Correspondence to: Suresh Subramani, Department of Biology, University of California San Diego, Bonner Hall, Room 3230, 9500 Gilman Drive, La Jolla, CA 92093-0322., ssubramani{at}ucsd.edu (E-mail), (619) 534-2327 (phone), (619) 534-0053 (fax)
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Abstract |
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We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A pex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.
Key Words: organelle, peroxin, peroxisome, protein transport, yeast
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Introduction |
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PEROXISOMES are single-membranebound organelles present in all eukaryotic cells. They contain enzymes that are responsible for such metabolic pathways as hydrogen peroxide metabolism, ß-oxidation of long-chain fatty acids, synthesis of plasmalogens, cholesterol, and bile acids, and degradation of purines and amino acids (for review see
Matrix-localized enzymes contain either one of two peroxisome-targeting signals (PTSs)1. PTS1 is located at the extreme COOH terminus of peroxisomal proteins. It consists of three amino acids and has the sequence SKL or some variants of it. PTS2 is present at the NH2 terminus and has a consensus sequence of R/K-L/V/I-X5-H/Q-L/A (for review see
There is little known of the mechanism for targeting peroxisomal membrane proteins. Different consensus sequences for peroxisomal membrane targeting have been proposed (
Another interesting protein involved in peroxisomal protein transport is Pex4p. Pex4p was shown to be highly homologous to ubiquitin-conjugating enzymes (UBCs) (
In this study we have characterized a novel peroxin, Pex22p, from P. pastoris. We analyzed the subcellular localization, targeting signal, topology, interacting partners, and the functions of this protein. Our data shed light on the roles of Pex4p and Pex22p in peroxisomal matrix protein import and show that these proteins are also conserved in S. cerevisiae.
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Materials and Methods |
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Strains and Media
P. pastoris strains used in this study were as follows: parental wild-type strains PPY1, PPY12 (PPY1 arg4, his4), and SMD1163 (his4, pep4, prb1); STK10 (PPY12, pex22.1, ARG4::pTW84 (PTS2-GFP [green fluorescent protein] (S65T)/GAPDH, PTS2-BLE/GAPDH); STK11 (PPY12, pex22::Zeocin); STK12 (SMD1163,
pex22::Zeocin); STK13 (PPY12,
pex4::Zeocin); STK14 (SMD1163,
pex4::Zeocin); STK15 (PPY12, pTK51 [PEX4p::NH-PEX4, Zeocinr]). S. cerevisiae strains used: BJ1991 (MAT
, leu2, trp1, ura3-251, prb1-1122, pep4-3); STK16 (BJ1991,
yaf5); L40 (MATa, his3
200, trp1-901, leu2-3,112, ade2, LYS2::(lexAop)4-HIS3, URA3::(lexAop)8-lacZ). The Escherichia coli strain used for cloning procedures was JM109 and for protein expression, SG13009. Yeast media were as described in
Cloning Procedures
Standard cloning procedures were used (
Isolation of pex Mutants and Cloning of PEX22
The isolation of pex mutants was performed according to
Construction of Disruptions
To disrupt PEX22, the 5' and 3' regions of the gene were amplified with PCR (TK45 and TK46 for the 5' region and TK47 and TK48 for the 3' region). The 5' fragment was cloned as a BamHI-SmaI fragment into pBluescriptSKII (Stratagene). The 3' fragment was then ligated as an EcoRI-SmaI fragment into this vector. The resulting plasmid was cut with SmaI and a blunt-ended HaeII-BamHI Zeocin fragment (cut out from plasmid pPICZ A; Invitrogen) was inserted. The resulting plasmid, pTK29, was cut with BamHI and EcoRI and transformed into PPY12 and SMD1163. The disruptions were confirmed by PCR.
The 5' and 3' regions of the PEX4 gene were amplified with PCR (primers TK41 and TK42 for the 5' region and TK43 and TK44 for the 3' region). The 5' fragment was cloned as a BamHI-SmaI fragment into pBluescriptSKII. The 3' fragment was then ligated as an HindIII-SmaI fragment into this vector (cut with HindIII-SmaI). The resulting fragment was then cut with SmaI and a blunt-ended HaeII-BamHI Zeocin fragment was inserted. The resulting plasmid, pTK35, was cut with BamHI and HindIII and transformed into PPY12 and SMD1163. The disruptions were confirmed by PCR.
