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Article |
Address correspondence to Dr. Stephen J. Gould, Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: (410) 955-3424. Fax: (410) 955-0215. email: sgould{at}jhmi.edu
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Abstract |
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Key Words: PMP import; temperature-sensitive mutants; Zellweger syndrome; organelle biogenesis; protein import
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Introduction |
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Two pathways of PMP import have been described (Jones et al., 2004). The class I pathway of PMP import is dependent upon PEX19, which binds a wide variety of PMPs (Gloeckner et al., 2000; Sacksteder et al., 2000; Snyder et al., 2000; Fransen et al., 2001; Brosius et al., 2002), and then binds their transmembrane domains, stabilizes them during their transit through the cytoplasm, and releases them before or during their insertion into the peroxisome membrane (Jones et al., 2004). PEX19 also binds to the targeting signals (mPTSs) of numerous class I PMPs (Jones et al., 2001, 2004; Brosius et al., 2002), is required for class I mPTS function in vivo, and is essential for the import of class I PMPs, though it is not required for import of peroxisomal matrix enzymes or a class II PMP (Jones et al., 2004). Taken together, these results indicate that PEX19 is a cytoplasmic chaperone and import receptor for newly synthesized class I PMPs.
The class I PMPs are a diverse group and include metabolite transporters such as PMP22, PMP34, and PMP70, peroxins involved in peroxisome division such as PEX11, peroxins involved in peroxisomal matrix protein import such as PEX2 and PEX13, and even one peroxin involved in peroxisome membrane synthesis, PEX16 (Sacksteder et al., 2000; Jones et al., 2001, 2004; Brosius et al., 2002). In fact, the only PMP that is known to be imported independently of PEX19 is PEX3, which currently defines the class II PMP import pathway (Jones et al., 2004).
The observation that PEX19 binds newly synthesized class I PMPs in the cytoplasm and then transports them to the peroxisome indicates that there is at least one docking factor for PEX19 on the peroxisome membrane. A docking factor for PEX19 can be expected to have certain properties. For example, it must bind to PEX19 and it must reside in the peroxisome membrane. However, these properties are also shared by all integral PMPs that depend on PEX19 for their import (Sacksteder et al., 2000; Jones et al., 2004). More stringent criteria for the PEX19 docking factor are necessary and some are as follows. First, loss of a putative docking factor must block the recruitment of PEX19 to peroxisomes. Second, a docking factor should interact with a region of PEX19 that (a) differs from its PMP-binding domain, (b) is sufficient for docking, and (c) requires the docking factor for its recruitment to peroxisomes. Third, mislocalization of a PEX19 docking factor to another organelle should be sufficient to dock PEX19 at the heterologous organelle. Fourth, the docking factor for PEX19 should be required for class I PMP import but dispensable for class II PMP import and peroxisomal matrix protein import. Here we show that PEX3 meets all of these criteria.
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Results |
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To determine whether PEX3 is required for PEX19 to dock at the peroxisome membrane we tested whether transient depletion of PEX3, using RNA interference (RNAi) technology, affected PEX19's association with peroxisomes. Wild-type (WT) human fibroblasts transfected with small interfering RNAs (siRNAs) specific for the PEX3 gene show a significant reduction in PEX3 protein levels at 23 d after transfection, with levels reduced 90% on day 2 (Fig. 1 A). At this time point, PEX3 can no longer be detected on peroxisomes by immunofluorescence (Fig. 1 B). Control RNAi directed against TRIP8b (a gene that has no role in peroxisome biogenesis; Chen et al., 2001) or PEX5 (a gene that functions only in peroxisomal matrix protein import; Dodt et al., 1995; Chang et al., 1999a) had no effect on PEX3 abundance within the cell.
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The mislocalization of PEX3 to other subcellular organelles in cells that lack PEX19 (and therefore lack peroxisomes) makes it possible to ask whether PEX3 is sufficient to recruit PEX19 to organelle surfaces. PEX19-deficient cells were cotransfected with plasmids designed to express the docking domain of PEX19 (3xHA-PEX19aa156) and either WT PEX33xmyc or the L125P/N134D mutant form of PEX33xmyc. Both PEX3 proteins are imported into heterologous organelles, including mitochondria and small unidentifiable small vesicles. WT PEX3 is able to recruit the docking domain of PEX19 to these other organelles (Fig. 6, A and B) but the L125P/N134D mutant form of PEX3myc is unable to recruit the docking domain of PEX19 to these organelles (Fig. 6, C and D).
