Figure 6.
Figure 6. Pex3Bp and Pex3p interact directly with the cargo-binding tail of Y. lipolytica class V myosin. (A) Peroxisome inheritance is reduced by overexpression of the Y. lipolytica class V myosin cargo-binding tail. Wild-type strain E122 expressing genomically encoded Pot1p-GFP to fluorescently label peroxisomes was transformed with the empty plasmid pUB4 or with pUB4 expressing the globular tail domain (amino acids 1,092–1,594) of Y. lipolytica class V myosin under the control of the oleic acid–inducible POT1 promoter. Cells were grown in YPD supplemented with hygromycin B and then transferred to and incubated in oleic acid–containing YPBO supplemented with hygromycin B for 6 h. Fluorescent images of randomly chosen fields of cells were acquired as a stack by confocal microscopy and then deconvolved. Buds were sized as “small” (0–39% of mother cell volume) or “large” (40–61% of mother cell volume; see Materials and methods). The percentages of buds containing peroxisomes in each size category are presented. Quantification was performed on at least 50 budded cells from each category. Graphic results are the means and SEM of three independent experiments. Bar, 5 µm. (B) Split-ubiquitin membrane yeast two-hybrid analysis. Cells of the S. cerevisiae strain DSY-1 synthesizing Cub protein fusions to Pex3Bp or Pex3p and NubG protein fusions to Pex3Bp, Pex3p, or the globular tail of the class V myosin of Y. lipolytica (amino acids 1,092–1,594) were tested for their ability to interact with each other by a β-galactosidase filter detection assay. A positive interaction was detected by the production of blue color. The color intensities of positive (+) and negative (−) controls are indicated. (C) Glutathione sepharose beads containing GST fused to the cargo-binding tail of the class V myosin of Y. lipolytica (GST-YlMyoV); the cargo-binding tail of the class V myosin, Myo2p, of S. cerevisiae (GST-ScMyoV); or GST alone were incubated with extracts of E. coli synthesizing MBP, MBP-Pex3p, MBP-Pex3Bp, MBP-YALI0E03124p, MBP-ScInp2p, or MBP-ScVam6p. Bound proteins, as well as 10% of input proteins, were analyzed by immunoblotting with anti-MBP antibodies. Total GST-YlMyoV, GST-ScMyoV, or GST protein levels were visualized by immunoblotting with anti-GST antibodies. Arrowheads indicate MBP or MBP fusion proteins. (D) Overexpression of PEX3B delivers peroxisomes preferentially to buds. pex3BΔ cells containing peroxisomes labeled with Pot1p-GFP and the plasmid pUB4 expressing PEX3B under the control of the oleic acid–inducible POT1 promoter were grown for 16 h in YPD supplemented with hygromycin B, then transferred to oleic acid–containing YPBO supplemented with hygromycin B for 6 h, and visualized at 23°C with an LSM 510 confocal microscope specifically modified for 4D in vivo microscopy (see Materials and methods). Representative frames from Video 4 show the specific movements of peroxisomes and their inheritance from mother cell to bud. At 0 min, two large peroxisome clusters are initially located next to the mother-bud neck. By 1 h 49 min, these peroxisomes have been transferred to their respective buds, and by 4 h 5 min, the cycle is repeated, with the peroxisomes now residing in the granddaughters of the original mother cells. De novo synthesis of peroxisomes can also be detected by the reappearance of fluorescent punctae in mother cells that had transferred their original peroxisome complement to their buds. These de novo formed peroxisomes are also vectorially transferred to newly formed buds (6 h 20 min). The formation of peroxisomes and subsequent transfer to buds continued (6 h 56 min). Bar, 5 µm.

Pex3Bp and Pex3p interact directly with the cargo-binding tail of Y. lipolytica class V myosin. (A) Peroxisome inheritance is reduced by overexpression of the Y. lipolytica class V myosin cargo-binding tail. Wild-type strain E122 expressing genomically encoded Pot1p-GFP to fluorescently label peroxisomes was transformed with the empty plasmid pUB4 or with pUB4 expressing the globular tail domain (amino acids 1,092–1,594) of Y. lipolytica class V myosin under the control of the oleic acid–inducible POT1 promoter. Cells were grown in YPD supplemented with hygromycin B and then transferred to and incubated in oleic acid–containing YPBO supplemented with hygromycin B for 6 h. Fluorescent images of randomly chosen fields of cells were acquired as a stack by confocal microscopy and then deconvolved. Buds were sized as “small” (0–39% of mother cell volume) or “large” (40–61% of mother cell volume; see Materials and methods). The percentages of buds containing peroxisomes in each size category are presented. Quantification was performed on at least 50 budded cells from each category. Graphic results are the means and SEM of three independent experiments. Bar, 5 µm. (B) Split-ubiquitin membrane yeast two-hybrid analysis. Cells of the S. cerevisiae strain DSY-1 synthesizing Cub protein fusions to Pex3Bp or Pex3p and NubG protein fusions to Pex3Bp, Pex3p, or the globular tail of the class V myosin of Y. lipolytica (amino acids 1,092–1,594) were tested for their ability to interact with each other by a β-galactosidase filter detection assay. A positive interaction was detected by the production of blue color. The color intensities of positive (+) and negative (−) controls are indicated. (C) Glutathione sepharose beads containing GST fused to the cargo-binding tail of the class V myosin of Y. lipolytica (GST-YlMyoV); the cargo-binding tail of the class V myosin, Myo2p, of S. cerevisiae (GST-ScMyoV); or GST alone were incubated with extracts of E. coli synthesizing MBP, MBP-Pex3p, MBP-Pex3Bp, MBP-YALI0E03124p, MBP-ScInp2p, or MBP-ScVam6p. Bound proteins, as well as 10% of input proteins, were analyzed by immunoblotting with anti-MBP antibodies. Total GST-YlMyoV, GST-ScMyoV, or GST protein levels were visualized by immunoblotting with anti-GST antibodies. Arrowheads indicate MBP or MBP fusion proteins. (D) Overexpression of PEX3B delivers peroxisomes preferentially to buds. pex3BΔ cells containing peroxisomes labeled with Pot1p-GFP and the plasmid pUB4 expressing PEX3B under the control of the oleic acid–inducible POT1 promoter were grown for 16 h in YPD supplemented with hygromycin B, then transferred to oleic acid–containing YPBO supplemented with hygromycin B for 6 h, and visualized at 23°C with an LSM 510 confocal microscope specifically modified for 4D in vivo microscopy (see Materials and methods). Representative frames from Video 4 show the specific movements of peroxisomes and their inheritance from mother cell to bud. At 0 min, two large peroxisome clusters are initially located next to the mother-bud neck. By 1 h 49 min, these peroxisomes have been transferred to their respective buds, and by 4 h 5 min, the cycle is repeated, with the peroxisomes now residing in the granddaughters of the original mother cells. De novo synthesis of peroxisomes can also be detected by the reappearance of fluorescent punctae in mother cells that had transferred their original peroxisome complement to their buds. These de novo formed peroxisomes are also vectorially transferred to newly formed buds (6 h 20 min). The formation of peroxisomes and subsequent transfer to buds continued (6 h 56 min). Bar, 5 µm.

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