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Address correspondence to S.J. Gould, Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: (410) 955-3085. Fax: (410) 955-0215. email: sgould{at}jhmi.edu
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Abstract |
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Key Words: peroxisome; protein import; posttranslational; Zellweger syndrome; PEX3
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Introduction |
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A common view of protein targeting signals is that they are independent elements that have little or no role in the protein's ultimate structure or function. It is difficult to reconcile this view with two well-established properties of targeting signals for integral PMPs. First, many polytopic PMPs contain multiple, nonoverlapping peroxisomal targeting signals, any one of which is sufficient to direct proteins into the peroxisome membrane (Biermanns and Gartner, 2001; Jones et al., 2001; Wang et al., 2001; Brosius et al., 2002). Second, PMP targeting signals are large, 50100 aa in length, and the aggregate size of the targeting signals in several PMPs comprises a significant fraction of the entire protein (Sacksteder et al., 2000; Biermanns and Gartner, 2001; Jones et al., 2001; Wang et al., 2001; Brosius et al., 2002).
We recently developed a hypothesis that can explain both the large size and functional redundancy of PMP targeting signals in the context of posttranslational PMP import (Jones et al., 2001). Specifically, we proposed the existence of a cytoplasmic PMP chaperone and import receptor that (a) binds to multiple sites along polytopic PMPs; (b) prevents PMP misfolding, aggregation, and destruction in the cytoplasm by masking their transmembrane domains; and (c) directs newly synthesized PMPs to the peroxisome membrane, and therefore plays a specific and essential role in PMP import. Although previous works have concluded that PEX19 cannot function as a PMP import receptor (Snyder et al., 2000; Fransen et al., 2001). We show here that PEX19 has all of the properties one would expect for a bifunctional PMP chaperone/import receptor. In addition, we establish the existence of two mechanistically distinct PMP import pathways: one that requires PEX19 and mediates the import of multiple PMPs; and one that is PEX19 independent and mediates the import of PEX3.
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Results |
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Immunoblot analysis revealed that integral PMPs were much more abundant in peroxisome-deficient cells that express PEX19 than in cells that do not express PEX19 (Fig. 1 A). These results reflect the status of soluble, cytoplasmic PMPs in detergent-free, membrane-free lysates. This was confirmed by immunofluorescence studies which revealed that all three PMPs shared the same cytoplasmic accumulation and nuclear exclusion as PEX19 (Fig. 1 B). In contrast, these PMPs could only rarely be detected in PEX19-deficient cells, and what little could be detected was mislocalized to the mitochondrion (unpublished data).
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To determine whether PEX19 stabilized PMPs through physical interaction in the cytosol, we asked whether these cytoplasmic, stable PMPs could be recovered in PEX19 immunoprecipitates (IPs). We coexpressed each integral PMP with PEX19 and an epitope-tagged form of PEX19, 3xHA-PEX19. 1 d later, the cells were lysed and subjected to immunoprecipitation with anti-HA antibodies. The IPs were then separated by SDS-PAGE and immunoblotted with anti-myc antibodies. A higher level of each PMP was detected in the anti-HA IPs from cells expressing 3xHA-PEX19 (Fig. 1 E). Furthermore, the analysis of PMP levels in the samples before and after immunoprecipitation indicated that the majority of each PMP was associated with PEX19 (Fig. 1 F).
If PEX19 is a cytoplasmic PMP chaperone, it should also bind newly synthesized PMPs. To test this prediction we used PBD399 (PEX19-deficient) cells that stably express 3xHA-PEX19 at approximately the level of PEX19 in wild-type cells (these cells contain normal peroxisomes and import PMPs; unpublished data). These PBD399/3xHA-PEX19 cells were transiently transfected with pcDNA3-PMP34/13xmyc, pulse labeled with [35S]methionine and chased with excess cold methionine for varying amounts of time. At each time point, half of the cells were lysed, membranes were discarded, and the samples were subjected to immunoprecipitation with an anti-HA mAb. The IPs were then solubilized with SDS, diluted, and subjected to a second immunoprecipitation with an anti-myc mAb. The other half of the cells were solubilized in 1% Triton X-100 and subjected to immunoprecipitation using an anti-myc mAb. After separation of the samples by SDS-PAGE, the labeled PMPs were detected by autoradiography (Fig. 1 G). Densitometric analysis of these films revealed that the half-time of association between cytosolic PMP34/13xmyc and PEX19 was 15 min, whereas the half-life of total PMP34/13xmyc was
300 min. Thus, PEX19 displays a kinetically restricted interaction with PMPs, preferentially binding newly synthesized PMPs in the cytosol.