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These experiments revealed that inhibition of PEX3 induced a selective defect in import of class I PMPs but had no detectable effect on the import of a class II PMP or a peroxisomal matrix protein (Fig. 7). In addition to the immunofluorescence microscopy images, which provide a visual representation of these results (Fig. 7 A), we assessed import capabilities of hundreds of randomly selected cells from each population. The statistical analysis of this data confirms that inhibition of PEX3 resulted in a specific defect in class I PMP import (Fig. 7, BD).
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To determine whether PEX3 played essential roles in PMP import, peroxisomal matrix protein import, or both, we assayed both processes in YFY1 cells carrying either a WT PEX3 expression plasmid or the pex3-G6 plasmid, which appeared to confer the "tightest" conditional phenotype of the three mutant plasmids we obtained. These two strains, which constitutively express CFP-PTS1, were then transformed, individually, with plasmids that carried the galactose-inducible GAL1 promoter driving the expression of four peroxisomal marker proteins: (a) YFP-PTS1, a peroxisomal matrix protein (Kalish et al., 1996), (b) YFP-PEX10, a YFP-tagged form of a PMP that is involved in peroxisome biogenesis (Kalish et al., 1995), (c) YFP-PEX15, another YFP-tagged form of a PMP that is involved in peroxisome biogenesis (Elgersma et al., 1997), and (d) ANT1-YFP, a YFP-tagged form of a PMP that is involved in ATP translocation across the peroxisome membrane and plays no role in peroxisome biogenesis (Palmieri et al., 2001). The resulting strains were grown at the permissive temperature in minimal glucose medium to repress expression of the YFP marker proteins. Each strain was then transferred to galactose medium to induce expression of the YFP marker proteins and either maintained at the permissive temperature or shifted to the restrictive temperature. 4 h after the temperature shift the cells were fixed and examined by epifluorescence microscopy to determine the subcellular distribution of the proteins as marked by CFP and YFP. As expected, YFY1 cells containing the WT PEX3 gene imported both YFP-PTS1 and the YFP-PMP fusion proteins into peroxisomes at both the permissive and restrictive temperatures (Fig. 9 A). The temperature-sensitive pex3-G6 strain also imported all four peroxisomal marker proteins at the permissive temperature. However, when the pex3-G6 strain was shifted to 37°C, it was unable to import YFP-PEX10, ANT1-YFP, or YFP-PEX15 proteins into peroxisomes, even though it continued to import the peroxisomal matrix protein marker YFP-PTS1 (Fig. 9 B). Similar results were obtained in the pex3-A8 and pex3-B5 strains (Fig. 10). Thus, transient inhibition of yeast PEX3 results in a specific defect in PMP import with no apparent defect in peroxisomal matrix protein import. Previous studies have also reported variable and aberrant fates for PMPs in cells that lack PEX3 activity (Hettema et al., 2000; South et al., 2000), and these are reflected in our inability to detect YFP-PEX10 and YFP-PEX15 and the mislocalization of ANT1-YFP to nonperoxisomal structures in cells that lack PEX3 activity.
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Discussion |
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The most widely accepted model of peroxisome biogenesis is that peroxisomes are formed by the growth and division of preexisting peroxisomes (Lazarow and Fujiki, 1985). Under this model, any significant defect in PMP import would be expected to cause the loss of peroxisomes from the cell. This is precisely the phenotype of pex3- and pex19-null mutants in both humans and S. cerevisiae (Matsuzono et al., 1999; Hettema et al., 2000; Sacksteder et al., 2000; South et al., 2000). Although class I PMP import might require more protein factors than PEX19 and PEX3, it is also possible that it does not. Therefore, we should not exclude additional roles for PEX19 and PEX3 in PMP import. In fact, some studies have detected interactions between class I PMPs and both PEX19 and PEX3 at the peroxisome membrane (Snyder et al., 2000; Hazra et al., 2002) and these observations might reflect a role for PEX3 and/or PEX19 in the insertion of class I PMPs.
Neither PEX3 nor PEX19 are required for class II PMP import (Jones et al., 2004). Of the PMPs that have been tested, PEX3 is the only class II PMP yet identified. Given the essential role for PEX3 in PMP import and the phenotypes associated with the loss of PEX3 activity (loss of peroxisomes from the cell, rapid destruction of PMPs, and cytosolic accumulation of matrix enzymes in the cytosol), it is expected that loss of any factor involved in PEX3 import would result in phenotypes that are at least as severe as the pex3-null mutant. No such mutants are known in yeast but the pex16 mutant of humans does meet these criteria. Future studies that test whether PEX16 acts as a PEX3 import factor, as another class I PMP import factor, or as a critical player in some other aspect of peroxisome membrane biogenesis (lipid import, peroxisome division, etc.) are a high priority. In addition to searching for factors that might mediate PEX3 import, it might be possible that PEX3 does not require any specific protein factors for import into peroxisomes, perhaps autocatalyzing its import into peroxisomes. Interestingly, Haan et al. (2002) have observed that an engineered epitope appears exposed to the cytosol regardless of where it is inserted in PEX3, and on this basis have argued that PEX3 might not even be an integral PMP, even though it has the biochemical properties of being embedded in the membrane.