The ScYAF5 gene was disrupted according to
Construction of Plasmids
Plasmids used are in Table 1 and DNA primers are in Table 2. Plasmid p82.20 contains the 1.1-kb BamHI fragment of p82.13 in vector pSG560 (
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Plasmid pTK10, which expresses the PEX22 gene from the alcohol oxidase (AOX) promoter, was cloned as follows: the gene was amplified by PCR using primer TK31 and TK40, thereby introducing a BamHI site immediately upstream of the ATG. The PEX22 gene was excised with BamHI and EcoRI and cloned into pPIC3K (Invitrogen) cut with BamHI and EcoRI.
Yeast two-hybrid plasmids were made by fusing appropriate gene fragments downstream of the DNA binding (DB) domain of LexA or the activation domain (AD) of VP16. PEX22 fusions were generated by cloning, in-frame, parts of or the full-length PEX22 fused to either domains in plasmids pKNSD55 (pBTM116 based) or pKNSD52 (pVP16 based) (
Plasmids containing PEX4 (as a BamHI-EcoRI fragment made by PCR with primers KNF13 and KNF14) in pKNSD55 or pKNSD52 (
Plasmids expressing GFP-SKL (pTW51) and PTS2-GFP (pTW66) were as described (
Plasmid pTK50 expressing a NH-tagged Pex4p from the acyl-CoA oxidase (ACO) promoter was cloned as follows: a BamHI-EcoRI fragment containing the full-length PEX4 was cloned into plasmid pM22 cut with BamHI-EcoRI (pex4 strain (PPY12,
pex4::ARG4). Arginine minus and Zeocin resistant colonies were checked for their expression of NH-Pex4p.
A fragment of ScYAF5 containing the full-length gene was amplified with primers TK52 and TK62 on genomic S. cerevisiae DNA. The resulting EcoRI fragment was cloned into pRS306, cut with EcoRI to yield plasmid pTK45. The two-hybrid vectors with ScYAF5 were made as follows: ScYAF5 was amplified with PCR with primers TK52 and TK53. The resulting BamHI-EcoRI fragment was cloned into either pKNSD55 (to yield plasmid pTK46) or pKNSD52 (pTK47). Plasmids for the two-hybrid experiment expressing ScPEX4 were made by amplifying ScPEX4 with primers TK67 and TK68. The resulting fragment was cloned as a BamHI-EcoRI fragment into pKNSD55 (to yield pTK48) and pKNSD52 (to yield pTK49).
Production of Antibodies
For the construction of a 6HIS-Pex22p, a BamHI-EcoRI fragment of PEX22 produced by PCR with primers TK35 and TK40 was cloned into a BamHI-EcoRI cut plasmid pQE30 (Qiagen) creating plasmid pTK20. This plasmid, expressing Pex22p missing the first 25 amino acids, was transformed into E. coli SG13009 and the gene induced with isopropyl ß-D-thiogalactopyranoside (IPTG). The protein was purified under native and denaturing conditions on Ni2+-NTA beads according to the manufacturer's manual (Qiagen). The purified proteins were used to immunize rabbits. The antibodies were preabsorbed against an acetone powder extract from a pex22 strain. Basically, the deletion strain was grown in one liter of methanol medium to an OD of 1. The cells were pelleted and resuspended in PBS at a cell density of 20 OD. Zymolyase was added (1 mg) and the cells were incubated with gentle shaking for 20 min. The cells were placed on ice for 5 min. Cold acetone (four volumes) was added, followed by another incubation on ice for 30 min. The cells were rewashed with cold acetone and placed on ice for another 30 min. The cells were pelleted, put into a mortar and dried. The cells were subsequently ground to a fine powder using a pestle. This powder was incubated with undiluted sera at a concentration of 1% (wt/vol) at 4°C. After an overnight incubation, the tube was centrifuged for 10 min, the supernatant collected and used for further studies.
