Pex19 is a PMP chaperone. (A) PEX19 expression results in increased abundance of PMPs in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34myc, PEX11βmyc, PMP24/3xmyc, VSV-G, mycPTE1, and PEX19 (+PEX19). PEX19-deficient human fibroblasts were transfected with plasmids designed to express the same test proteins and vector alone (−PEX19). Equal total protein from a membrane-free lysate of each cell sample was analyzed by immunoblot using antibodies to the c-myc epitope or to VSV-G as appropriate. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 and either PMP34myc, PEX11βmyc, or PMP24/3xmyc were processed for indirect immunofluorescence using antibodies to the c-myc epitope (left) or PEX19 (right). Bar, 15 μM. (C) PEX19 stabilizes PMP34/13xmyc in the cytosol. PEX3-deficient human fibroblasts were transfected with plasmids designed to express PMP34/13xmyc and PEX19 (+PEX19); PEX19-deficient human fibroblasts were transfected with a plasmid designed to express PMP34/13xmyc and empty vector (−PEX19). Cells were pulsed for 15 min with [³⁵S]methionine and chased with excess cold methionine for the times indicated. A membrane-free cell lysate of each sample was subjected to immunoprecipitation with anti-myc antibodies. Equal fractions of each IP were analyzed by autoradiography (³⁵S) and immunoblot using anti-myc antibodies (anti-myc). (D) Relative ³⁵S-signal intensities for each time point in B versus time with best fit exponential curves. (□, solid line), +PEX19; (♦, dashed line), −PEX19; t_{1/2 (+PEX19)} = 300 min; t_{1/2 (−PEX19)} = 15 min. (E) PEX19 binds multiple PMPs in the cytosol. PEX3-deficient human fibroblasts transfected with plasmids designed to express PMP34/13xmyc, PEX11βmyc, PMP24/3xmyc, and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with anti-HA antibodies. IPs were analyzed by immunoblot using anti-myc antibodies. (F) The majority of cytosolic PMP is bound to PEX19. Equal amounts of lysates of 3xHA-PEX19–expressing cells from E sampled before (Pre-IP) and after (Post-IP) immunoprecipitation were analyzed by immunoblot using anti-myc antibodies. (G) PEX19 interacts with newly synthesized PMPs. PEX19-deficient human fibroblasts stably expressing 3xHA-PEX19 were transfected with a plasmid designed to express PMP34/13xmyc. Cells were pulsed with [³⁵S]methionine for 10 min, chased with excess methionine for the indicated times, and split into two fractions. Membrane-free lysates of cells in the first fraction were subjected to immunoprecipitation with anti-HA antibodies. The samples were solubilized in SDS and subjected to a second immunoprecipitation with anti-myc antibodies. Immunoprecipitations were analyzed by autoradiography (PEX19-associated, ³⁵S) and immunoblot using anti-myc antibodies (PEX19-associated, anti-myc). Cells from the second fraction were solubilized in 1% Triton X-100 and subjected to immunoprecipitation using anti-myc antibodies; and PMP34/13xmyc was detected by autoradiography (whole-cell IP, ³⁵S). For PEX19-PMP34/13xmyc association, t_1/2 = 15 min. For the lifetime of PMP34/13xmyc in the whole cell, t_1/2 = 300 min.

PEX19 binds the transmembrane region of the PMP34 mPTS. PEX3-deficient human fibroblasts expressing 3xNLS-PEX19 and myc-tagged mutant forms of the COOH-terminal PMP34 mPTS were processed for indirect immunofluorescence using antibodies to the myc epitope and PEX19. (A, left) Diagram showing amino acid range of each truncation mutant studied. Darkened regions indicated relative position of the putative transmembrane domain. Light vertical bars indicate relative positions of the engineered point mutations. All proteins fused to three tandem c-myc epitopes at the COOH terminus. (Right) Bar graph of relative binding efficiency of each mutant. Relative binding efficiency is the proportion of cells expressing both 3xNLS-PEX19 and PMP34 mPTS mutant in which the PMP34 mPTS mutant is seen in the nucleus. n = 100 cells for each sample. Values are normalized. (B) Amino acids 270-307 of PMP34. Putative transmembrane domain is boxed, sites of engineered point mutations are in bold and underlined. (C) Immunofluorescence images of cells expressing selected PMP34 mPTS mutants from the experiment in A. (Left) Anti-myc staining; (right) anti-PEX19 staining. Bar, 15 μM.

