|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/2001//1141 $5.00
The Journal of Cell Biology, Volume 153, Number 6,
, 2001 1141-1150
Original Article |
Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins
sgould{at}jhmi.edu
Peroxisomal proteins are synthesized on free polysomes and then transported from the cytoplasm to peroxisomes. This process is mediated by two short well-defined targeting signals in peroxisomal matrix proteins, but a well-defined targeting signal has not yet been described for peroxisomal membrane proteins (PMPs). One assumption in virtually all prior studies of PMP targeting is that a given protein contains one, and only one, distinct targeting signal. Here, we show that the metabolite transporter PMP34, an integral PMP, contains at least two nonoverlapping sets of targeting information, either of which is sufficient for insertion into the peroxisome membrane. We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information. These results challenge a major assumption of most PMP targeting studies. In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34. Together, these results raise the interesting possibility that PMP import may require novel mechanisms to ensure the solubility of integral PMPs before their insertion in the peroxisome membrane, and that PEX19 may play a central role in this process.
Key Words: peroxisome • organelle biogenesis • PEX19 • targeting signal • PMP34
© 2001 The Rockefeller University Press
 |
Introduction |
|---|
 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
|---|
Less is known about the import of proteins destined for insertion into the peroxisome membrane. Like matrix proteins, integral peroxisomal membrane proteins (PMPs) are synthesized on free polyribosomes and imported posttranslationally from the cytosol (Fujiki et al. 1984; Suzuki et al. 1987; Diestelkotter and Just 1993; Imanaka et al. 1996), though their import does not require the hydrolysis of ATP (Diestelkotter and Just 1993). In addition, integral PMPs lack functional PTS1 or PTS2 signals, and their import is independent of the PTS1 and PTS2 receptors (Chang et al. 1999; Hettema et al. 2000). Integral PMPs are therefore thought to be imported into peroxisomes by a distinct targeting mechanism from that used by peroxisomal matrix proteins.
Multiple studies have attempted to define the targeting information in integral PMPs. These include studies of Candida boidinii PMP47 (McCammon et al. 1994; Dyer et al. 1996), PEX3 from Pichia pastoris, Hansenula polymorpha, and humans (Baerends et al. 1996, Baerends et al. 2000; Wiemer et al. 1996; Kammerer et al. 1998; Soukupova et al. 1999), P. pastoris PEX2, PEX10, PEX13, PEX17, and PEX22 (Snyder et al. 2000), rat PMP22 (Pause et al. 2000), and human PMP70 and PEX11β (Sacksteder et al. 2000). Of these studies, only the examination of C. boidinii PMP47 defined PMP targeting information at the level of functional residues. In that study, Dyer et al. 1996 reported that the PMP47 targeting information lies between transmembrane domains 4 and 5 in a positively charged intraperoxisomal loop. Although these studies have all advanced our understanding of PMP targeting, they have not yet yielded a clear model of what constitutes a PMP targeting signal. In addition, virtually all of the above studies have assumed that PMPs contain one and only one set of targeting information.
Here, we report an examination of the targeting information in PMP34, the human homologue of C. boidinii PMP47. We demonstrate that PMP34 contains not one but at least two targeting signals, either of which is sufficient for targeting to peroxisomes. We also show that two nonoverlapping segments of PEX13 target to peroxisomes, indicating that the presence of multiple independent targeting regions within a single PMP is not unique to PMP34. Last, we demonstrate that the minimal targeting regions of PMP34 both bind to PEX19. The implications of these results for PMP recognition and targeting are discussed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Indirect Immunofluorescence and Fluorescence Microscopy
Indirect immunofluorescence studies were performed on normal human skin fibroblasts (GM 5756-T) or the PEX10-deficient fibroblast cell line, PBD100, which has been described previously (Warren et al. 1998). Cell lines were cultured and transfected as described (Chang et al. 1997). After transfection, cells were transferred to cover glasses and incubated in complete medium for 1 d. The cells were then fixed in 3% formaldehyde, permeabilized with 1% Triton X-100, and processed for indirect immunofluorescence as described (Chang et al. 1997). The anti–c-myc mouse monoclonal antibody was derived from tissue culture medium of the mouse hybridoma line 1-9E10 (Roche Molecular Biochemicals). Sheep anti-PMP70 and rabbit anti-PEX19 antibodies (Sacksteder et al. 2000) and rabbit anti-HAOX3 antibodies (Jones et al. 2000) have been described. Fluorescent secondary antibodies were obtained from commercial sources.