PEX19 is an import receptor for multiple integral PMPs
Our hypothesis that PMP import requires a bifunctional PMP chaperone/import receptor led us to next ask whether PEX19 also functioned as a PMP import receptor. We first tested whether PEX19 binds to the mPTSs of multiple PMPs. We tested the c-terminal mPTS of PMP34 (Jones et al., 2001), an mPTS from PEX11ß (Sacksteder et al., 2000), and both reported mPTSs of PMP22 (Brosius et al., 2002). We also tested two distinct mPTSs from PEX16, one reported previously (Fransen et al., 2001) and one (amino acids 221336) identified in our laboratory (unpublished data). Finally, we examined three reported mPTSs of PMP70 (Sacksteder et al., 2000; Biermanns and Gartner, 2001). PEX3-deficient PBD400-TI cells, which lack peroxisomes, were cotransfected with plasmids designed to express the mPTS-containing proteins together with either PEX19 or 3xHA-PEX19. The next day the cells were lysed in hypotonic buffer and membranes were discarded after centrifugation. The resulting soluble protein lysates were subjected to immunoprecipitation with antibodies to the HA epitope tag, followed by immunoblot with antibodies specific for the mPTS-containing proteins. Each mPTS-containing protein was precipitated with 3xHA-PEX19, demonstrating the ability of PEX19 to bind these diverse mPTSs (Fig. 2 A).
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PEX19 interacts with the transmembrane region of PMP targeting signals
The hypothesis that PMP import requires a bifunctional PMP chaperone/import receptor is rooted in the assumption that the hydrophobic transmembrane domains of PMPs must be masked as PMPs move through the cytoplasm to the peroxisome membrane. If this is true, and if PEX19 is the PMP chaperone/import receptor of this hypothesis, then PEX19 should bind to subregions of mPTSs that contain transmembrane domains. We tested this prediction using the COOH-terminal mPTS of PMP34, which contains a single transmembrane domain. We generated a series of mutations in this mPTS and examined their effects on PEX19 interaction in vivo. Truncation mutants that retained the transmembrane domain retained their interaction with PEX19, whereas those that lacked part of the transmembrane domain were no longer efficiently bound by PEX19 (Fig. 3). The smallest fragment that retained full interaction with PEX19, PMP34aa270-307, contains a long hydrophobic stretch that is interrupted by a pair of charged residues (E289 and K290). Replacement of these charged residues with leucines inhibited the fragment's interaction with PEX19, as did replacement of three hydrophobic residues with hydrophilic amino acids (LMF283-285KKK). Replacing the flanking basic residues at the COOH-terminal side of the putative transmembrane domain with acidic residues (KR302-303EE) had no substantive effect on mPTSPEX19 interaction. These results indicate that PEX19 interacts with the transmembrane domain of the PMP34 mPTS, and that the distribution of hydrophobic and charged residues within the transmembrane domain are important for mPTSPEX19 interaction.
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Next, we tested whether the observed PMP import defect in cells treated with PEX19 siRNA would hold true for mPTSs. We treated wild-type cells with PEX19 and TRIP8b siRNAs, waited 60 h, and transfected the cells with plasmids that express HA-PTE1 and either PMP34aa244-307/3xmyc or PEX16aa221-336/3xmyc. Next, we assayed for import of the mPTS-containing proteins by identifying cells that had imported HA-PTE1 and scoring the distribution of the mPTS-containing protein in those cells as either peroxisomal (if any peroxisomes were identified), nonperoxisomal (if no peroxisomes were identified), or not seen (Fig. 5 B). Scoring those cells that effectively imported HA-PTE1 ensured that only those cells with peroxisomes able to import matrix proteins were counted in this analysis of PMP import. Both PMP34aa244-307/3xmyc and PEX16aa221-336/3xmyc were imported into peroxisomes in the majority of control cells (88% and 91%, respectively). In PEX19 siRNA-treated cells, however, both mPTS-containing proteins were imported poorly. Specifically, only 12% of cells imported PMP34aa244-307/3xmyc and only 22% imported PEX16aa221-336/3xmyc. Of special interest here is that, rather than being degraded like full-length PMP34myc, PMP34aa224-337/3xmyc was mistargeted to other compartments of the cell in the majority of PEX19 siRNA-treated cells. This result provides direct evidence that inhibition of PEX19 function disrupts PMP import not only by destabilizing PMPs but also by preventing appropriate PMP targeting.
