative targeting information,
ScPmp22 and ScPex15p, and one ER membrane
protein, ScSec61p, respectively, are shown. Charged residues
are in boldface text. All four sequences immediately flank a
TMD. B, representative pairs of fluorescence images from
cells doubly transformed with DsRed-AKL and domain exchange mutants and
cultured in glucose-containing medium. Pmp22-Pmp47,
ScPmp22-(1-19)-Pmp47-(95-267)-GFP;
Pex15p-Pmp47,
ScPex15p-(301-331)-Pmp47-(95-267)-GFP;
Sec61p-Pmp47,
ScSec61p-(1-32)-Pmp47-(95-267)-GFP. Scale bar,
2 µm.
|
|
 |
DISCUSSION |
In this work we have analyzed some important characteristics of
the Pmp47 basic cluster and have identified a second area consisting of
a transmembrane domain and adjacent cytoplasmic sequence within Pmp47
that is important for targeting. A quantitative localization assay
allows us to estimate that a minimal GFP fusion protein consisting of
little more than these regions and an anchoring membrane span localizes
to peroxisomes with about 50% efficiency compared with wild type
protein and with full fidelity (i.e. no detectable mistargeting).
Judging Localization in Vivo--
Results from our GFP assay
correlated well with previous data derived from immunofluorescence and
cell fractionation studies (9, 24). Thus, matrix loop 2 was found to be
essential for targeting, and substitution of amino acids in block II
had by far the most severe effect compared with changes in the other two blocks.
We measured both localization efficiency (approximated by the strength
of the fluorescence signal and reflected in the localization score) and
fidelity (the degree of correspondence of green fluorescent spots with
those from the peroxisomal marker). The use of two distinct but
complementary assays allowed us to dissect the basic cluster and
analyze other areas of Pmp47 for targeting signals with confidence. The
behavior of several Pmp47-GFP fusion proteins that targeted to
nonperoxisomal punctate structures in oleate-cultured cells serves to
emphasize the importance of a good colocalization assay.
There are limitations inherent in our in vivo system,
however. It is possible that the rates of synthesis among the fusion proteins are different, although such difference should be minor because the same promoter is used for all fusion proteins. Another potential limitation is that the dynamic range may not fully reflect the extent of targeting; the assay might reach a maximum value at low
actual targeting efficiency. However, fluorescence of our positive
control (Pmp47-(1-267)-GFP) is very dim relative to that of a GFP
peroxisomal matrix marker and we can detect fluorescence in only 70%
of cells with this fusion protein, whereas GFP-AKL is detected in over
90% of cells (data not shown), suggesting that our assay for
peroxisomal membrane targeting does not saturate. However, targeting of
fusion proteins with very weak signals may be missed by our assay,
although it is probably detected by organelle fractionation and
sensitive immunoblotting.
A more substantive caveat of the assay is that aberrant protein folding
or low stability, rather than a weak targeting signal, would be
reflected as a low localization score. This is a problem with most
in vivo targeting assays as well as our own. Therefore, we
have interpreted low localization scores with caution. The strength of
the assay lies in identification of fusion proteins with high sorting
scores, indicating that they must contain strong targeting signals.
The Role of the Basic Cluster--
The mPTSs of several
peroxisomal membrane proteins contain basic clusters (10-13). Our data
show that basicity is an important functional attribute of the cluster.
However, the loss of two of the four positive charges has only a minor
effect. This is in agreement with studies of a basic cluster that
directs Pex3p to peroxisomes (10). The fact that both KIKQQ and AAAKR
localize well (scores of 70 and 68, respectively) indicates that no
particular specific residue within KIKKR is required. But not all
substitutions with similar total charge behave similarly: AAKKA
localizes much worse that AAAKR (scores of 26 and 68, respectively).
Likewise, AAAAR localizes worse than AAAKA (scores of 27 and 8, respectively). This suggests that the position of the residues, in
addition to the total charge of the cluster, governs targeting in some
way. The ability of glutamines to substitute for all basic residues and
sort better than a construct with alanines (both higher localization score and better colocalization with thiolase and DsRed-AKL) further suggests that the side chains of residues in the basic cluster contribute significantly to the binding to a receptor. This is also the
case for the binding of the classical NLS to importin
, where the
binding is stabilized by the methylene bridges of the basic residues
(25).