Plasmid pTK37, expressing a 6HIS-tagged NH2 terminus of Pex4p, was made by cloning the BamHI-SspI fragment of pTK23 into pQE30, which was cut with BamHI-SmaI. This plasmid was transformed into strain SG13009 and the protein induced with IPTG. Expressed protein was purified under denaturing conditions according to the manufacturers procedure (Qiagen). The pure protein was then injected into rabbits for antibody production. The resulting antibody was then further purified using an affinity-purification protocol according to
Differential Centrifugation, Nycodenz Gradient, Floatation Gradient, Membrane Extraction, and Protease Protection
Differential centrifugation and Nycodenz gradients were done as described (
Binding of Pex22p to Pex4p
A pex4 strain (STK14) was transformed with plasmid pTK36, expressing a 6HIS-Pex4p. This strain and SMD1163 as a control were grown in methanol and spheroplasts were prepared. Cross-linking of cell extracts was performed as previously described (
Fluorescence and Electron Microscopy
Fluorescence microscopy for the detection of GFP-tagged proteins was done as described by -Pex3p and
-AOX were used at a dilution of 1:10,000. Microscopy for immunofluorescence was as described (
Miscellaneous
TCA lysates were made as follows: 2 OD of cells were collected by centrifugation, resuspended in 10% TCA and incubated on ice for >30 min. The suspension was centrifuged and the pellet washed three times with acetone. The pellet was resuspended in sample buffer and glass beads added. The tube was vortexed for 1 min and heated at 100°C for 1 min. This procedure was repeated four times. The sample was separated from the glass beads and loaded on gels.
Digitonin permeabilization was done according to -Sccatalase, 1:10,000;
-Scthiolase, 1:10,000;
-ScG6PDH (glucose-6-phosphate dehydrogenase), 13,000;
-F1ß subunit of mitochondrial ATPase, 1:10,000;
-PpPex3p, 1:10,000;
-PpPex4p, 1:1,000;
-PpPex5p, 10,000;
-PpPex7p, 1:10,000;
-PpPex22p, 1:2,000;
-GFP, 1:2,000.
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Results |
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Isolation of Peroxisomal Protein Import Mutants
The screen employed for the isolation of import mutants was based on a positive screening procedure (
Cloning of PEX22
The pex22.1 mutant (STK10) was transformed with a wild-type genomic library and plasmids (p82.2, p82.3, p82.9, p82.13, and p82.15) from colonies that grew on methanol medium were isolated and rechecked for their ability to restore growth on methanol and oleate. The five inserts contained an overlapping fragment of 1.1 kb which was isolated from p82.13 as a BamHI fragment and subcloned into the pSG560 vector (pex22 strain grew normally on glucose, but not on methanol and oleate, for which growth was complemented upon reintroduction of PEX22 (pTK10; Figure 1 B).
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The pex22 Strain Does Not Import PTS1- and PTS2-Containing Proteins
The pex22 (STK11) strain was transformed with GFP constructs to determine the ability of this strain to import peroxisomal matrix proteins. The GFP constructs used were shown to be properly localized to peroxisomes in wild-type cells (
pex22 strain was not targeted into peroxisomes when grown in methanol medium but was localized in the cytosol (Figure 3). A PTS2-GFP (expressing the first 17 amino acids of S. cerevisiae thiolase fused to GFP; pTW61) was also not targeted to peroxisomes when grown in oleate but was localized in the cytosol (Figure 3). However, immunofluorescence with Pex3p antibody showed that this peroxisomal membrane protein localized to punctate structures in the cytosol in the mutant strain, suggesting that the
pex22 strain retains the ability to target peroxisomal membrane proteins to some peroxisome-like structures, so called remnants (Figure 3). Electron microscopy revealed that in wild-type cells, the peroxisomes were clearly present in both methanol (Figure 4 A) and oleate (Figure 4 B) grown cells. In
pex22 cells, no normal peroxisomes could be observed (Figure 4C and Figure D). However, in both growth media, small single-membrane organelles could be observed, suggesting that
pex22 cells contain peroxisome remnants.