A second PMP import pathway
The hypothesis that PEX19 is a PMP chaperone and import receptor contrasts with previous observations that PEX19 does not bind to the mPTS of PEX3, another integral PMP (Kammerer et al., 1998; Soukupova et al., 1999; Snyder et al., 2000; Fransen et al., 2001). Therefore, we tested whether PEX19 functioned as a chaperone and import receptor for PEX3 (Fig. 6). Using the same assays that showed strong interaction between PEX19 and other mPTSs, we found that the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) is neither bound by PEX19 (Fig. 6 B) nor is its subcellular distribution affected by PEX19 or 3xNLSPEX19 (Fig. 6 C). Rather, the PEX3 mPTS was targeted to mitochondria in cells that lack peroxisomes, as reported previously for PEX3 and the PEX3 mPTS (Soukupova et al., 1999; Sacksteder et al., 2000).
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Discussion |
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PEX19 is a predominantly cytoplasmic, partly peroxisomal protein (Gotte et al., 1998; Matsuzono et al., 1999; Sacksteder et al., 2000); and we have shown here that PEX19 binds and stabilizes newly synthesized PMPs in the cytoplasm by interacting with their hydrophobic membrane-spanning domains. We have also shown that PEX19 binds the targeting signals of class 1 PMPs and is required for class 1 PMP import. These results demonstrate a direct and specific role for PEX19 in PMP import, that of a cytoplasmic PMP chaperone and a PMP import receptor.
This conclusion is supported by the prior observations that PEX19PMP interactions can be detected with purified PMPs (Sacksteder et al., 2000) and in yeast two-hybrid systems (Gotte et al., 1998; Sacksteder et al., 2000; Snyder et al., 2000; Fransen et al., 2001), as well as the observation that PEX19 binds to all PMPs regardless of their function (Gotte et al., 1998; Sacksteder et al., 2000; Snyder et al., 2000; Fransen et al., 2001). Furthermore, PEX19 function appears to be specific to PMP import, as transient inhibition of PEX19 has no effect on peroxisomal matrix protein import and permanent loss of PEX19 in yeast and human cells generates peroxisome-specific cellular phenotypes (Gotte et al., 1998; Matsuzono et al., 1999; Sacksteder et al., 2000).
In this paper, we found that PEX19 does not bind the mPTS of PEX3 and that inhibition of PEX19 has no effect on its import into peroxisomes. These results suggest that PEX3 uses a distinct type of mPTS and a distinct import mechanism from the majority of PMPs. The fact that PEX3 contains a distinct mPTS may be related to its essential role in peroxisome membrane biogenesis. In both human cells and yeast, loss of PEX3 results in the absence of detectable peroxisomes and the rapid proteolysis of integral PMPs (Hettema et al., 2000; South et al., 2000). This and other observations have fueled speculation that PEX3 might mediate peroxisome formation from some other organelle, perhaps the ER (Baerends et al., 1996; Salomons et al., 1997; Kammerer et al., 1998; Kunau and Erdmann, 1998; Titorenko and Rachubinski, 1998), or might even catalyze de novo peroxisome synthesis (South et al., 2000, 2001). These models all imply that PEX3 occupies an early and central role in peroxisome membrane biogenesis, and our observation that PEX3 is imported by a mechanism that is distinct from that of several other PMPs is consistent with this possibility. Whether PEX3 is the sole class 2 PMP remains to be determined. PEX16 is the only other PMP known to be essential for peroxisome membrane biogenesis, but PEX19 does interact with both mPTSs from PEX16 and is required for the peroxisomal import of at least one of them.
The identification of PEX19 as a chaperone and import receptor for class-1 PMPs opens several areas for future investigation. For example, does PEX19 recognize newly synthesized PMPs cotranslationally at the ribosome or does a more general chaperone mediate the transfer of nascent PMPs from the protein synthesis machinery to PEX19? Also, what is the precise nature of PEX19mPTS interactions? The extreme hydrophobicity of the known mPTSs has retarded our development of a quantitative in vitro PEX19mPTS interaction assay, but such assays are clearly needed before we can truly understand the PEX19mPTS interaction. The observation that a small fraction of PEX19 is found at peroxisome membranes (Sacksteder et al., 2000; Snyder et al., 2000) suggests that PEX19, together with other factors, might also participate in the insertion of PMPs into the peroxisome membrane. Of chief interest will be the improvement of our understanding of the relationship between PEX19 and PEX3.