Our data indicate that the functional equivalent of the basic cluster
in other peroxisomal membrane proteins need not contain more than two
basic charges, or may function with even fewer in the proper context.
Thus, the first matrix loop of Pmp47, containing three pairs of basic
residues, can substitute for the second one in our minimal construct
without significant loss of targeting. At least one matrix domain with
a dibasic sequence or multiple basic residues in a cluster is present
in all peroxisomal integral membrane proteins with known topology
(11-14, 26-30). These all may serve a similar targeting role.
However, the spacing between these basic clusters and their preceding
TMDs are quite different. The fact that deletion of block I in
Pmp47-(1-243)-GGG-GFP does not obliterate targeting suggests that this
spacing is not critical. These data all support the hypothesis that the
matrix-facing basic cluster serves as a targeting motif.
Additional Amino-terminal Signals Are Required for Sorting--
A
number of experiments motivated us to search for targeting signals
upstream of matrix loop 2. First, residual sorting was observed even
when the basic cluster KIKKR was replaced with AAAAA, which suggested
that additional signals existed elsewhere. Second, fusion proteins
missing large regions of the amino-terminal half of Pmp47 have been
unstable and unable to target (24). Third, although we had previously
shown that a fusion protein consisting of only GFP and matrix loop 2 comigrated on a Nycodenz gradient with peroxisomes, the extent of
targeting was poor and could not be independently verified by
fluorescence microscopy (9). Finally, Pmp47-GFP fusion proteins
containing the second matrix loop flanked only with TMD4, TMD5, or both
(Pmp47-(176-243)-GFP, Pmp47-(226-267)-GFP, and Pmp47-(203-267)-GFP,
respectively) do not show peroxisomal localization at all (Table II and
data not shown).
Our analysis indicates that peroxisomal localization is affected when
more than 69 amino acids are deleted from the amino terminus,
implicating the end of the first cytoplasmic loop in peroxisomal
targeting. Our substitution experiments with cytoplasmic regions of
Pmp22 and Pex15p suggest that these regions may fulfill similar
functions. A conserved motif,
YX3LX3PX3(K/Q/N), was
identified in proteins in the Pmp22 family that may serve a targeting
function (14). This motif, however, is not found in the Pmp47
cytoplasmic loop, suggesting that the shared targeting signal may be
related to secondary versus primary sequence structure. Our
experiments show that Pmp22 and Pex15p cytoplasmic sequences were only
able to complement in cells grown in glucose, not oleate. This may reflect a quantitative or qualitative difference in targeting to basal
versus induced peroxisomes. There may be enhanced cytosolic or matrix proteolysis in oleate-grown cells, such that poorly folded
proteins are more likely to be degraded before or after arrival at
peroxisomes. Perhaps the signals on Pmp47 form internal interactions
that are not optimally reproduced with the Pex15p or Pmp22
substitutions. Another possibility is that there is an additional
targeting step involved requiring a hierarchy of signals to reach
induced peroxisomes, perhaps related to the vesicular intermediates
identified in Yarrowia lipolytica (31), and the mixed
Pex15p-Pmp47 or Pmp22-Pmp47 signals are not sufficiently strong to
allow passage through this critical step.
TMD2, adjacent to the important region of cytoplasmic loop 1, is
necessary for the targeting of Pmp47. The evidence supporting the
importance of TMD2 in targeting stems by its necessity for targeting
and the inability to be substituted with either TMD4 or spans of
analogous mitochondrial carrier proteins. TMD2 has a low hydrophobicity
score, in contrast to TMD4 and TMD5 (32). Fusion proteins lacking TMD4
and TMD5 are soluble in the cell (24). Therefore, we added TMD5 to
anchor our minimal fusion protein to the membrane. Stabilization of
TMD2 by TMD5 within the membrane may occur through an ionic interaction
between E97 in TMD2 and K252 in TMD5. We detect no sequence similarity
of Pmp47 TMD2 and transmembrane domains of other peroxisomal membrane proteins. The TMD2 signal may be more related to its secondary structure, or it may depend on interactions with the adjacent region of
the cytoplasmic loop during targeting. It would be interesting to
assess whether these two adjacent regions can be separated while
maintaining targeting function.