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Differential centrifugation experiments confirmed the results obtained with the GFP fusions. Wild-type cells, SMD1163 (for control), and the pex22 strain (STK12) were grown in oleate to induce peroxisomes. Post-nuclear supernatants (PNS) from these strains were centrifuged at 27,000 g (27 k). The supernatant was spun further at 100,000 g (100 k). Equal portions of these fractions (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) were analyzed by immunoblotting. Both catalase and thiolase, which are PTS1- and PTS2-containing proteins, respectively, in yeasts and mammals, were localized in the 27-k pellet in the wild-type strain, whereas in the
pex22 strain these proteins were cytosolic (100-k supernatant) (Figure 5 A). Pex3p, however, was localized in the 27-k pellet in both strains. To check if the pelletable Pex3p is membrane bound, the 27-k pellet was resuspended in 65% sucrose and overlaid with layers of 50% and 30% sucrose, respectively. After centrifugation, fractions were collected from the top and analyzed. Immunoblots showed that in both strains, Pex3p floated to the middle or top of the gradient, as did a mitochondrial marker (F1ß-ATPase), suggesting that Pex3p is membrane-bound in the
pex22 strain (Figure 5 B). Together, these data suggest that both PTS1- and PTS2-containing proteins are not properly targeted in a
pex22 strain, whereas peroxisomal membrane proteins (Pex3p) are targeted to membrane structures, most likely the peroxisome remnants seen by immunofluorescence and electron microscopy.
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Pex22p Is Localized to Peroxisomes
Antibodies raised against Pex22p (see Materials and Methods) specifically detected a protein of ~23 kD in cells grown on oleate and methanol (Figure 5 A). Cells grown in glucose only showed a faint band corresponding to Pex22p (data not shown). No band was apparent in pex22 strains as expected (Figure 5 A). The same fractions as above (PNS, 27-k pellet, 100-k pellet, and 100-k supernatant) taken from the wild-type strain were checked for the presence of Pex22p by immunoblotting. Pex22p was localized to the 27-k pellet, suggesting an organellar localization for this protein (Figure 5 A). The PNS of the wild-type strain was fractionated on a linear Nycodenz gradient and analyzed by immunoblotting. Catalase and thiolase migrated, although with some trailing most likely due to rupture of some peroxisomes, near the bottom of the gradient, as did Pex3p (Figure 5 C). Pex22p colocalized with the peroxisomal markers catalase, thiolase, and Pex3p. Further evidence that Pex22p is a peroxisomal protein was obtained by immunoelectron microscopy. Sections of methanol- and oleate-grown cells were decorated with Pex22p antibodies followed by incubation with gold-conjugated protein A. The gold particles almost exclusively decorated the peroxisomal membrane in the wild-type (Figure 6B and Figure D), but not the
pex22 strain (Figure 6 A). Sometimes, Pex22p was localized to patches on peroxisomes (Figure 6 C).
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Pex22p Is a Peroxisomal Membrane Protein with Its COOH Terminus Facing the Cytosol
The topology of Pex22p within the peroxisomal membrane was analyzed by organelle subfractionation. The wild-type strain, SMD1163, was grown in oleate and the 27-k pellet was fractionated into soluble and insoluble fractions after treatment with 0.1 M Na2CO3, pH 11.5, 10 mM Tris, pH 8.5 (no salt), 1 M NaCl in 10 mM Tris, pH 8.5 (high salt), and 0.1% Triton X-100. Pex22p behaved like Pex3p, a peroxisomal membrane protein (
The First 25 Amino Acids of Pex22p Contain an mPTS
Sequence analysis of Pex22p did not reveal an obvious mPTS. There is, however, a stretch of positively charged amino acids near the extreme NH2 terminus of Pex22p which does not completely fit the consensus sequence for an mPTS (pex22 mutant for growth on oleate and methanol), a second fusion with the first 25 amino acids of Pex22 (Pex22(125)-GFP; pTK32), containing the transmembrane domain, a third fusion with only the transmembrane domain (Pex22(825)-GFP; pTK44), and a fourth fusion with the first seven amino acids of Pex22 (Pex22(17)-GFP; pTK34), not containing the transmembrane domain. These constructs were transformed into PPY12 and the resulting strains induced on methanol. The constructs expressing full-length Pex22-GFP and Pex22(125)-GFP showed colocalization with alcohol oxidase, a bona fide peroxisomal matrix protein (Figure 7 A), proving that these constructs get targeted to peroxisomes, whereas the other two constructs (Pex22(17)-GFP, Pex22(825)-GFP) were localized in the cytosol (data not shown). The Pex22(125)-GFP fusion protein could also be shown to colocalize with peroxisomes when an organelle fraction was separated on Nycodenz gradients (data not shown). Furthermore, this fusion was organelle associated since the fusion protein (Pex22(125)-GFP) only leaked from cells at digitonin concentrations that released membrane proteins (Figure 7 B). The cytosolic protein, G6PDH, was released into the supernatant at low concentrations (25 µg/ml), whereas the peroxisomal matrix protein GFP-SKL started to leak at digitonin concentrations of 50100 µg/ml, and release was not complete until the concentration of digitonin was 500 µg/ml. Pex3p, a peroxisomal membrane protein, was only fully released into the supernatant at digitonin concentrations exceeding 1,000 µg/ml. The Pex22(125)-GFP fusion protein was released into the medium at very high concentrations (1,0001,500 µg/ml), or when the cells were treated with 0.2% Triton X-100 (Figure 7 B). These results show that the Pex22(125)-GFP construct is targeted to peroxisomal membranes.