It should be noted that some previous works have claimed that PEX19 cannot function as either a cytosolic chaperone or a PMP import receptor. Our results allow a fresh interpretation of many of the data used to support this opposing conclusion. Snyder et al. (2000) claimed that PEX19 cannot function as a cytosolic chaperone for newly synthesized PMPs. They based this conclusion on their inability to detect PEX19PMP interactions in steady-state cross-linking experiments from cytosolic fractions. Aside from the general problems inherent in making conclusions solely from negative results, Snyder et al. (2000) failed to consider the differences in the steady-state abundance of PMPs in the cytoplasm as opposed to their steady-state abundance in the peroxisome membrane. This difference reflects the short half-time of import for PMPs (3 min; Imanaka et al., 1996) relative to their half-life (
5 h; Fig. 1 G) and may be sufficient to explain the Snyder et al. (2000) result.
Reports have also claimed that PEX19 cannot function as an import receptor for newly synthesized PMPs (Snyder et al., 2000; Fransen et al., 2001). This claim is based largely on the observations that PEX19 does not bind all known mPTSs and that PEX19 has been observed to bind PMPs at locations distinct from known mPTSs. However, without additional information, this data can only be interpreted as evidence against a role for PEX19 as an import receptor if one assumes (a) that there is only one PMP import mechanism and therefore only one class of mPTS; and (b) that PMPs contain one and only one mPTS. Both of these assumptions are problematic. The first assumption flies in the face of many precedents in the field of cell biology, including the existence of two peroxisomal targeting signals and receptors for peroxisomal matrix proteins, and is refuted by the finding of two distinct classes of mPTSs in this paper. It is also now clear that many PMPs contain multiple mPTSs. Thus, the binding of PEX19 to a region of a PMP other than a known mPTS may simply suggest the presence of an additional, not yet identified, mPTS. As for the reported discordance between PEX19 binding sites and mPTSs in the yeast PMPs PEX10, PEX13, PEX22, and PEX17 (Snyder et al., 2000), and the human PMP PEX12 (Fransen et al., 2001), this discordance is based only on uncontrolled, negative results from two-hybrid assays using large protein fusions of unknown integrity. Thus, it is unclear whether these proteins lack a class 1 mPTS and are imported independently of PEX19 or whether more rigorous investigations would reveal them to also be class I PMPs. We are hopeful that the siRNA assays developed here for mammalian cells, or similar assays using temperature-sensitive mutants in yeast, will serve to distinguish between those PMPs whose import is PEX19 dependent and those whose import is PEX19 independent.
Another argument used to claim that PEX19 is not an import receptor for PMPs is that PEX19 sometimes binds to fragments of proteins or mutations thereof that do not function as mPTSs (Snyder et al., 2000; Fransen et al., 2001). However, this argument is also flawed, for it assumes (a) that the binding of a PMP to its import receptor is sufficient to mediate all facets of its localization, including both its targeting to the peroxisome surface and its insertion into the peroxisome membrane; and (b) that there is only one form of PEX19PMP interaction. There is no evidence to support the first assumption, nor is there any a priori basis for this assumption. In fact, the mechanistic differences between import receptor-ligand binding and membrane protein insertion processes provide a compelling a priori basis for the opposite conclusion: correct PMP targeting likely requires mPTSs that possess multiple functional domains, only one of which mediates its binding to its cognate import receptor. For instance, other functional domains might mediate interaction with factors at the peroxisome surface or might be required for proper insertion in the peroxisome membrane. Disruption of these elements during a mutation analysis study would result in a failure to import, although receptor binding might be maintained. As for the second assumption, it seems likely that an import receptor, in addition to its interactions with its cargo, would also interact with factors at the peroxisome membrane required for its appropriate docking and the subsequent steps of membrane protein insertion. Therefore, the assumption that all of the interactions of a PMP import receptor must be with cargo molecules is unlikely to be true.
In summary, there is no credible evidence or argument that PEX19 does not or cannot function as a cytosolic chaperone for newly synthesized PMPs or as an import receptor for PMPs. Rather, the data presented in this paper offer significant mechanistic evidence that PEX19 does function as bifunctional chaperone/import receptor for class I PMPs. In addition, the data presented here also suggest the existence of a second, PEX19-independent PMP import pathway.