The unique targeting pathway for PEX3 and its conserved role in the import of most PMPs can also be interpreted in the context of more speculative models of peroxisome biogenesis. For example, several researchers believe that peroxisomes arise, directly or indirectly, from the ER (Mullen et al., 2001; Titorenko and Rachubinski, 2001a, b; Tabak et al., 2003). A critical requirement of such models is the existence of a peroxin that is essential for the formation of peroxisome membranes but traffics to peroxisomes by a mechanism that is fundamentally distinct from that used by the vast majority of PMPs. Moreover, Faber et al. (2002) have claimed that the overexpression of nonfunctional forms of PEX3 in a pex3-null mutant can stimulate the formation of "preperoxisomal" vesicles from the ER and that these vesicles subsequently mature into fully functional peroxisomes upon the expression of WT PEX3. Our observation that PEX3 is imported differently than most other PMPs might be interpreted by some as indirect support for both an ER-to-peroxisome route for PEX3 biogenesis and a direct role for the ER in the genesis of peroxisomal vesicles.
However, we do not feel that such a conclusion is warranted at this time, and for several reasons. First, the kinetics of PEX3-mediated peroxisome synthesis during the complementation of pex3-null mutants is one to two orders of magnitude slower than the kinetics of PEX3 import into peroxisomes of WT cells (South et al., 2000). This result means that virtually all PEX3 is imported into preexisting peroxisomes long before it even has the chance to mediate "de novo" peroxisome synthesis. These considerations raise serious doubts as to whether any observations of peroxisome synthesis in the absence of preexisting peroxisomes are physiologically relevant. Second, the one study that has attempted to follow the biogenesis of PEX3 in vivo found no evidence for PEX3 migration through the ER to the peroxisome in WT cells or in cells that lack peroxisomes and instead detected PEX3 first in the peroxisome (South et al., 2000). Third, PEX3-mediated peroxisome synthesis occurs independently of the SEC61 and SSH1 protein translocons in the ER membrane, independently of the COPII coat complex, which is essential for vesicular traffic from the ER, and independently of the COPI coat complex, which is required for other vesicle-mediated trafficking events in the early secretory pathway (South et al., 2001, 2000). These concerns and observations, coupled with the unique function of PEX3 in peroxisome biogenesis and its enigmatic route of import, make it more important than ever to resolve the details of PEX3 biogenesis through direct analysis of endogenously expressed PEX3 in normal cells.
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Materials and methods |
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Immunofluorescence images were captured on a BH2-RFCA microscope (Olympus) with an Olympus SplanApo 60x 0.40 oil objective and a Sensicam QE (Cooke) digital camera using IPLab 3.6 software (Scanalytics) at room temperature, and processed with Adobe Photoshop 7.0 software (Adobe Systems, Inc.). Contrast, brightness, and gamma values were adjusted to approximate the original IPLab image.
SiRNA treatment and immunoblots
RNA oligonucleotides were obtained from Dharmacon Research, Inc. Four pairs of RNA oligonucleotides were used in this study: PEX5: 5'-AGAAGCUACUCCCAAAGGCdTdT-3', 5'-GCCUUUGGGAGUAGCUUCUdTdT-3'; TRIP8b: 5'-GCAGGGAAAAGGCUCUAGGdTdT-3', 5'-CCUAGAGCCUUUUCCCUGCdTdT-3'; PEX3: 5'-CGGACAGAUCCAUUCAGUUdTdT-3', 5'-AACUGAAUGGAUCUGUCCGdTdT-3'; PEX16: 5'-UGACGGGAUCCUACGGAAGdTdT-3', 5'-CUUCCGUAGGAUCCCGUCAdTdT-3'. To test the effect of siRNA on protein levels, GM 5756-TI cells from each confluent 75-cm2 flask were suspended in 500 µl Hepes-buffered saline (21 mM Hepes, pH 7.15, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose), mixed with 50 µl siRNA (20 µM in annealing buffer: 100 mM KAc, 30 mM Hepes-KOH, pH 7.4, 2 mM MgAc), and electroporated in a BTX ECM600 Electroporation System at 230 V, 1,500 µF, 129. After transfection, cells were grown for 5 d. Every 24 h, cells were fixed with 3% formaldehyde for indirect immunofluoresence study or lysed with SDS-loading buffer for immunoblot study, respectively. An equal amount of total protein from each sample was subjected to SDS-PAGE gel. The proteins were then transferred to PVDF membrane (Millipore), followed by incubation with primary antibodies and HRP-conjugated secondary antibodies sequentially, and visualized by using ECL detection reagents (Amersham Biosciences).