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Pex22p Interacts with Pex4p
To determine interactions of Pex22p with other Pex proteins, the yeast two-hybrid system was employed. PEX22 was fused to the DB domain of LexA, or the AD of VP16. All published P. pastoris PEX genes (PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX8, PEX10, PEX12, and PEX13) were also fused to these domains. These plasmids were then transformed in combination into the S. cerevisiae strain L40 and interaction of these proteins was assessed by the production of ß-galactosidase activity. Only the combination of Pex22p with Pex4p, a ubiquitin-conjugating enzyme, produced any detectable enzyme activity. Almost the whole Pex22p protein (construct Pex22.1) was needed for interaction with Pex4p, whereas the COOH-terminal 39% of Pex4p (construct Pex4.2) interacted with Pex22p (Figure 8 A). Control experiments performed by exchanging the backbone vectors confirmed our findings (data not shown). We were also able to show that these two fragments of Pex22p (Pex22.1) and Pex4p (Pex4.2) interacted with each other (data not shown).
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To show that Pex22p and Pex4p interact in vivo, 6HIS-Pex4p was expressed from the GAPDH promoter (plasmid pTK36). This plasmid was then transformed into the pex4 strain (STK14). The 6HIS-Pex4p complemented the disrupted strain as assessed by growth on methanol and oleate (data not shown). This strain was grown in methanol, and spheroplasts were prepared. The cross-linker dithiobix(succinimidylpropionate) (DSP) was added to the lysates to cross-link neighboring proteins. 6HIS-Pex4p and associated proteins were precipitated with Ni2+-NTA beads. Bound proteins were run on an SDS gel, blotted onto nitrocellulose and checked for the presence of Pex4p, Pex22p, and Pex3p. The 6HIS-Pex4p specifically bound Pex22p in the presence of the cross-linker DSP (Figure 8 B), whereas no Pex22p could be detected in the sample without DSP. Pex3p, another peroxisomal membrane protein, did not bind to the beads or to 6HIS-Pex4p. Pex22p and Pex4p did also not bind to the beads, as seen in the wild-type strain, not expressing any 6HIS-tagged protein. These experiments confirm the specific interaction between Pex4p and Pex22p by two different methods.
pex4 and
pex22 Strains Share Similar Phenotypes
PpPex4p was previously characterized as a ubiquitin-conjugating enzyme, similar to ScPex4p (pex4 strain (STK14) behaved similarly in differential centrifugation, as did a
pex22 strain (data not shown). TCA lysates were made from strains (STK12 and STK14) grown in methanol and oleate. Equal amounts of cells were loaded on a gel and blotted for the presence of Pex3p, Pex4p, Pex5p, Pex7p, and Pex22p. As shown in Figure 9 A, all the strains showed similar amounts of Pex3p, whereas strains deleted for
pex4 and
pex22 did not contain any detectable Pex5p. However, Pex7p was present in wild-type amounts in all the strains and was induced by oleate relative to methanol growth. Interestingly, we were unable to detect any Pex4p in a
pex22 strain.
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Pex22p Anchors Pex4p at the Peroxisomal Membrane
NH-Pex4p expressed from its own promoter (strain STK15) complemented a pex4 strain and was localized in the 27-k pellet during differential centrifugation (Figure 9 B). The controls, Pex3p and G6PDH, were exclusively in the 27-k pellet and 100-k supernatant, respectively (Figure 9 B). We were interested in seeing whether the localization of Pex4p is disturbed in a
pex22 strain. We overexpressed the NH-tagged Pex4p from the ACO promoter in wild-type (PPY12) and
pex22 strains and performed a differential centrifugation with oleate-induced cells. Interestingly, the wild-type Pex4p was undetectable in these strains (data not shown). In PPY12, the overexpressed NH-Pex4p was localized to the 27-k pellet and 100-k supernatant, whereas in a
pex22 strain, all of the NH-tagged Pex4p was in the cytosol (Figure 9 B). This experiment suggests that Pex22p anchors Pex4p at the peroxisomal membrane.