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Materials and methods |
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Cell lines, transfections, and immunofluorescence studies
The cell lines used in this paper were wild-type human fibroblasts (GM 5756-TI), the PEX3-deficient fibroblast line PBD400-TI, and the PEX19-deficient fibroblast line PBD399-TI. These are immortalized derivatives of 5756-T, PBD400-T (South et al., 2000), and PBD399-T (Sacksteder et al., 2000) cells, and were generated by stable transfection with a vector that constitutively expresses human telomerase (pBabePuro/hTERT; Hahn et al., 1999). Cells were cultured, transfected, and processed for immunofluorescence as described (Chang et al., 1997). We used the following antibodies: an antic-myc mAb from the mouse hybridoma line 1-9E10 (Roche), sheep anti-PMP70 antibodies (South et al., 2000), rabbit anti-PEX19 antibodies (Sacksteder et al., 2000), anti-catalase antibodies (The Binding Site), and polyclonal anti-HA antibodies (Santa Cruz Biotechnology, Inc.). Fluorescent secondary antibodies used either FITC or Texas red fluorochromes and were from Jackson ImmunoResearch Laboratories.
Immunofluorescence images were obtained on an BH2-RFCA microscope (Olympus) with an Olympus SplanApo 60x 0.40 oil objective and a Sensicam QE (Cooke) digital camera using IPLab 3.6.3 software (Scanalytics, Inc.) at room temperature. Images were imported into Photoshop 7.0 software (Adobe Systems, Inc.) for figure use, and contrast was adjusted to approximate the original IPLab image.
Immunoblots, cell lysates, immunoprecipitations, pulse-chase studies
We used the Bradford protein assay (Bio-Rad Laboratories) for protein quantification and CompleteTM protease inhibitor cocktail (Roche Diagnostics) for protease inhibition. For immunoprecipitation experiments, tubes were pretreated with 10 mg/ml BSA and preclear was with 15 µl of protein G agarose for 30 min at 4°C. Immunoblots were performed using standard protocols and the following antibodies: rabbit anti-PEX19 antibodies (Sacksteder et al., 2000), rabbit anti-PEX5 antibodies (Dodt et al., 1995), rabbit anti-c-myc antibodies (Santa Cruz Biotechnology, Inc.), and mouse monoclonal antiVSV-G antibodies obtained from C. Machamer (The Johns Hopkins University School of Medicine). Rabbit anti-PEX13 antibodies were generated against the COOH-terminal 13 aa of human PEX13.
For comparison of cytosolic PMP levels, PEX3-deficient PBD400-TI cells were transfected with the appropriate PMP expression plasmid and pcDNA3-PEX19. PEX19-deficient PBD399-TI cells were transfected with the appropriate PMP expression plasmid and vector alone. 1 d later, cells were washed in PBS, lysed by harsh resuspension in 400 µl hypotonic lysis buffer (10 mM Tris HCl, pH 7.5, 1 mM EDTA, protease inhibitors), and centrifuged at 200,000 g for 30 min. Equal amounts of total protein from supernatant were analyzed by immunoblot.
For immunoprecipitations, PBD400-TI cells were transfected with the appropriate PMP or mPTS expression plasmid and either pN3xHA-PEX19 or pcDNA3-PEX19. Cells were washed, lysed, and centrifuged as in the previous paragraph. Equal total protein from each supernatant was brought to a final volume of 1 ml with PBS. Lysates were precleared and samples were incubated for 3 h at 4°C with 15 µl of anti-HA mAb agarose beads (Santa Cruz Biotechnology, Inc.). Beads were washed three times in PBS and PMPs were detected by immunoblot.
For the determination of PEX19's affect on the stability of PMP34/13xmyc, PEX3-deficient PBD400-TI cells were transfected with pcDNA3-PMP34/13xmyc and pcDNA3-PEX19, whereas PEX19-deficient PBD399-TI cells were transfected with pcDNA3-PMP34/13xmyc and vector alone. Cells were divided into five equal fractions, incubated overnight in complete medium, incubated for 1 h in methionine-free medium, pulsed for 15 min with 2 ml of methionine-free medium containing 0.5 mCi 35S-labeled methionine per fraction, and chased with excess cold methionine. Cells were washed in cold PBS, lysed, and centrifuged as above. The entire supernatant from each sample was brought to 1 ml with PBS plus 0.5% Triton X-100. Lysates were precleared then incubated for 3 h at 4°C with 15 µl of anti-myc antibody agarose beads (Santa Cruz Biotechnology, Inc.). Beads were washed three times in PBS plus 0.5% Triton X-100 and analyzed by autoradiography and immunoblot.
