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YHR150w and YDR479c encode peroxisomal integral membrane proteins involved in the regulation of peroxisome number, size, and distribution in Saccharomyces cerevisiae.

Vizeacoumar FJ, Torres-Guzman JC, Tam YY, Aitchison JD, Rachubinski RA - J. Cell Biol. (2003)

Bottom Line: Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening.Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae.Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Medical Sciences Building 5-14, Edmonton, Alberta T6G 2H7, Canada.

ABSTRACT
The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid-containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.

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Peroxisomes isolated from yhr150Δ/ydr479Δ cells are less dense than isolated wild-type peroxisomes and retain a clustered phenotype. (A) The wild-type (WT) strain BY4742 and the mutant strain yhr150Δ/ydr479Δ were grown overnight in YPD medium, transferred to oleic acid–containing YPBO medium, and incubated in YPBO medium for 8 h. A PNS fraction was prepared from cells of each strain and divided by centrifugation into 20KgS and 20KgP fractions. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a continuous 30–45% Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting with antibodies to the PTS1, Ser-Lys-Leu, to detect peroxisomes. (B) Electron micrographs of peak peroxisomal fractions from cells of the wild-type strain (WT) BY4742 (fraction 1) and the mutant strain yhr150Δ/ydr479Δ (fraction 8). Bar, 0.5 μm.
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fig6: Peroxisomes isolated from yhr150Δ/ydr479Δ cells are less dense than isolated wild-type peroxisomes and retain a clustered phenotype. (A) The wild-type (WT) strain BY4742 and the mutant strain yhr150Δ/ydr479Δ were grown overnight in YPD medium, transferred to oleic acid–containing YPBO medium, and incubated in YPBO medium for 8 h. A PNS fraction was prepared from cells of each strain and divided by centrifugation into 20KgS and 20KgP fractions. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a continuous 30–45% Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting with antibodies to the PTS1, Ser-Lys-Leu, to detect peroxisomes. (B) Electron micrographs of peak peroxisomal fractions from cells of the wild-type strain (WT) BY4742 (fraction 1) and the mutant strain yhr150Δ/ydr479Δ (fraction 8). Bar, 0.5 μm.

Mentions: We next investigated the ultrastructure of cells incubated in oleic acid–containing YPBO medium by transmission EM. Wild-type cells (Fig. 5 A) consistently showed individual peroxisomes well separated from one another. In contrast, cells of the yhr150Δ (Fig. 5 B), ydr479Δ (Fig. 5 C), and, particularly, yhr150Δ/ydr479Δ (Fig. 5 D) strains contained peroxisomes that exhibited clustering. 28.0, 19.2, and 20.4% of peroxisomes of cells of the yhr150Δ/ydr479Δ, ydr479Δ, and yhr150Δ strains, respectively, showed clustering, in contrast to 4.0% of peroxisomes of wild-type cells (a cluster of peroxisomes was operationally defined as three or more adherent peroxisomes). The clustered peroxisomes often showed evidence of membrane thickening between adjacent peroxisomes in the cluster. Morphometric analysis showed that cells of the deletion strains contained a greater number of peroxisomes than wild-type cells and that, on average, these peroxisomes were smaller in size than those of wild-type cells (Table I). Cells of the deletion strains contained much greater numbers of peroxisomes with areas of 0.02 μm2 or less than wild-type cells (Fig. 5 E). Nycodenz density gradient centrifugation analysis showed that peroxisomes purified from yhr150Δ/ydr479Δ cells have a greatly reduced density (peak fraction 8, 1.19 g/cm3) compared with peroxisomes from wild-type cells (peak fraction 1, 1.22 g/cm3) (Fig. 6 A). Peroxisomes isolated from cells deleted for YHR150w or YDR479c were also less dense than wild-type peroxisomes, although the differences in density were less than that observed between peroxisomes from yhr150Δ/ydr479Δ cells and wild-type peroxisomes (unpublished data). EM analysis showed that the peroxisomes purified from yhr150Δ/ydr479Δ cells still exhibited clustering and evidence of thickened peroxisomal membranes, whereas peroxisomes purified from wild-type cells were largely well separated from one another, with no evidence of membrane thickening (Fig. 6 B).