Cell Lysates, Na2CO3 Extraction, Immunoprecipitation, and Immunoblots
Normal human skin fibroblasts (GM5756-T) or PEX10-deficient PBD100 fibroblasts were cultured and transfected with the appropriate expression plasmids as described (Chang et al. 1997). 1 d after transfection, cells were washed twice in PBS (GIBCO BRL) and harvested by gentle scraping. For whole cell lysates, transfected cells were briskly resuspended in PBS plus 1% Triton X-100. Protein content of lysates was determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Equal quantities of total protein from each lysate were separated by PAGE, transferred to membranes, and analyzed by immunoblot using standard protocols (Crane et al. 1994).
For membrane extraction experiments, cells were resuspended in hypotonic lysis buffer (10 mM Tris HCl, pH 7.5, 1 mM EDTA, CompleteTM protease inhibitor cocktail [Boerhinger]) and lysis was achieved by passing cell slurries through a 20-gauge syringe needle five times. Cellular membranes were harvested by centrifugation at 100,000 g for 1 h. Supernatant and pellet were separated, pellets were resuspended in 100 mM Na2CO3 at a final protein concentration of 0.4 mg/ml, and the suspensions were incubated for 30 min on ice with gentle agitation. Membranes were pelleted by centrifugation at 100,000 g for 1 h. Extraction samples were diluted to the initial volume of lysate, and equal volumes of the samples were analyzed by immunoblot using standard techniques (Crane et al. 1994). Detection of the c-myc epitope was performed by a commercially available anti–c-myc polyclonal antibody (Santa Cruz Biotechnology, Inc.), and detection of catalase was performed by a commercially available sheep anticatalase antibody (The Binding Site). Anti-PEX13 antibodies were raised in rabbit against a synthetic peptide corresponding to the COOH-terminal 14 amino acids of human PEX13 (Gould et al. 1996).
For immunoprecipitation experiments, cells expressing either PMP34aa1-147/13xmyc or PMP34aa244-307/3xmyc in combination with either HA-PEX19 or HA-PTE1 were lysed in 500 µl hypotonic lysis buffer as above. Lysates were then subjected to centrifugation at 25,000 g for 15 min to pellet large organelles such as peroxisomes. The resulting supernatant was brought to 1 ml final volume by addition of PBS (Life Technologies) plus CompleteTM protease inhibitor cocktail (Boehringer). 20 µl of a 50% slurry of protein A agarose (Sigma-Aldrich) in PBS was added and the mixture, incubated with mixing at 4°C for 30 min. The protein A agarose was pelleted, and the supernatant was removed to a new tube. 15 µl of a 50% slurry of anti-HA monoclonal antibodies coupled to agarose beads (Santa Cruz Biotechnology, Inc.), which had been preincubated in 0.1 mg/ml BSA in PBS for 30 min, was added to each sample, and the mixtures were incubated with mixing at 4°C for 3 h. The anti-HA beads were recovered by centrifugation, washed 4 times with 1 mL PBS, and resuspended in SDS-PAGE sample buffer. Corresponding proportions of lysate and immunoprecipitate samples were analyzed by immunoblot using the anti–c-myc polyclonal antibody as above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To distinguish between endogenously expressed PMP34 and PMP34 proteins expressed from plasmids, the plasmid-encoded proteins all contained the c-myc epitope at their COOH terminus. The full-length PMP34myc fusion protein was expressed in wild-type human fibroblasts (Fig. 1 a) and was efficiently targeted to peroxisomes (b and c). The Zellweger syndrome fibroblast cell line PBD100 is unable to import peroxisomal matrix proteins but imports PMPs normally (Warren et al. 1998). PMP34myc was expressed in PBD100 cells (Fig. 1 a) and was targeted to peroxisomes (d and e), demonstrating that PMP34myc is targeted to peroxisomes by a membrane protein targeting pathway, as previously suggested (Wylin et al. 1999).