Membrane Orientation May Affect Requirements for Targeting
Signals--
There are conflicting reports about the nature of the
signal that directs proteins to the peroxisomal membrane. Although
matrix-oriented basic clusters have been shown to be important for many
peroxisomal membrane proteins, the critical signal on Pmp22 appears to
be a cytoplasmic sequence adjacent to a TMD. All membrane proteins require a TMD for membrane interaction, yet a synthetic TMD might function to some extent (13).
Do these data imply several different targeting pathways and receptor
systems for peroxisomal membrane proteins or can the observations
described above be reconciled into a simpler model? We propose that
targeting signals depend in part on the topology of proteins across the
peroxisomal membrane (Fig. 6). According to this model type I membrane proteins require a matrix-oriented basic
cluster and transmembrane span for targeting and integration. We
hypothesize that the transmembrane span has specific targeting information as well as a membrane-anchoring role, because it seems improbable that a basic cluster has sufficient information for targeting by itself. A test of this hypothesis will require a demonstration of peroxisomal colocalization of a single span protein with a synthetic TMD substitution.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Peroxisomal targeting signals may depend on
protein orientation. The peroxisomal targeting signals for three
different types of membrane proteins (type I, type II, and multispan
transmembrane proteins, respectively) are shown. According to this
model, all of them contain an important basic cluster facing the matrix
and membrane span containing targeting information. Type II proteins
and multispanning proteins may require additional information on the
cytoplasmic side of the membrane. Targeting information is indicated in
black. Listed are proteins with known topology and
identified targeting regions. See text for further discussion.
References: PpPex22p, PpPex17p,
PpPex13p, PpPex2p (30); ScPex15p (11);
GhpAPX (13); CbPmp47, this study;
RnPmp22 (14); HsALDP (26).
|
|
In contrast to type I proteins, our model predicts that type II and
multispanning proteins require additional information within
cytoplasmic-oriented sequences. This may reflect a more complex pathway
and different kinetics of membrane insertion compared with those of
type I proteins. This is reasonable, because the targeting signal of
type I proteins would become available to the receptor machinery sooner
during translation. Proteins in the other two classes may require
additional chaperones or receptor components that bind earlier.
The model stipulates that the functionality of the matrix basic cluster
is conserved in all peroxisomal integral membrane proteins. Although
removal of the basic cluster in loop 1 of Pmp22 has no effect on
targeting, a minimally sufficient fragment retains the sequence KMR
from the second matrix loop (14), which may suffice for a basic
cluster, suggesting a redundant function. Redundancy in targeting
signals on multispanning peroxisomal membrane proteins is also shown in
our substitution of matrix loop 1 for matrix loop 2 in our minimal
fusion proteins. Both Pmp47 minimal proteins have similar localization
scores. The score for Pmp47-(1-267)-GFP is double that of the minimal
proteins, which may reflect the presence of both basic matrix loops,
other redundant signals yet undetected, or a more stable protein
structure in the membrane.
Beyond our model, there may be another level of complexity of targeting
signals, if peroxisomal membrane proteins can enter a series of
vesicular precursors at different points in the pathway (31).
Determining the signals that direct proteins to specific peroxisomal
organelle precursors awaits a more complete understanding of this novel pathway.
The identification of discrete sorting regions on peroxisomal membrane
proteins is an important step to understanding the mechanism of
targeting and membrane integration. Although Pex19p has been implicated
as a receptor for peroxisomal membrane proteins (33), absence of
overlap between binding and targeting domains on several peroxisomal
membrane proteins suggest that other factors must also be involved
(30). We hope the identification of the important targeting elements
within Pmp47 will lead to dissection of the precise function of each
signal in peroxisomal membrane assembly.