The effect of PEX3 on the localization of 3xHA-PEX19. (A) GM5756-TI cells were transfected with TRIP8bsiRNAs, PEX3siRNAs, or PEX5siRNAs, respectively. Cells were lysed each day following the siRNA treatment, and equivalent amounts of protein from each sample were processed for immunoblot with antibodies against PEX3 and PEX5. (B) On day 2 after the siRNA treatment, cells were processed for double indirect immunofluorescence microscopy with anti-PEX3 and anti-PMP70 antibodies. (C and D) 3xHA-PEX19/PBD399-TI cells (untreated) and 3xHA-PEX19/PBD399-TI cells transfected with TRIP8bsiRNAs or PEX3siRNAs were grown for 2 d and processed for double indirect immunofluorescence microscopy with antibodies against (C) the HA epitope and PEX3, or (D) the HA epitope and PMP70. Bars, 20 μm.

Characterization of the docking domain of PEX19. (A–D) GM5756-TI cells were transfected with (A and B) the 3xHA-PEX19aa1–56 or (C and D) 3xHA-PEX19aa57–299 expression plasmids, respectively, grown overnight and processed for double indirect immunofluorescence using (A and C) anti-HA and (B and D) anti-PMP70 antibodies. (E) PBD399-TI cells were cotransfected with the plasmids designed to express either 3xNLS-PEX19aa1–56–3xHA or 3xNLS-PEX19aa57–299–3xHA, together with plasmids designed to express PMP22aa1–94–3xmyc, PMP34aa244–307–3xmyc, PEX16aa221–336–3xmyc, and PEX3–3xmyc. 24 h after the transfection, cells were processed for double indirect immunofluorescence using anti-HA and anti-myc antibodies. Bars, 20 μm.

Characterization of one PEX19-interacting motif of PEX3. (A) Selected segments of PEX3 tagged with 3xmyc were coexpressed in PBD399-TI cells with either 3xNLS-PEX19 or 3xNLS-HAOX3 followed by double indirect immunofluorescence microscopy. The interaction strength was calculated by counting cells in which PEX3 segments accumulated in the nucleus (++++, >80% cells concentrated the PEX3 segment in the nucleus; +++, 60–79%; ++, 40–59%; +, 15–39%; +/−, <15%; −, 0%). (B–E) PBD399-TI cells were cotransfected with plasmids to express (B, C) 3xmyc-PEX3aa120–136 and 3xNLS-PEX19, or (D, E) 3xmyc-PEX3aa120–136 and 3xNLS-HAOX3. 24 h after the transfection, cells were processed for double indirect immunofluorescence microscopy with (B) anti-PEX19 and (C) anti-myc antibodies, or (D) anti-HAOX3 and (E) anti-myc antibodies. (F–I) PBD399-TI cells were cotransfected with plasmids designed to express (F and G) 3xNLS-PEX19 and PEX3L125P/N134D-3xmyc, or (H and I) 3xNLS-PEX19 and PEX3–3xmyc, grown overnight, and processed for double indirect immunofluoresence microscopy with (F and H) anti-PEX19 and (G and I) anti-myc antibodies. (J–M) GM5756-TI cells were transfected with (J and K) pcDNA3-PEX3L125P/N134D-3xmyc or (L and M) pcDNA3-PEX3–3xmyc, respectively, and grown overnight. The localization of PEX3L125P/N134D-3xmyc and PEX3–3xmyc was examined by double indirect immunofluoresence with antibodies against (J and L) the myc epitope and (K and M) endogenously expressed PMP70. Bar, 20 μm.

The effect of PEX3 depletion on class I and II PMP import. (A) GM5756-TI cells were electroporated with PEX3, TRIP8b, or PEX5 siRNAs, respectively, grown for 2–3 d, retransfected with a pair of plasmids designed to express (a) HA-PTE1 and (b) each of the following myc-tagged proteins: PMP34myc, PEX11βmyc, the mPTS of PEX16 (PEX16aa221–336–3xmyc), and PEX3(PEX3aa1–50–6xmet3xmyc). 2 h later, cells were processed for double indirect immunofluoresence microscopy with antibodies against the HA and myc epitopes. Bar, 20 μm. (B–D) Cells showing peroxisomal staining (import of either marker) were counted as to whether they imported HA-PTE1 only (matrix only), PMPs-myc only (membrane only), or both (both) in the populations transfected with (B) PEX3siRNAs, (C) TRIP8bsiRNAs, and (D) PEX5siRNAs. Each experiment was done in triplicate and the mean and standard deviation are presented.