|
|
|
Dyer et al. 1996 found that the peroxisomal targeting signal of C. boidinii PMP47 resided in the loop between transmembrane spans 4 and 5. To determine whether the corresponding loop in human PMP34 (Fig. 2, *) is also important for targeting to peroxisomes, we assayed the targeting ability of several proteins containing this region. Although the loop alone (amino acids 183–199) failed to express to levels detectable by immunofluorescence, we found that two larger constructs containing this segment (amino acids 183–225 and amino acids 183–243) failed to target to peroxisomes.
The Minimal Targeting Regions in PMP34 Are Sufficient for Membrane Insertion
Peroxisomal localization of the two PMP34 fragments could reflect insertion into the peroxisome membrane. However, it could also have been caused by interaction with a binding partner at the peroxisome surface or by cryptic import into the peroxisome matrix. To address these concerns, fibroblasts expressing the NH2- and COOH-terminal targeting elements of PMP34 were lysed in hypotonic buffer. Soluble proteins were separated from membranes by centrifugation, the membranes were extracted with alkaline sodium carbonate, and the resulting suspension was separated into a membrane pellet and a soluble supernatant. These fractions were then assayed by immunoblot. Proteins containing either the NH2-terminal PTS (PMP34aa1-147/3xmyc) or the COOH-terminal PTS (PMP34aa244-307/3xmyc) remained in the membrane even after carbonate extraction, demonstrating that these signals direct proteins into the peroxisome membrane (Fig. 4). As expected, control immunoblots showed that the peroxisomal matrix enzyme catalase was released to the supernatant by hypotonic lysis and that the integral PMP PEX13 was resistant to carbonate extraction.
|
|
We used the nuclear mislocalization assay to determine whether PEX19 might also interact with the two targeting signals of PMP34. A mutant version of the PEX19 cDNA was constructed that encoded three tandem copies of the nuclear localization signal (NLS) from SV-40 large T antigen (Adam and Gerace 1991) at the 5' end of the PEX19 ORF. The two small targeting elements of PMP34, PMP34aa1-147/3xmyc, and PMP34aa244-307/3xmyc were coexpressed with 3xNLS-PEX19 in normal human fibroblasts, and their subcellular distribution was determined by immunofluorescence microscopy. Both fragments of PMP34 were efficiently targeted to the nucleoplasm when coexpressed with 3xNLS-PEX19 (Fig. 6, a–d). In contrast, coexpression of these PMP34 fragments with a control protein, 3xNLS-HAOX3, had no effect on their trafficking to peroxisomes (Fig. 6e–h; HAOX3 is a peroxisomal oxidase that is not normally expressed in fibroblasts and is not involved in PMP import [Jones et al. 2000]).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Properties of the PMP34 Targeting Signals
The two PTSs we identified correspond to amino acids 1–147 and 244–307 of PMP34. An inspection of these sequences failed to reveal any common sequences with each other or with previously identified PTSs from human PMP70 (Sacksteder et al. 2000), human PEX11β (Sacksteder et al. 2000), human PEX14 (Sacksteder et al. 2000), human PEX3 (Kammerer et al. 1998; Soukupova et al. 1999), rat PMP22 (Pause et al. 2000), or PTSs identified in fungal forms of PMPs (Baerends et al. 1996, Baerends et al. 2000; Wiemer et al. 1996; Dyer et al. 1996; Snyder et al. 2000). This was not altogether surprising, given that these previously described PMP targeting signals lack significant sequence similarities. We have also considered the possibility that there may be multiple types of PMP targeting signals, just as there are at least two types of peroxisomal matrix protein targeting signals. However, our efforts to identify subgroups of PMP targeting signals that share conserved sequence motifs have also failed.