ScYaf5p Is a Homologue of PpPex22p
PpPex22p was run against protein databases (SwissProt, SGD) with Blast and Fasta searches. No high-scoring homologue could be found. Only several low-scoring proteins could be found in the Saccharomyces Genome Database (SGD) database. Out of these, only ScYaf5p (open reading frame YAL055w) is of about similar size and exhibits a transmembrane region at the NH2 terminus similar to Pex22p, although it starts at amino acid 1432 (Figure 10 A). To determine if ScYaf5p is the real Pex22p homologue, the entire open reading frame of ScYAF5 was replaced by a PCR-generated kanMX2 cassette (Scyaf5 strains grew on glucose like wild-type cells, whereas they did not grow on oleate. A
Scyaf5 strain transformed with a plasmid expressing ScYAF5 from a catalase promoter complemented the growth defect on oleate (data not shown).
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GFP-SKL is targeted to peroxisomes in wild-type cells, whereas in the Scyaf5 strain this construct was localized in the cytosol (Figure 10 B). To test the interaction between ScYaf5p and ScPex4p, the genes encoding these proteins were cloned into the two-hybrid vectors and transformed into strain L40. As seen in Figure 10 C, only strains containing both constructs showed ß-galactosidase activity. These results indicate that ScYaf5p is the functional homologue of Pex22p. However, overexpression of ScYAF5 from an alcohol oxidase promoter could not complement the growth phenotype of a P. pastoris
pex22 strain on methanol. This could be explained by the fact that ScYaf5p does not interact in a two-hybrid experiment with PpPex4p (data not shown).
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Discussion |
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Pex22p Is a Peroxisomal Integral Membrane Protein
The newly discovered peroxin, Pex22p, described in this study behaves like a peroxisomal integral membrane protein by several criteria. It is pelletable in differential centrifugations (Figure 5 A) and colocalizes with peroxisomal markers in Nycodenz gradients (Figure 5 C). In immunoelectron microscopy experiments, the protein was associated with the peroxisomal membrane (Figure 6, BD). The protein was not extracted from the membrane by buffers of low ionic strength, high salt or by alkaline sodium carbonate, indicating that it is an integral membrane protein (Figure 5 D). Finally, most of the Pex22p is degraded upon addition of proteases, even in the absence of detergent, under conditions where thiolase, a matrix marker, is resistant (Figure 5 E). These results, when combined with the prediction of a single transmembrane domain near the NH2 terminus of Pex22p, are consistent with a topology in which the NH2 terminus of Pex22p is in the peroxisomal matrix and the COOH terminus is in the cytosol. This topology makes it possible for the COOH terminus of Pex22p to be involved in protein interactions with the peroxisomal peripheral membrane protein, Pex4p, as discussed later.
We do not understand why Pex22p is localized in some immunoelectron microscopy pictures to patches at the peroxisomes. This is not seen in all the sections. It is possible that Pex22p clusters are required for its normal functions which are discussed later. The same behavior has also been observed for Pex14p in Hansenula polymorpha (
The mPTS of Pex22p Resides Within the NH2-terminal 25 Amino Acids
Pex22p contains a signal at the NH2 terminus that is sufficient for peroxisome targeting (Figure 7 A). Fusing GFP to the first 25 amino acids of Pex22p targets the resulting fusion protein to peroxisomes. This conclusion is supported by the colocalization of this fusion protein with peroxisomal markers in a Nycodenz gradient (data not shown), by fluorescence microscopy showing colocalization of the fusion with a peroxisomal marker (Figure 7 A), and by the release of the fusion protein from cells only with high concentrations of digitonin or by Triton X-100 (Figure 7 B). Other experiments designed to show that the GFP portion of the fusion protein faces the cytosol failed because GFP is highly resistant to proteases (data not shown;
At present, we are unable to decipher why some mPTSs require transmembrane domains to function while others do not. In the case of Pex22p, the seven amino acids fused to GFP could be buried and inaccessible to the putative receptor. That would explain why this fusion protein is seen in the cytosol. Another possibility is that the targeting signal requires some amino acids that are located in the transmembrane domain of Pex22p. Experiments to determine the important amino acids of the mPTS are underway. Comparison of the different mPTSs found so far shows that there is a predominance of positively charged amino acids. Pex22(125)-GFP fusions with alanine substitutions in two of the three positively charged amino acids of the sevenamino acid lumenal stretch (K(2)A and R(6)
A) do not properly localize to the peroxisome (data not shown). This result suggests that at least these two positive charges are important for proper targeting of the fusion protein.
