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1.
Figure 1

Figure 1. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Nucleotide sequence of the PEX20 gene and deduced amino acid sequence of Pex20p. These sequence data are available from EMBL/GeneBank/DDBJ under accession number AF054613.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
2.
Figure 4

Figure 4. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Ultrastructure of wild-type and pex20 mutant strains. Wild-type strain E122 (WT) and mutant strains pex20-1 and pex20KO grown for 9 h in YPBO were fixed in KMnO4 and processed for EM. P, peroxisome; M, mitochondrion; N, nucleus. Arrowheads, peroxisomes having a “relative area of peroxisome section (%)” of 0.05–0.2%. Bar, 1 μm.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
3.

Figure 8. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Double-labeling, indirect immunofluorescence analysis of wild-type and mutant strains. Wild-type strain E122 (WT) and mutant strains pex20-1 and pex20KO were grown in YPBO. Cells were processed for immunofluorescence microscopy with guinea pig anti-THI and rabbit anti-SKL; rabbit anti-MLS and guinea pig anti–SKL; guinea pig anti-Pex2p and rabbit anti-SKL; and rabbit anti-MLS and guinea pig anti-Pex2p primary antibodies. Primary antibodies were detected with fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated donkey anti-guinea pig IgG secondary antibodies. Arrows in B indicate punctate structures in pex20KO cells that are decorated with anti-MLS (leftmost panels) or anti-Pex2p (middle panels) antibodies but not with anti-SKL antibodies.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
4.
Figure 7

Figure 7. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Isolation of LSP peroxisomes from wild-type and mutant strains. Wild-type strain E122 (WT) and mutant strains pex20-1 and pex20KO were grown in YPBO. Peroxisomes were isolated from the 20KgP of each strain by isopycnic centrifugation on a discontinuous sucrose density gradient. Equal amounts of protein were loaded onto each gradient. Sucrose density (g/cm3) of gradient fractions, and the percent recovery of loaded protein and of CAT (peroxisome) and cytochrome c oxidase (CCO) (mitochondria) marker enzyme activities in gradient fractions are presented. Equal volumes of gradient fractions were analyzed by immunoblotting with antibodies to peroxisomal matrix (AOX, ICL, SKL, MLS, THI) and membrane (Pex2p) proteins.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
5.

Figure 5. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Subcellular localization of peroxisomal matrix and membrane proteins in wild-type and pex20 mutant strains. The recoveries of the matrix proteins AOX, 62-kD SKL, 64-kD SKL, ICL, CAT, MLS, and THI, and of the membrane proteins Pex2p and Pex16p, in different subcellular fractions of the wild-type strain E122 (WT) and of the mutant strains pex20-1 and pex20KO are presented. Strains were grown for 9 h in YPBO and subjected to subcellular fractionation. Equal portions of the PNS, 20KgP, 20KgS, 200KgP, and 200KgS were analyzed by immunoblotting with antibodies to the indicated proteins. The activity of CAT was assayed enzymatically. Values for CAT activity in the 20KgP, 20KgS, 200KgP, and 200KgS fractions are relative to CAT activity in the PNS fraction of the indicated strain. Values for enzymatic activities are the means ± SD from three independent experiments.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
6.
Figure 10

Figure 10. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Pex20p and THI in the 200KgP fraction do not form a complex. Wild-type E122 cells grown in YPBO for 9 h were pulse labeled with l-[35S]methionine and chased for 5 min with unlabeled l-methionine. Cells were subjected to subcellular fractionation, and the 200KgP fraction was divided into equal aliquots that were subjected to immunoprecipitation under native conditions with anti-Pex20p (1) or anti-THI (6) antibodies. Proteins that did not bind to protein A–Sepharose and were recovered in the flow-through after native immunoprecipitation with either anti-Pex20p or anti-THI antibodies were subjected to a second immunoprecipitation under denaturing conditions using anti-Pex20p (2, 7), anti-THI (3, 8), anti-SKL (4, 9) or anti-MLS (5, 10) antibodies. These immunoprecipitates were resolved by SDS-PAGE and were analyzed by fluorography and immunoblotting with the indicated antibodies.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
7.
Figure 3