Although PMP targeting signals lack recognizable amino acid sequence motifs, they do have some common features. One is that they are relatively long. A targeting signal in human PMP70 is 61 amino acids long (Sacksteder and Gould 2000), a targeting signal from PEX11β is 47 amino acids long (Sacksteder and Gould 2000), a targeting signal in rat PMP22 is 97 amino acids long (Pause et al. 2000), and the targeting signals we identified in human PMP34 are 147 and 64 amino acids long. In fact, the shortest PMP targeting signals yet identified are the first 33 amino acids of PEX3, which functions only inefficiently (Soukupova et al. 1999), and the first 25 amino acids of P. pastoris PEX17 (Snyder et al. 2000). The long length of PMP targeting signals contrasts sharply with the small size of the targeting signals for peroxisomal matrix proteins, the PTS1 and PTS2, which are three and nine amino acids long, respectively (Gould et al. 1987, Gould et al. 1989b; Swinkels et al. 1991). This difference in targeting signal size may reflect the nature of protein targeting assays and the fundamental differences between membrane and matrix protein targeting mechanisms. All protein targeting assays measure two processes, the targeting of the protein to its destination and the retention of the protein at its destination. In the case of peroxisomal matrix protein targeting, retention is provided by the passive barrier of the peroxisome membrane and the targeting signals correspond to the minimal sequence necessary for binding the PTS1 and PTS2 receptors, PEX5 and PEX7 (Rehling et al. 1996; Zhang and Lazarow 1996; Gatto et al. 2000). However, the only retention mechanism for PMPs is insertion into the peroxisomal membrane. PMP insertion likely requires a transmembrane domain on the targeted protein, but additional sequences may also be required for the insertion process. Thus, it is not surprising that the second common property of PMP targeting signals is that they contain at least one membrane-spanning domain.
Another feature of the PMP34 targeting signals that warrants discussion is their relationship to the mPTS of PMP47. PMP47 was the subject of the first PMP targeting signal studies (McCammon et al. 1994; Dyer et al. 1996), which concluded that its targeting signal, the mPTS, resided within a hydrophilic loop between membrane spanning domains 4 and 5 (Dyer et al. 1996). We assumed that the targeting signal of human PMP34 would be similar, particularly since this loop is relatively well conserved between human PMP34 and C. boidinii PMP47 (Wylin et al. 1999). This is not the case. The two PTSs we identified in human PMP34 differ in many respects from the mPTS of C. boidinii PMP47. The most obvious differences are that the two targeting signals of human PMP34 do not correspond in position or sequence to the mPTS of PMP47. Also, the region of human PMP34 that does correspond to the mPTS of PMP47 does not function as a PMP targeting signal. Another notable difference is that the PMP34 targeting signals both contain at least one putative membrane-spanning domain, whereas the mPTS of PMP47 lacks a membrane-spanning domain. Another difference worth discussing is the role of a basic patch of amino acids in PMP targeting. A basic patch of amino acids was thought to be important for the mPTS of PMP47 (Dyer et al. 1996), and much attention has been given to the importance of basic amino acid patches in targeting of other PMPs (Baerends et al. 2000; Pause et al. 2000). We identified a small patch of basic amino acids (amino acids 255–259) in the COOH-terminal PMP34 targeting signal. However, a smaller fragment of PMP34 lacking these basic residues (PMP34aa260–307/3xmyc) retained peroxisomal targeting, indicating that a basic patch of amino acids may not be an essential feature of all PMP targeting signals. While this work was in progress, Wang et al. 2001 revisited the peroxisomal targeting of C. boidinii PMP47 and found that PMP47 does require a transmembrane domain for targeting and that multiple fragments containing the mPTS identified by Dyer et al. 1996 did not target to peroxisomes. Furthermore, additional sequences far upstream of the mPTS facilitated peroxisomal targeting of PMP47, though there is as yet no data on whether PMP47 contains more than one functional peroxisomal targeting signal.