Requirement of Pex22p for Import of Peroxisomal Matrix, but Not Membrane Proteins
Pex22p is important for peroxisome biogenesis and for growth of P. pastoris on methanol and oleate (Figure 1). Functional peroxisomes are not formed in a pex22 strain (Figure 4C and Figure D). Both exogenously expressed and endogenous PTS1- and PTS2-containing proteins accumulate in the cytosol (Figure 3 and Figure 5 A), whereas the membrane protein, Pex3p, is targeted to pelletable membranous structures that float in sucrose gradients (Figure 5A and Figure B) and likely correspond to the peroxisomal remnants observed using fluorescence (Figure 3) and electron microscopy (Figure 4C and Figure D).
Pex22p Interacts with Pex4p and Anchors It at the Peroxisomal Membrane
Yeast two-hybrid experiments performed with Pex22p and all published peroxins of P. pastoris show that it only interacts with Pex4p, a UBC enzyme that is localized to the cytosolic face of peroxisomal membranes (Figure 8 A). The COOH-terminal cytosolic domain (amino acids 26187) of Pex22p interacts with the COOH terminus (amino acids 125204) of Pex4p. Although this domain of Pex4p includes the active site Cys (C133), Pex4p constructs containing Ala (C133A) or Ser (C133S) substitutions (
The interaction between Pex22p and Pex4p sheds light on the function of Pex22p. One possibility is that Pex22p is the elusive substrate for ubiquitination by Pex4p. However, this seems unlikely as Pex22p migrates in SDS gels at the predicted molecular mass (23 kD) and not as a protein with mono- or poly-ubiquitin modifications (Figure 5 and Figure 9 A). The molecular mass of Pex22p is also unchanged throughout oleate induction (data not shown).
An alternative possibility suggested by several experiments is that Pex22p anchors Pex4p on the peroxisomal membrane. First, Pex4p is a peripheral peroxisomal membrane protein facing the cytosol and is tightly associated with the peroxisomal membrane even though it has no transmembrane segment of its own (pex22 strain (Figure 9 A). Fourth, NH-Pex4p is mislocalized to the cytosol in the
pex22 strain (Figure 9 B). Many of these points are reminiscent of the relationship between Ubc7p and Cue1p in S. cerevisiae. Cue1p, an integral membrane protein of the ER, is essential for the localization of Ubc7p, a UBC enzyme, to the cytosolic face of the ER, and both these proteins are required for the degradation of aberrant proteins in the ER membrane and for the retrograde transport of lumenal substrates out of the ER (
cue1 strain, Ubc7p could not be found and a myc-tagged Ubc7p, when overexpressed in this strain, was found in the cytosol. Pex4p is unstable in a
pex22 strain and NH-Pex4p, when overexpressed from the acyl-CoA oxidase promoter, is localized to the cytosol in this strain. NH-Pex4p, in a wild-type strain, is localized equally in the 27-k pellet and 100-k supernatant, whereas wild-type levels of NH-Pex4p are localized solely to the 27-k pellet (Figure 9 B). This shows that there is a saturable binding site for Pex4p on membranes. These results are consistent with the idea that Pex22p provides the binding site for Pex4p. Based on these data, we propose that Pex22p is the anchor protein at the peroxisomal membrane that recruits and holds Pex4p at this location. We are not able to explain why in the strains overexpressing NH-Pex4p, Pex3p is not only present in the 27-k pellet but also in the 100-k pellet and 100-k supernatant (Figure 9 B).