Inhibition of PEX19 causes a specific PMP import defect. (A) Inhibition of PEX19 disrupts import of PMP34 but not import of the peroxisomal matrix protein, PTE1. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA (top), PEX19 siRNA (middle), or PEX5 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and PMP34myc, and processed for indirect immunofluorescence with antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells showing peroxisomal import were counted and scored for import of HA-PTE1 or PMP34myc (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 314; PEX19 siRNA n = 303; PEX5 siRNA n = 67. Bar, 15 μM. (B) Inhibition of PEX19 disrupts mPTS targeting. Wild-type human fibroblasts were subjected to electroporation with TRIP8b siRNA or PEX19 siRNA followed by transfection with plasmids designed to express HA-PTE1 and either PMP34aa244-307/3xmyc (top) or PEX16aa221-336/3xmyc (bottom). Cells were processed for immunofluorescence using anti-HA (left) or anti-myc (middle) antibodies. Cells that imported HA-PTE1 into peroxisomes were scored as to whether PMP34aa244-307/3xmyc or PEX16aa221-336/3xmyc was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. PMP34aa244-307/3xmyc: TRIP8b siRNA n = 306, PEX19 siRNA n = 305; PEX16aa221-336/3xmyc: TRIP8b siRNA n = 301, PEX19 siRNA n = 306. Bar, 15 μM.

PEX19 binds multiple PMP targeting signals. (A) Multiple mPTSs coprecipitate with PEX19. PEX3- deficient human fibroblasts were transfected with plasmids designed to express PMP34aa244-307/3xmyc, PEX11βaa181-259/3xmyc, PEX16aa221-336/3xmyc, PEX16aa59-219/3xmyc, PMP22aa1-94/3xmyc, PMP22aa95-195/3xmyc, PMP70aa1-61/13xmyc, PMP70aa61-160/13xmyc, PMP70aa1-124/3xmyc, and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with anti-HA antibodies. IPs were analyzed by immunoblot using anti-myc antibodies. (B) PEX3-deficient human fibroblasts transfected with plasmids designed to express PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using antibodies against PEX19. Bar, 15 μM. (C) PEX19 controls the subcellular distribution of multiple mPTSs. PEX3- deficient human fibroblasts transfected with plasmids designed to express the mPTSs from A and either PEX19 (left) or 3xNLS-PEX19 (right) were processed for indirect immunofluorescence using anti-myc antibodies. Bar, 15 μM.

Peroxisomes persist for multiple days after inhibition of PEX19. (A) Treatment with PEX19 and PEX5 siRNA mediates specific reductions in PEX19 and PEX5 levels. Wild-type human fibroblasts were subjected to electroporation with PEX19 siRNA (top) or PEX5 siRNA (bottom). Equal amounts of protein from daily cell samples after siRNA treatment were separated by SDS-PAGE and processed for immunoblot using antibodies to PEX19, PEX5, and PEX13. (B) Peroxisomes persist for >3 d despite inhibition of PEX19. Daily cell samples after treatment with PEX19 siRNA (top) or PEX5 siRNA (bottom) were processed for indirect immunofluorescence using antibodies to the peroxisomal membrane marker PMP70. Bar, 15 μM.

The mPTS of PEX3 functions via a PEX19-independent pathway. (A) The first 50 aa of PEX3 are sufficient for peroxisomal targeting. Wild-type human fibroblasts were transfected with a plasmid designed to express the PEX3 mPTS (PEX3aa1-50/6xmet3xmyc) and cells were processed for immunofluorescence using antibodies to the c-myc epitope (left) or PMP70 (right). Bar, 15 μM. (B) The mPTS of PEX3 does not coprecipitate with PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either 3xHA-PEX19 (+3xHA-PEX19) or PEX19 (+PEX19). Equal total protein from a membrane-free lysate of each cell sample was subjected to immunoprecipitation with antibodies to the HA epitope. IPs (top) and whole cell lysates (bottom) were processed for immunoblot using anti-myc antibodies. (C) The subcellular distribution of the PEX3 mPTS is not affected by PEX19. PEX3-deficient human fibroblasts were transfected with plasmids designed to express the PEX3 mPTS and either PEX19 (top) or 3xNLS-PEX19 (bottom). Cells were processed for indirect immunofluorescence using anti-myc (left) or anti-PEX19 (right) antibodies. Bar, 15 μM. (D) Inhibition of PEX19 does not affect peroxisomal targeting of the PEX3 mPTS. Wild-type human fibroblasts were subjected to electroporation with either TRIP8b siRNA (top) or PEX19 siRNA (bottom). Cells were transfected with plasmids designed to express HA-PTE1 and the PEX3 mPTS. Cells were processed for immunofluorescence using antibodies to the HA epitope (left) or the c-myc epitope (middle). Cells importing HA-PTE1 were scored as to whether the PEX3 mPTS was seen in peroxisomes, seen only in nonperoxisomal compartments, or not seen (right). Means and SD of three independent trials are presented. TRIP8b siRNA n = 311; PEX19 siRNA n = 294. Bar, 15 μM.