While our study was in progress, an independent study of PMP34 targeting information was reported by Honsho and Fujiki 2001. However, their report concluded that at least three transmembrane domains were necessary for targeting, that PMP34 contained a single targeting signal, and that the loop between transmembrane domains 4 and 5 of PMP34 was essential for targeting. Given that our study and the Honsho and Fujiki study (2001) both examined the same protein (human PMP34) using a very similar protein targeting assay in very similar cells, we feel compelled to address the significant differences between the two studies. Although there are some differences in the experimental results, the major difference between the two reports is in the interpretation of the data. Our conclusions are drawn entirely from PMP34 fragments that target to peroxisomes. In contrast, Honsho and Fujiki based many of their conclusions on PMP34 fragments that failed to be targeted. For example, Honsho and Fujiki concluded that the NH2-terminal half of PMP34 lacks targeting information because a single fusion protein containing amino acids 1–186 of PMP34 did not localize to peroxisomes, whereas we showed that even smaller NH2-terminal fragments did target to peroxisomes. Their conclusion ignores other reasonable explanations, such as aggregation, insolubility, or the masking of protein targeting signals in the artificial fusion protein. Given the many possible reasons that a mutant form of a protein might fail to target, we feel that there is a logical basis for relying on positive experimental results in the interpretation of these types of experiments, and prior studies of the PTS1 support this view (Gould et al. 1987).
Why Do Some PMPs Have Multiple Targeting Signals?
Previous studies of PMP targeting, including ours, have assumed that there is one and only one targeting signal per PMP (Baerends et al. 1996, Baerends et al. 2000; Dyer et al. 1996; Kammerer et al. 1998; Pause et al. 2000; Sacksteder et al. 2000; Snyder et al. 2000). Our data show that this assumption is flawed. We detected two distinct PTSs in PMP34 and showed that two nonoverlapping fragments of PEX13, another integral PMP, are targeted to peroxisomes. PMP70 also appears to have multiple targeting signals; we have identified a PTS at the protein's NH2 terminus (amino acids 1–61 [Sacksteder et al. 2000]), and Imanaka et al. 1996 demonstrated that another PTS exists in a COOH-terminal 50-kD fragment of PMP70. Because these targeting signals are sufficient to direct proteins to and into the peroxisome membrane, the identification of multiple targeting signals in several PMPs warrants further discussion.
We can envision two general hypotheses that might explain our observations. One is that the presence of multiple targeting signals in PMP34, PEX13, and PMP70 reflects a random meaningless event, namely the unselected acquisition of a second set of targeting information in these proteins. This hypothesis suggests that all or most other PMPs will be found to have one and only one PMP targeting signal and that our results do not reveal anything significant about the mechanism of PMP import. Although we cannot exclude this possibility, it seems unlikely that multiple proteins would acquire and maintain superfluous targeting information. Thus, we must also consider the alternative hypothesis that the presence of multiple peroxisomal targeting signals in PMP34, PEX13, and PMP70 is meaningful and reflects the properties of the underlying PMP import mechanism. But what mechanism would require multiple targeting signals per PMP?
Current paradigms of protein import systems do not provide an obvious answer to this question. Both co- and posttranslation import systems are known that rely on a single set of targeting information. In addition, import mechanisms that are heavily reliant on maintaining import substrates in the unfolded state, such as translocation through the endoplasmic reticulum and mitochondrial membranes (Deshaies et al. 1988), are mediated by a single set of targeting information, as is peroxisomal matrix protein import, which appears to be less reliant on protein unfolding before import (Subramani 1993). In fact, there is only one substantive difference we can see between existing paradigms of protein import and PMP import. PMP import involves the posttranslational import of proteins that contain membrane-spanning domains (Lazarow and Fujiki 1985).