For the detection of PEX19PMP interaction during PMP biogenesis, PEX19-deficient PBD399-TI cells were made to stably express 3xHA-PEX19 by transfection with pN3xHA-PEX19 and selection with 400 µg/ml G418. Cells were transfected with pcDNA3-PMP34/13xmyc, separated into eight equal fractions, incubated overnight in complete medium, incubated in methionine-free medium for 1 h, pulsed for 10 min with 0.66 mCi per sample, and chased with excess methionine. Cells were washed twice in ice-cold PBS and resuspended in cold STE (250 mM sucrose, 10 mM Tris, pH 7.5, 1 mM EDTA, protease inhibitors). Cells were lysed using a ball bearing homogenizer and centrifuged at 3,000 g for 5 min. Supernatants were centrifuged at 25,000 g for 30 min and the resulting supernatants were diluted 1:1 in PBS and centrifuged at 200,000 g for 30 min. The resulting supernatants were precleared and the samples were incubated with 15 µl of anti-HA antibody beads for 16 h at 4°C. Beads were pelleted, washed in PBS, resuspended in 100 µl of release buffer (1% SDS, 50 mM Tris, pH 7.5, and 1 mM EDTA), and heated in boiling water for 10 min. The beads were pelleted and the supernatant was brought to 1 ml in 2% BSA, 50 mM Tris, pH 7.5, 1 mM EDTA. Samples were incubated with 15 µl of anti-myc antibody beads for 3 h at 4°C. The beads were pelleted, washed once in 0.1% SDS, 2% BSA, 50 mM Tris, pH 7.5, 1 mM EDTA, twice in PBS plus 1% Triton X-100, and analyzed by autoradiography and immunoblot.
Studies using siRNA-mediated inhibition
The following RNA oligonucleotide pairs (Dharmacon Research, Inc.) were used in the creation of siRNA duplexes according to the manufacturer's instructions: PEX19, 5'-GAGAUCGCCAGGAGACACUdTdT-3', 5'-AGUGUCUCCU-GGCGAUCUCdTdT-3'; PEX5, 5'-AGAAGCUACUCCCAAAGGCdTdT-3', 5'-GCC-UUUGGGAGUAGCUUCUdTdT-3'; TRIP8b, 5'-GCAGGGAAAAGGCUCUAG-GdTdT-3', 5'-CCUAGAGCCUTTTCCCUGCdTdT-3'. 50 µl of a 20 µM RNA duplex stock in annealing buffer (100 mM KAc, 30 mM Hepes-KOH, pH7.4, and 2 mM MgAc) was mixed with normal human fibroblasts (GM 5756-TI) from a confluent 75 cm2 flask in 500 ml Hepes buffered saline (21 mM Hepes, pH 7.15, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM dextrose). Cells were electroporated (model BTX ECM600; Genetronics) at 220 V, 1,500 µF, 129 . For characterization of siRNA effect on cell phenotype, cells were grown for 5 d after siRNA treatment. Every 24 h, samples of cells were either fixed in 3% formaldehyde or stored at 20°C for study by immunofluorescence or immunoblot.
For assay of peroxisomal protein import in siRNA-treated cells, cells were treated a second time with siRNA at 16 h after initial treatment to optimize treatment efficiency. At 60 h after initial siRNA treatment, cells were transfected with pNHA-PTE1 and either pcDNA3-PMP34myc, pcDNA3-PMP34aa244-307/3xmyc, pcDNA3-PEX16aa221-336/3xmyc, or pcDNA3-PEX3aa1-50/6xmet3xmyc. 100 min after this transfection cells were processed for immunofluorescence. For comparison of PEX19 and PEX5 siRNA effects on import, all cells showing import of either marker were scored as to whether they imported HA-PTE1, PMP34myc, or both. For the comparison of PEX19 siRNA effects on mPTS targeting, cells importing HA-PTE1 were scored as to whether the mPTS-containing protein was imported into peroxisomes, was only seen in nonperoxisomal compartments, or was not seen. This methodology ensured that only those cells with intact, import-competent peroxisomes were scored.
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Acknowledgments |
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Submitted: 25 April 2003
Accepted: 15 October 2003
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