SiRNA treatment and peroxisomal protein import assay
GM5756-TI cells were transfected with PEX3, PEX5, and TRIP8b siRNAs three times at 24-h intervals, respectively. At 72 h after the initial treatment, cells were cotransfected with (a) pNHA-PTE1 and (b) pcDNA3-PMP34myc, pcDNA3-PEX11ßmyc, pcDNA3-PEX16aa2213363xmyc, or pcDNA3-PEX3aa1506xmet3xmyc. 2 h after the DNA transfection, cells were processed for double indirect immunofluoresence microscopy using anti-myc and anti-HA antibodies. To quantify the import assay, all cells showing peroxisomal staining (import of either marker) were counted as to whether they imported HA-PTE1, PMP34myc (PEX11ßmyc, PEX3aa150-6xmet3xmyc, PEX16aa221336-3xmyc), or both. Each experiment was done in triplicate and the mean and standard deviation were presented.
Yeast strains and screening for temperature-sensitive mutants of PEX3
The genotype of the S. cerevisiae strain BY4733 used in this study is MATa, his3200, leu2
0, met15
0, trp1
63, ura3
0. The YFY1 strain was generated by one-step PCR-mediated disruption of PEX3 in BY4733 with URA3 as the selectable marker (Baker-Brachmann et al., 1998), followed by transforming the cells with pRS315-PGK1-CFP-PTS1. To screen for the temperature-sensitive mutants of PEX3, YFY1 cells were cotransformed with the linearized gap repair plasmid pRS314-scPEX3UTR (digested by SalI and NotI) and the error-prone PCR product of PEX3 flanked by 5' 102 bps and 3' 120 bps. Transformants were selected on minimal S medium (0.17% yeast nitrogen base without ammonium sulfate [Sigma-Aldrich], 0.5% ammonium sulfate) with 2% glucose lacking uracil, leucine, and tryptophan. Each transformant was spotted onto duplicate plates, which were then incubated at 25° and 37°C for 3 d, and then examined by epifluorescence microscopy to determine the subcellular distribution of CFP-PTS1. Those that imported CFP-PTS1 at 25°C but accumulated CFP-PTS1 in the cytoplasm at 37°C were considered temperature-sensitive mutants of PEX3.
PMP import assay in WT and pex3ts strains
The YFY1 strain was transformed with pRS314-PEX3 as WT control for pex3ts strains. Next, the WT and pex3ts strains, which constitutively expressed CFP-PTS1, were retransformed with the plasmids: pRS313-GAL1-YFP-PTS1, pRS313-GAL1-YFP-PEX10, pRS313-GAL1-ANT1-YFP, pRS313-GAL1-YFP-PEX15. The YFP-tagged peroxisomal proteins were expressed under the control of the galactose-inducible GAL1 promoter. Cells were grown at 25°C on minimal glucose medium lacking uracil, leucine, histidine, and tryptophan to repress expression of the YFP marker proteins. For the import assay of YFP-PTS1, YFP-PEX10, YFP-PEX15, pex3-WT, and pex3-G6 cells were transferred to minimal medium with 2% galactose at 25°C for 23 h, and then either maintained at 25°C or shifted to 37°C for four more hours. For the import assay of ANT1-YFP, pex3-WT and pex3-G6 cells grown in glucose minimal medium were shifted to 37°C for 2.5 h, and then transferred to minimal galactose (2%) medium for two and a half more hours. For the import assay of YFP-PTS1 and YFP-PEX10 in pex3-A8 and pex3-B5 strains, cells were transferred to minimal galactose medium at 25°C for 23 h, and then either maintained at 25°C or shifted to 37°C for four more hours. Cells with CFP-PTS1 imported to peroxisome were scored as to whether YFP imported or not. Then the calculated percentage of import of each protein at 37°C was normalized to that at 25°C, respectively. Each experiment was performed in triplicate and the mean and standard deviation are presented.
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Acknowledgments |
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Submitted: 25 November 2003
Accepted: 13 January 2004
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References |
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