It is generally appreciated that hydrophobic membrane-spanning domains cannot be exposed to the aqueous environment of the cytoplasm without deleterious affects on protein folding and solubility. Thus, it may well be that posttranslational import of PMPs requires special mechanisms to prevent exposure of their transmembrane domains to the cytoplasm. This could be accomplished by a specific protein or protein complex that binds hydrophobic regions of PMPs during and/or after PMP synthesis and before insertion of the PMP into the peroxisome membrane. Furthermore, PMPs with multiple transmembrane domains such as PMP34, PEX13, and PMP70 might be expected to contain multiple sites for interaction with such a PMP binding factor.
A priori, the existence of such a PMP binding factor does not demand the presence of multiple targeting signals in a polytopic PMP. However, we can imagine one circumstance in which it would. If the interaction between a PMP and the PMP binding factor is sufficient to direct the PMP into the peroxisomal import pathway, then we would expect that the sites along the PMP that interact with the binding factor would function as PMP targeting signals. PMPs with multiple membrane-spanning domains would be expected to have multiple interaction sites for the PMP binding factor and would therefore be expected to contain multiple nonoverlapping targeting signals.
Although this speculative model remains to be tested, it is interesting to consider known proteins that might fulfill the role of the proposed PMP binding factor. Molecular chaperones are clear candidates since they are known to mask hydrophobic domains and prevent protein aggregation. In addition to Hsp70 and Hsp40 molecules that have been implicated in peroxisome biogenesis, a recent study has suggested that the multicomponent cytosolic ring chaperone, TriC, may interact with newly synthesized PMPs (Pause et al. 1997). However, it is difficult to imagine how these somewhat generic chaperones, which affect proteins destined for many functions and subcellular compartments, would specify entry into the PMP import pathway. Our hypothesis may make more sense if the PMP binding factor acts in a pathway-specific manner, and one such candidate has been identified. PEX19 is required for PMP import, interacts with all known human PMPs, and interacts with the PMP targeting signals of PMP70 and PEX11β (Sacksteder et al. 2000). We have shown here that PEX19 also interacts with the two targeting signals of PMP34. Mislocalization of PEX19 to the nucleus results in the nuclear accumulation of proteins containing either PMP34 targeting signal. Also, we detected coimmunoprecipitation of PEX19 and the PMP34 targeting signals. A previous study of PEX19–PMP interactions in the yeast P. pastoris concluded that PEX19 does not interact with PMPs before their import and therefore could not act as the proposed PMP binding factor (Snyder et al. 2000). However, in this study, we established that PEX19 does interact with PMPs in the cytosol, since our coimmunoprecipitation experiments were performed with cytosolic lysates that lack peroxisomal membranes. Although our results are consistent with the hypothesis that PEX19 may function as a chaperone and/or import receptor for newly synthesized PMPs, a direct test of this hypothesis remains to be performed.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health grant DK45787 to S.J. Gould.
Submitted: 2 October 2000
Revised: 9 April 2001
Accepted: 24 April 2001
Abbreviations used in this paper: HA, hemagglutinin; NLS, nuclear localization signal; PMP, peroxisomal membrane protein; PTS, peroxisome-targeting signal.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Adam S.A. & Gerace L.. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import, Cell, 66, 1991, 837–847.[Medline]
Baerends R.J.S., Rasmussen S.W., Hilbrands R.E., van der Heide M., Faber K.N., Reuvekamp P.T.W., Kiel J.A.K.W., Cregg J.M., van der Klei I.J. & Veenhuis M.. The Hansenula polymorpha PER9 gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity, J. Biol. Chem., 271, 1996, 8887–8894.
Baerends R.J., Faber K.N., Kram A.M., Kiel J.A., van der Klei I.J. & Veenhuis M.. A stretch of positively charged amino acids at the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into the peroxisomal membrane, J. Biol. Chem, 275, 2000, 9986–9995.