Figure 3. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Trafficking of Pex20p. (A) Wild-type strain E122 grown in YPBO for 9 h was pulse-labeled with l-[35S]methionine and chased with unlabeled l-methionine. Samples were taken at the indicated times after chase. Cells were subjected to subcellular fractionation to yield 20KgP, 200KgP, and 200KgS (cytosolic) fractions. Pex20p was immunoprecipitated from the fractions. Immunoprecipitates were resolved by SDS-PAGE and visualized by fluorography. (B) Equal portions of the 200KgP fraction isolated from YPBO-grown wild-type cells pulse-labeled with l-[35S]methionine and chased for 5 min with unlabeled l-methionine, were incubated with 0, 4, 8, or 20 μg trypsin in the absence (−) or presence (+) of 1.0% (vol/vol) Triton X-100 for 60 min on ice. Reactions were terminated by addition of TCA to 10%. Samples were subjected to immunoprecipitation with anti-Pex20p or anti-THI antibodies, and immunoprecipitates were resolved by SDS-PAGE and then visualized by fluorography.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
8.

Figure 6. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Trafficking of peroxisomal matrix and membrane proteins in wild-type and pex20KO cells. Wild-type strain E122 (WT) (A and C) and mutant strain pex20KO (B) grown in YPBO for 9 h were pulse labeled with l-[35S]methionine and chased with unlabeled l-methionine. Samples were taken at the indicated times after chase. Cells were subjected to subcellular fractionation to yield 20KgP, 200KgP, and 200KgS (cytosolic) fractions. AOX, MLS, Pex2p, and THI were immunoprecipitated from the fractions. Immunoprecipitates were resolved by SDS-PAGE and visualized by fluorography. Fluorograms were quantitated by densitometry. The maximal level of a protein in a particular fraction is set at 100%. Half-times for the exit from, and for the import into, a particular fraction by a protein in either wild-type or pex20KO cells were calculated. (C) Conversion of the precursor form of THI (pTHI) to the mature form of THI (mTHI) occurs in the 200KgP fraction of wild-type cells. 200KgP fractions were isolated from wild-type cells subjected to pulse labeling with l-[35S]methionine and chase with unlabeled l-methionine. pTHI and mTHI were immunoprecipitated from the 200KgP fractions isolated from cells harvested at the indicated times after chase, resolved by SDS-PAGE, and then visualized by fluorography.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
9.

Figure 12. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

The oligomerization of ICL and AOX is unaffected in the pex20KO mutant. The wild-type strain E122 and the mutant strain pex20KO grown in YPBO for 9 h were pulse labeled with l-[35S]methionine and chased with unlabeled l-methionine. One aliquot of cells of each strain was taken at chase time 0 and then subjected to subcellular fractionation to yield a 200KgS (cytosolic) fraction. The second aliquots of the wild-type and pex20KO mutant cells were chased for 30 min, and LSP peroxisomes were isolated from the 20KgP fraction. Cytosolic and peroxisomal proteins were treated with increasing concentrations of the crosslinkers DSS (A, B, G, and H) or DSP (C–F, I–L) and subjected to immunoprecipitation with anti-ICL (A–F) or anti-AOX (G–L) antibodies under denaturing conditions. Immunoprecipitates were resolved by SDS-PAGE and subjected to fluorography. Immunoprecipitated proteins treated with the noncleavable crosslinker DSS were analyzed by SDS-PAGE only under reducing conditions (A, B, G, and H). Immunoprecipitated proteins treated with the thiol-cleavable cross-linker DSP were analyzed by SDS-PAGE under nonreducing (C, E, I, and K) and reducing (D, F, J, and L) conditions. The positions of the monomeric and oligomeric forms of ICL and AOX are indicated by arrowheads (right).

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
10.