The effect of PEX3 activity on PMP import. A pex3-null mutant that constitutively expresses CFP-PTS1 and (A) WT PEX3 allele or (B) pex3-G6 allele was transformed with plasmids designed to express (a–f) YFP-PTS1, (g–l) YFP-PEX10, (m–r) ANT1-YFP, or (s–x) YFP-PEX15 under the control of galactose. The resulting strains were grown on minimal glucose medium at 25°C, transferred to minimal galactose medium, and maintained at (a–c, g–i, m–o, and s–u) 25°C, or shifted to (d–f, j–l, p–r, and v–x) 37°C for 2.5–4 h, followed by examination by phase contrast (a, d, g, j, m, p, s, and v), and epifluorescence microscopy specific for (b, e, h, k, n, q, t, and w) CFP and (c, f, i, l, o, r, u, and x) YFP. Bars, 5 μm.

The localization of 3xHA-PEX19 in other human pex mutants. Human fibroblasts from peroxisome biogenesis disorder patients that carry inactivating mutations in the PEX1, PEX2, PEX5, PEX6, PEX7, PEX10, PEX12, PEX13, and PEX26 genes were transfected with 3xHA-PEX19 expression plasmid, grown overnight, and processed for indirect immunofluorescence microscopy with anti-HA and anti-PMP70 antibodies. GM5756-TI cells were also transfected with PEX16siRNAs and the 3xHA-PEX19 expression plasmid. 2 d after the transfection, at a time when PEX16 activity is impaired (unpublished data), these cells (PEX16-) were subjected to double indirect immunofluorescence microscopy using anti-HA and anti-PMP70 antibodies. Bar, 20 μm.

The effect of PEX3 depletion on the localization of the PEX19 docking domain. GM5756-TI cells were cotransfected with a plasmid designed to express 3xHA-PEX19aa1–56 and siRNAs specific for either (A and B) TRIP8b or (C–F) PEX3. On day 2 after transfection, cells were processed for double indirect immunofluorescence microscopy with (A and C) anti-HA antibody and (B and D) anti-PEX3 antibodies. The same samples were also processed for double indirect immunofluorescence assay with (E) anti-HA and (F) anti-PMP70 antibodies. Bar, 20 μm.

The effect of PEX3L125P/N134D on PEX19 docking. PBD399-TI cells were cotransfected with (A and B) pcDNA3–3xHA-PEX19aa1–56 and pcDNA3-PEX3–3xmyc or (C and D) pcDNA3–3xHA-PEX19aa1–56 and pcDNA3-PEX3L125P/N134D-3xmyc, grown overnight, and processed for double indirect immunofluorescence microscopy using (A and C) anti-myc and (B and D) anti-HA antibodies. Bar, 20 μm.

Characterization of conditional pex3 mutants. A pex3-null strain that constitutively expresses CFP-PTS1 was transformed with plasmids that express the WT PEX3 allele (a–d), pex3-A8 allele (e–h), pex3-B5 allele (i–l), or pex3-G6 allele (m–p). The resulting strains were grown at either (a, b, e, f, i, j, m, and n) 25°C or (c, d, g, h, k, l, o, and p) 37°C for 3 d, fixed, and examined by phase contrast (a, c, e, g, i, k, m, and o) and epifluorescence microscopy specific for CFP (b, d, f, h, j, l, n, and p). Bar, 5 μm.

The effect of other two conditional pex3 mutants on PMP import. A pex3-null mutant, which constitutively expresses CFP-PTS1 and (A) pex3-A8 allele or (B) pex3-B5 allele, was transformed with plasmids designed to express (a–f) YFP-PTS1, or (g–l) YFP-PEX10 under the control of galactose. After being transferred to minimal galactose medium for 3 h at 25°C, the resultant strains were either maintained at (a–c and g–i) 25°C, or shifted to (d–f and j–l) 37°C for four more hours, and examined by phase contrast (a, d, g, and j) and epifluorescence microscope specific for (b, e, h, and k) CFP and (c, f, i, and l) YFP. Bars, 5 μm. (C) All cells with peroxisomal CFP-PTS1 were scored as to whether YFP was 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 done in triplicate and the mean and standard deviation are shown.