Bjorkman J., Stetten G., Moore C.S., Gould S.J. & Crane D.I.. Genomic structure of PEX13, a candidate peroxisome biogenesis disorder gene, Genomics, 54, 1998, 521–528.[Medline]
Chang C.C., Lee W.H., Moser H.W., Valle D. & Gould S.J.. Isolation of the human PEX12 gene, mutated in group 3 of the peroxisome biogenesis disorders, Nat. Genet., 15, 1997, 385–388.[Medline]
Chang C.-C., South S., Warren D., Jones J., Moser A.B., Moser H.W. & Gould S.J.. Metabolic control of peroxisome abundance, J. Cell Sci, 112, 1999, 1579–1590.[Abstract]
Crane D.I., Kalish J.E. & Gould S.J.. The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly, J. Biol. Chem., 269, 1994, 21835–21844.
Deshaies R.J., Koch B.D., Werner-Washburne M., Craig E.A. & Schekman R.. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides, Nature, 332, 1988, 800–805.[Medline]
Diestelkotter P. & Just W.W.. In vitro insertion of the 22 kD peroxisomal membrane protein into isolated rat liver peroxisomes, J. Cell Biol, 123, 1993, 1717–1725.
Dyer J.M., McNew J.A. & Goodman J.M.. The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop, J. Cell Biol., 133, 1996, 269–280.
Fujiki Y., Rachubinski R.A. & Lazarow P.B.. Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes, Proc. Natl. Acad. Sci. USA., 81, 1984, 7127–7131.
Gatto G.J. Jr., Geisbrecht B.V., Gould S.J. & Berg J.M.. Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5, Nat. Struct. Biol., 7, 2000, 1091–1095.[Medline]
Geisbrecht B.V., Collins C.S., Reuber B.E. & Gould S.J.. Disruption of a PEX1-PEX6 interaction is the most common cause of the neurologic disorders zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, Proc. Natl. Acad. Sci. USA., 95, 1998, 8630–8635.
Gould S.G., Keller G.A. & Subramani S.. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase, J. Cell Biol, 105, 1987, 2923–2931.
Gould S.J., Keller G.A., Hosken N., Wilkinson J. & Subramani S.. A conserved tripeptide sorts proteins to peroxisomes, J. Cell Biol., 108, 1989, 1657–1664a.
Gould S.J., Subramani S. & Scheffler I.E.. Use of the DNA polymerase chain reaction for homology probingisolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species, PNAS, 86, 1989, 1934–1938b.
Gould S.J., Kalish J.E., Morrell J.C., Bjorkman J., Urquhart A.J. & Crane D.I.. PEX13p is an SH3 protein in the peroxisome membrane and a docking factor for the PTS1 receptor, J. Cell Biol., 135, 1996, 85–95.
Hettema E.H., Girzalsky W., van Den Berg M., Erdmann R. & Distel B.. Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins, EMBO J., 19, 2000, 223–233.[Medline]
Honsho M. & Fujiki Y.. Topogenesis of peroxisomal membrane protein requires a short, positively charged intervening-loop sequence and flanking hydrophobic segments. Study using human membrane protein pmp34, J. Biol. Chem, 276, 2001, 9375–9382.
Imanaka T., Shiina Y., Hashimoto T. & Osumi T.. Insertion of the 70-kDa peroxisomal membrane protein into peroxisomal membranes in vivo and in vitro, J. Biol. Chem., 271, 1996, 3706–3713.
Jones J.M., Nau K., Geraghty M.T., Erdmann R. & Gould S.J.. Identification of peroxisomal acyl-CoA thioesterases in yeast and humans, J. Biol. Chem., 274, 1999, 9216–9223.
Jones J.M., Morrell J.C. & Gould S.J.. Identification and characterization of HAOX1, HAOX2, and HAOX3, three human peroxisomal 2-hydroxy acid oxidases, J. Biol. Chem., 275, 2000, 12590–12597.