Figure 11. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Mutations in the PEX20 gene affect oligomerization of THI in the cytosol and prevent the formation of a heterotetrameric complex between Pex20p and THI. Wild-type strain E122 and mutant strains pex20-1 and pex20KO grown in YPBO for 9 h were pulse labeled with l-[35S]methionine for 1.5 min and chased with unlabeled l-methionine. 200KgS (cytosolic) fractions were isolated from each strain at chase time 0. A 200KgP fraction (200KgP) enriched for HSP peroxisomes was isolated from wild-type cells chased for 7 min. LSP peroxisomes (Peroxisome) were isolated from the 20KgP of wild-type cells chased for 30 min. Cytosolic and peroxisomal proteins were treated with increasing concentrations of the cross-linkers DSS (A–D, F–H, J) or DSP (E, I, K) and subjected to immunoprecipitation with anti-THI (A, E, F–K) or anti-Pex20p (C) antibodies under denaturing conditions. Immunoprecipitates were resolved by SDS-PAGE and subjected to fluorography. Proteins that did not bind to protein A–Sepharose during immunoprecipitation of DSS-treated cytosolic proteins from wild-type cells with anti-THI (A) or anti-Pex20p (C) antibodies, were further immunoprecipitated with anti-Pex20p (B) or anti-THI (D) antibodies under denaturing conditions. Immunoprecipitates were resolved by SDS-PAGE and subjected to fluorography. Immunoprecipitated proteins treated with the noncleavable crosslinker DSS were analyzed by SDS-PAGE only under reducing conditions (A–D, F–H, J), whereas immunoprecipitated proteins treated with the thiol-cleavable cross-linker DSP were analyzed by SDS-PAGE under both nonreducing (E, I, K, left panels) and reducing (E, I, K, right panels) conditions. The positions of the monomeric and oligomeric forms of THI, of the monomeric form of Pex20p, and of the complex between Pex20p and THI are indicated by arrowheads (right).

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
11.
Figure 9

Figure 9. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

The association of Pex20p with newly synthesized THI in the cytosol is independent of the PTS2 of THI. (A) Wild-type E122 cells grown in YPBO for 9 h were pulse-labeled with l-[35S]methionine for 1.5 min and immediately subjected to subcellular fractionation to yield a 200KgS (cytosolic) fraction. The 200KgS fraction was divided into three equal aliquots, and Pex20p (1), THI (6), and anti-SKL–reactive proteins (11) were immunoprecipitated under native conditions. Immunoprecipitated proteins were eluted from protein A–Sepharose with 100 mM glycine, pH 2.8, and eluates were each divided into equal aliquots that were resolved by SDS-PAGE. One aliquot was subjected to fluorography, whereas the other four were analyzed by immunoblotting with anti-Pex20p, anti-THI, anti-SKL, or anti-MLS antibodies. Unbound proteins recovered in the flow-through after native immunoprecipitation with anti-Pex20p, anti-THI, and anti-SKL antibodies were subjected to a second immunoprecipitation under denaturing conditions using anti-Pex20p (2, 7, 12), anti-THI (3, 8, 13), anti-SKL (4, 9, 14) or anti-MLS (5, 10, 15) antibodies. Immunoprecipitates were divided into equal aliquots that were resolved by SDS-PAGE. One aliquot was subjected to fluorography, whereas the other four were analyzed by immunoblotting using the antibodies indicated. (B) The wild-type strain E122 and the mutant strain pex5KO grown in YPBO for 9 h were pulse labeled with l-[35S]methionine and chased for 30 min with unlabeled l-methionine. Cells were subjected to subcellular fractionation and peroxisomes were purified from the 20KgP fraction. Pulse-labeled and chased peroxisomal matrix proteins were subjected to immunoaffinity chromatography with anti-THI antibodies linked to protein A–Sepharose. Immunoprecipitated mature (mTHI) and precursor (pTHI) forms of THI were eluted with 100 mM glycine, pH 2.8, and the eluates were divided into equal aliquots. One aliquot of each eluate was resolved by SDS-PAGE and subjected to fluorography (B), whereas the other aliquots were used as probes in a blot overlay assay. (C) Unlabeled proteins from the 200KgS fraction of wild-type cells were divided into equal aliquots. One aliquot was subjected to depletion of Pex20p by native immunoprecipitation with Pex20p antibodies (pre-IP: anti-Pex20p), whereas the second aliquot remained untreated. Unbound cytosolic proteins recovered in the flow-through after native immunoprecipitation with anti-Pex20p, unlabeled proteins from the untreated 200KgS fraction of wild-type cells, and unlabeled proteins from the 200KgS fractions of pex20-1 and pex20KO cells were divided into equal aliquots, resolved by SDS-PAGE, and transferred to nitrocellulose. After denaturation/renaturation of the transferred proteins, the blots were subjected to an overlay assay using mTHI (strips 1) or pTHI (strips 2) as probes. One set of blots was subjected to fluorography (C), whereas a second set was used for stripping of bound mTHI and pTHI with urea (D). (D) Radiolabeled mTHI and pTHI (lanes 3 and 4, respectively) bound to proteins present only in the untreated 200KgS fraction of the wild-type strain and stripped with urea, were resolved by SDS-PAGE and subjected to fluorography.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.
12.