This model would predict that Pex4p and Pex22p act together for import of peroxisomal matrix proteins. This hypothesis is supported by the observation that both the pex22 and
pex4 strains do not contain wild-type levels of Pex5p, have similar phenotypes such as inability to grow on methanol and oleate, and are impaired in the import of peroxisomal matrix proteins, but not membrane proteins (
pex4 strain has been observed by another group (Kalish, J.E., and S.J. Gould, 6th International Congress on Cell Biology, 1996, Abstract 2873) but this was not observed with H. polymorpha (
pex22 and
pex4 strains were directly attributable to the absence of Pex5p, PEX5 was overexpressed in the
pex4 and
pex22 strains expressing GFP-SKL. The introduction of the PEX5 plasmid enhanced the level of Pex5p protein to wild-type levels as assessed by immunoblotting, but these strains remained unable to grow on methanol or import GFP-SKL into peroxisomes (data not shown). It is unlikely that Pex4p is solely responsible for the stability of Pex5p as we were unable to restore wild-type levels of Pex5p in a
pex22 strain overexpressing Pex4p (data not shown). Therefore, the phenotypes seen in the
pex4 and
pex22 strains are not simply a consequence of Pex5p instability. This is supported by the fact that not only PTS1-mediated import, but also the import of PTS2-containing proteins is compromised in
pex4 and
pex22 strains (Figure 3, see also
Models for the Role of Pex22p/Pex4p in Peroxisomal Matrix Protein Import
Our data clearly support a role for Pex22p in the anchoring of Pex4p to the peroxisomal membrane. However, further experiments will be required to determine the role of this protein complex in peroxisome biogenesis. One possibility is that the Pex4pPex22p complex functions similar to the Cue1pUbc7p complex, regulating the proper assembly and/or correct stoichiometry of protein import complexes at the peroxisomal membrane. It is known that altered stoichiometry of peroxisomal integral or peripheral membrane proteins, Pex3p and Pex14p, can yield an import-deficient phenotype (pex4 or
pex22 strains (data not shown) and Pex5p is stable in a P. pastoris
pex13 strain (
A variation of this model, equally compatible with the available data, is that Pex4p, instead of directly acting on these peroxisomal membrane proteins, negatively regulates (by ubiquitination and degradation) a protease, which in turn degrades peroxisomal membrane complexes. It is hoped that these testable models may lead, in the near future, to the function of Pex4p.
Conservation of PpPex22p in Other Yeasts
Although database searches did not reveal any proteins highly homologous to Pex22p, we did find a protein of similar predicted size and topology in S. cerevisiae. The hypothetical protein, ScYaf5p (open reading frame YAL055w), appears to be the homologue of PpPex22p. Like PpPEX22, the ScYAF5 gene is essential for growth on oleate, and for the import of GFP-SKL, a fusion protein that is readily imported into peroxisomes in wild-type yeast. Furthermore, ScYaf5p interacts with ScPex4p in a two-hybrid experiment. The conservation of Pex22p and its interacting partner, Pex4p, in other yeasts suggests that the functions of these proteins are likely to be conserved in all organisms.
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Footnotes |
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Klaas Nico Faber's present address is University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Eukaryotic Microbiology, Kerklaan 30, 9751 NN Haren, The Netherlands.
Thibaut J. Wenzel's present address is Gist-Brocades, Food Specialities Division, Wateringseweg 1, 2600 MA Delft, The Netherlands.
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Acknowledgements |
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We thank the members of the lab for support and helpful discussions and S. Emr for use of the fluorescence microscope.
A. Koller was supported by a fellowship of the Swiss National Science Foundation (no. 8230-046677), W.B. Snyder by a fellowship from the American Cancer Society, K.N. Faber by a fellowship from the Human Frontier Science Program Organization, and T.J. Wenzel by a fellowship from the European Molecular Biology Organization. This work was supported by National Institutes of Health grant DK41737 to S. Subramani.
Submitted: March 4, 1999; Revised: May 17, 1999; Accepted: June 3, 1999.
1.used in this paper: AD, activation domain; AOX, alcohol oxidase; DB, DNA-binding domain; DSP, dithiobis(succinimidylpropionate); G6PDH, glucose-6-phosphate dehydrogenase; GFP, green fluorescent protein; IPTG, isopropyl ß-D-thiogalactopyranoside; mPTS, membrane peroxisome targeting signal; Pex, peroxin; PNS, post nuclear supernatant; PTS, peroxisome-targeting signal; UBC, ubiquitin-conjugating enzyme
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References |
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