Kammerer S., Holzinger A., Welsh U. & Roscher A.A.. Cloning and characterization of the gene encoding the human peroxisomal assembly protein Pex3p, FEBS Lett., 429, 1998, 53–60.[Medline]
Lazarow P.B. & Fujiki Y.. Biogenesis of Peroxisomes, Annu. Rev. Cell Biol., 1, 1985, 489–530.
Liu Y., Bjorkman J., Urquhart A., Wanders R.J.A., Crane D. & Gould S.J.. PEX13 is mutated in complementation group 13 of the peroxisome biogenesis disorders, Am. J. Hum. Genet., 65, 1999, 621–634.[Medline]
McCammon M.T., McNew J.A., Willy P.J. & Goodman J.M.. An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes, J. Cell Biol, 124, 1994, 915–925.
Pause B., Diestelkotter P., Heid H. & Just W.W.. Cytosolic factors mediate protein insertion into the peroxisomal membrane, FEBS Lett., 414, 1997, 95–98.[Medline]
Pause B., Saffrich R., Hunziker A., Ansorge W. & Just W.W.. Targeting of the 22-kDa integral peroxisomal membrane protein, FEBS Lett, 471, 2000, 23–28.[Medline]
Rehling P., Marzioch M., Wittke E., Veenhuis M. & Kunau W.H.. The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene, EMBO J., 15, 1996, 2901–2913.[Medline]
Sacksteder K.A. & Gould S.J.. The genetics of peroxisome biogenesis, Annu. Rev. Genet, 34, 2000, 623–652.[Medline]
Sacksteder K.A., Jones J.M., South S.T., Li X., Liu Y. & Gould S.J.. PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis, J. Cell Biol., 148, 2000, 931–944.
Snyder W.B., Koller A., Choy A.J. & Subramani S.. The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane, J. Cell Biol, 149, 2000, 1–7.
Soukupova M., Sprenger C., Gorgas K., Kunau W.H. & Dodt G.. Identification and characterization of the human peroxin PEX3, Eur. J. Cell Biol., 78, 1999, 357–374.[Medline]
Subramani S.. Protein import into peroxisomes and biogenesis of the organelle, Annu. Rev. Cell Biol., 9, 1993, 445–478.
Suzuki Y., Orii T., Takiguchi M., Mori M., Hijikata M. & Hashimoto T.. Biosynthesis of membrane polypeptides of rat liver peroxisomes, J. Biochem, 101, 1987, 491–496.
Swinkels B.W., Gould S.J., Bodnar A.G., Rachubinski R.A. & Subramani S.. A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase, EMBO J., 10, 1991, 3244–3262.
Wang X., Unruh M.J. & Goodman J.M.. Discrete targeting signals direct PMP47 to oleate-induced peroxisomes in Saccharomyces cerevisiae, J. Biol. Chem., 276, 2001, 10897–10905.
Warren D.S., Morrell J.C., Moser H.W., Valle D. & Gould S.J.. Identification of PEX10, the gene defective in complementation group 7 of the peroxisome-biogenesis disorders, Am. J. Hum. Genet., 63, 1998, 347–359.[Medline]
Wiemer E.A.C., Luers G.H., Faber K.N., Wenzel T., Veenhuis M. & Subramani S.. Isolation and characterization of Pas2p, a peroxisomal membrane protein essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris, J. Biol. Chem., 271, 1996, 18973–18980.
Wylin T., Baes M., Brees C., Mannaerts G.P., Fransen M. & Van Veldhoven P.P.. Identification and characterization of human PMP34, a protein closely related to the peroxisomal integral membrane protein PMP47 of Candida boidinii, Eur. J. Biochem., 258, 1999, 332–338.[Medline]
Zhang J.W. & Lazarow P.B.. Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, Type 2, peroxisomal targeting sequence of thiolasePeb1p itself is targeted to peroxisomes by an NH2-terminal peptide, J. Cell Biol., 132, 1996, 325–334.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