Figure 2. From: Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome .

Subcellular localization and regulation of synthesis of Pex20p. (A) Immunoblot analysis of whole cell lysates (3 × 108 cells) of wild-type strain E122 (WT), and pex20-1 and pex20KO mutant strains probed with anti-Pex20p antibodies. Strains were grown in YPBO for 9 h. (B) Wild-type strain E122 was grown in YEPD until the cell titer was 6.0 × 107 cells/ ml. Cells were transferred to YPBO and incubated at 30°C. Equal aliquots of cells were taken at the times indicated. The levels of Pex20p in whole cell lysates were determined by immunoblotting. Blots were probed with anti-Pex20p antibodies. (C) Equal portions of the indicated subcellular fractions from YPBO-grown wild-type cells were separated by SDS-PAGE and analyzed by immunoblotting with anti-Pex20p antibodies. (D) Release of intracellular proteins by progressive digitonin titration of YPBO-grown wild-type cells. Aliquots of spheroplasts were incubated with different concentrations of digitonin for 30 min at 4°C and then subjected to centrifugation at 20,000 g for 10 min at 4°C. Supernatants were analyzed for the release of proteins. The activities of the peroxisomal enzyme catalase (CAT) and the mitochondrial enzyme fumarase (FUM) were determined. The cytosolic marker G6PDH, the peroxisomal matrix marker THI, and Pex20p were determined by immunoblotting. Immunoblots were scanned densitometrically, and protein levels were quantitated. The amount of each protein released to the supernatant was normalized to the amount of that protein released to the supernatant at a concentration of 1,000 μg digitonin/ml. (E) Double-labeling, indirect immunofluorescence analysis of YPBO-grown wild-type cells using rabbit anti-THI and guinea pig anti-Pex20p primary antibodies. Primary antibodies were detected with fluorescein-conjugated goat anti–rabbit IgG and rhodamine-conjugated donkey anti–guinea pig IgG secondary antibodies. (F) The 200KgP fraction of the wild-type strain E122 grown in YPBO for 9 h was subjected to flotation on a two-step sucrose gradient. Sucrose density (g/cm3) and percent recovery of loaded protein in gradient fractions are presented. Equal volumes of gradient fractions were analyzed by immunoblotting with anti-AOX, anti-THI, and anti-Pex20p antibodies. (G) Equal portions of the 200KgP fraction of the wild-type strain E122 grown in YPBO for 9 h were treated with one of Ti8 buffer (10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM PMSF, 1 μg leupeptin/ml, 1 μg pepstatin/ml, 1 μg aprotinin/ml), 1 M NaCl, 1 M urea or 0.1 M Na2CO3. After incubation on ice for 45 min, samples were separated into supernatant (S) and pellet (P) fractions by centrifugation at 200,000 g for 1 h at 4°C, and then subjected to immunoblot analysis with anti-Pex20p antibodies. (H) Protease protection analysis. The 200KgP fraction (60 μg of protein) of the wild-type strain E122 grown in YPBO for 9 h was incubated with 0, 4, 8, or 20 μg trypsin in the absence (−) or presence (+) of 1.0% (vol/vol) Triton X-100 for 60 min on ice. Reactions were terminated by addition of TCA to 10%. Equal portions of the samples were subjected to immunoblot analysis with anti-Pex20p and anti-THI antibodies.

Vladimir I. Titorenko, et al. J Cell Biol. 1998 July 27;142(2):403-420.

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