YHR150w and YDR479c encode peroxisomal integral membrane proteins involved in the regulation of peroxisome number, size, and distribution in Saccharomyces cerevisiae.

Vizeacoumar FJ, Torres-Guzman JC, Tam YY, Aitchison JD, Rachubinski RA - J. Cell Biol. (2003)

Peroxisomes isolated from yhr150Δ/ydr479Δ cells are less dense than isolated wild-type peroxisomes and retain a clustered phenotype. (A) The wild-type (WT) strain BY4742 and the mutant strain yhr150Δ/ydr479Δ were grown overnight in YPD medium, transferred to oleic acid–containing YPBO medium, and incubated in YPBO medium for 8 h. A PNS fraction was prepared from cells of each strain and divided by centrifugation into 20KgS and 20KgP fractions. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a continuous 30–45% Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting with antibodies to the PTS1, Ser-Lys-Leu, to detect peroxisomes. (B) Electron micrographs of peak peroxisomal fractions from cells of the wild-type strain (WT) BY4742 (fraction 1) and the mutant strain yhr150Δ/ydr479Δ (fraction 8). Bar, 0.5 μm.
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fig6: Peroxisomes isolated from yhr150Δ/ydr479Δ cells are less dense than isolated wild-type peroxisomes and retain a clustered phenotype. (A) The wild-type (WT) strain BY4742 and the mutant strain yhr150Δ/ydr479Δ were grown overnight in YPD medium, transferred to oleic acid–containing YPBO medium, and incubated in YPBO medium for 8 h. A PNS fraction was prepared from cells of each strain and divided by centrifugation into 20KgS and 20KgP fractions. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a continuous 30–45% Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting with antibodies to the PTS1, Ser-Lys-Leu, to detect peroxisomes. (B) Electron micrographs of peak peroxisomal fractions from cells of the wild-type strain (WT) BY4742 (fraction 1) and the mutant strain yhr150Δ/ydr479Δ (fraction 8). Bar, 0.5 μm.
Mentions: We next investigated the ultrastructure of cells incubated in oleic acid–containing YPBO medium by transmission EM. Wild-type cells (Fig. 5 A) consistently showed individual peroxisomes well separated from one another. In contrast, cells of the yhr150Δ (Fig. 5 B), ydr479Δ (Fig. 5 C), and, particularly, yhr150Δ/ydr479Δ (Fig. 5 D) strains contained peroxisomes that exhibited clustering. 28.0, 19.2, and 20.4% of peroxisomes of cells of the yhr150Δ/ydr479Δ, ydr479Δ, and yhr150Δ strains, respectively, showed clustering, in contrast to 4.0% of peroxisomes of wild-type cells (a cluster of peroxisomes was operationally defined as three or more adherent peroxisomes). The clustered peroxisomes often showed evidence of membrane thickening between adjacent peroxisomes in the cluster. Morphometric analysis showed that cells of the deletion strains contained a greater number of peroxisomes than wild-type cells and that, on average, these peroxisomes were smaller in size than those of wild-type cells (Table I). Cells of the deletion strains contained much greater numbers of peroxisomes with areas of 0.02 μm2 or less than wild-type cells (Fig. 5 E). Nycodenz density gradient centrifugation analysis showed that peroxisomes purified from yhr150Δ/ydr479Δ cells have a greatly reduced density (peak fraction 8, 1.19 g/cm3) compared with peroxisomes from wild-type cells (peak fraction 1, 1.22 g/cm3) (Fig. 6 A). Peroxisomes isolated from cells deleted for YHR150w or YDR479c were also less dense than wild-type peroxisomes, although the differences in density were less than that observed between peroxisomes from yhr150Δ/ydr479Δ cells and wild-type peroxisomes (unpublished data). EM analysis showed that the peroxisomes purified from yhr150Δ/ydr479Δ cells still exhibited clustering and evidence of thickened peroxisomal membranes, whereas peroxisomes purified from wild-type cells were largely well separated from one another, with no evidence of membrane thickening (Fig. 6 B).

Bottom Line: Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening.Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae.Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Medical Sciences Building 5-14, Edmonton, Alberta T6G 2H7, Canada.

ABSTRACT
The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid-containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.

Show MeSH
Related in: MedlinePlus