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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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Yhr150p-prA and Ydr479p-prA are primarily integral peroxisomal membrane proteins. (A) A PNS fraction was divided by centrifugation into a supernatant (20KgS) fraction enriched for cytosol and a pellet (20KgP) fraction enriched for peroxisomes and mitochondria. Equivalent portions of each fraction were analyzed. Immunoblotting detected the protein A chimeras shown, including that of the peroxisomal protein Pex17p. (B) Yhr150p-prA and Ydr479p-prA cofractionate with peroxisomes. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a discontinuous Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting. Fractions enriched for peroxisomes and mitochondria were identified by immunodetection of the protein A chimera of Pex17p and Sdh2p, respectively. (C) Peroxisomes purified by isopycnic density gradient centrifugation were lysed by treatment with 10 mM Tris-HCl, pH 8.0, releasing matrix proteins to a supernatant fraction (Ti8S) after centrifugation. The membrane-containing pellet fraction (Ti8P) was treated with 0.1 M Na2CO3, pH 11.3, and then subjected to centrifugation to yield a supernatant fraction (CO3S) enriched for peripherally associated membrane proteins and a pellet fraction (CO3P) enriched for integral membrane proteins. Equal portions of the respective supernatant and pellet fractions were analyzed by immunoblotting. Immunodetection of thiolase, Pex17p-prA, and Pex3p-prA marked the fractionation profiles of a matrix, peripheral membrane, and integral membrane protein, respectively.
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fig4: Yhr150p-prA and Ydr479p-prA are primarily integral peroxisomal membrane proteins. (A) A PNS fraction was divided by centrifugation into a supernatant (20KgS) fraction enriched for cytosol and a pellet (20KgP) fraction enriched for peroxisomes and mitochondria. Equivalent portions of each fraction were analyzed. Immunoblotting detected the protein A chimeras shown, including that of the peroxisomal protein Pex17p. (B) Yhr150p-prA and Ydr479p-prA cofractionate with peroxisomes. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a discontinuous Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting. Fractions enriched for peroxisomes and mitochondria were identified by immunodetection of the protein A chimera of Pex17p and Sdh2p, respectively. (C) Peroxisomes purified by isopycnic density gradient centrifugation were lysed by treatment with 10 mM Tris-HCl, pH 8.0, releasing matrix proteins to a supernatant fraction (Ti8S) after centrifugation. The membrane-containing pellet fraction (Ti8P) was treated with 0.1 M Na2CO3, pH 11.3, and then subjected to centrifugation to yield a supernatant fraction (CO3S) enriched for peripherally associated membrane proteins and a pellet fraction (CO3P) enriched for integral membrane proteins. Equal portions of the respective supernatant and pellet fractions were analyzed by immunoblotting. Immunodetection of thiolase, Pex17p-prA, and Pex3p-prA marked the fractionation profiles of a matrix, peripheral membrane, and integral membrane protein, respectively.

Mentions: Subcellular fractionation and organelle extraction were used to establish if Yhr150p and Ydr479p are associated with peroxisomes and to determine their suborganellar locations. Cells expressing Yhr150p-prA, Ydr479p-prA, and Pex17p-prA were incubated in oleic acid–containing medium and subjected to subcellular fractionation to yield postnuclear supernatant (PNS) fractions. The PNS fractions were subjected to further centrifugation to yield a supernatant fraction (20KgS) enriched for cytosol and a crude organellar pellet fraction (20KgP). Equal portions of the PNS, 20KgS, and the 20KgP were analyzed by immunoblotting. Yhr150p-prA, Ydr479p-prA, and Pex17p-prA all preferentially localized to the 20KgP fraction (Fig. 4 A). Peroxisomes were isolated from the 20KgP fractions of each of the strains expressing Yhr150p-prA, Ydr479p-prA, and Pex17p-prA. The gradients were fractionated, and equal portions of each fraction were analyzed by immunoblotting (Fig. 4 B). Yhr150p-prA and Ydr479p-prA coenriched with the peroxisomal peroxin Pex17p-prA and not with the mitochondrial protein, Sdh2p. Therefore, both microscopic analysis and subcellular fractionation showed Yhr150p and Ydr479p to be peroxisomal proteins. Some amount of Ydr479p-prA was always present in the 20KgS fraction and in the lighter fractions during the gradient isolation of peroxisomes. Whether this represents a selective liberation of a soluble form of Ydr479p-prA during the isolation of peroxisomes remains undetermined. It is unlikely that Ydr479p-prA is found in a cellular compartment in addition to peroxisomes, as immunostaining for Ydr479p-prA yielded exclusively a punctate pattern corresponding to the punctate pattern of peroxisomes defined by DsRed–PTS1 (Fig. 3). In addition, this punctate compartment does not correspond to mitochondria, as the fluorescence pattern generated with the mitochondria-specific dye MitoTracker did not overlap with the punctate pattern generated by detection of Ydr479p-prA by immunofluorescence (unpublished data).


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)

Yhr150p-prA and Ydr479p-prA are primarily integral peroxisomal membrane proteins. (A) A PNS fraction was divided by centrifugation into a supernatant (20KgS) fraction enriched for cytosol and a pellet (20KgP) fraction enriched for peroxisomes and mitochondria. Equivalent portions of each fraction were analyzed. Immunoblotting detected the protein A chimeras shown, including that of the peroxisomal protein Pex17p. (B) Yhr150p-prA and Ydr479p-prA cofractionate with peroxisomes. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a discontinuous Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting. Fractions enriched for peroxisomes and mitochondria were identified by immunodetection of the protein A chimera of Pex17p and Sdh2p, respectively. (C) Peroxisomes purified by isopycnic density gradient centrifugation were lysed by treatment with 10 mM Tris-HCl, pH 8.0, releasing matrix proteins to a supernatant fraction (Ti8S) after centrifugation. The membrane-containing pellet fraction (Ti8P) was treated with 0.1 M Na2CO3, pH 11.3, and then subjected to centrifugation to yield a supernatant fraction (CO3S) enriched for peripherally associated membrane proteins and a pellet fraction (CO3P) enriched for integral membrane proteins. Equal portions of the respective supernatant and pellet fractions were analyzed by immunoblotting. Immunodetection of thiolase, Pex17p-prA, and Pex3p-prA marked the fractionation profiles of a matrix, peripheral membrane, and integral membrane protein, respectively.
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Related In: Results  -  Collection

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fig4: Yhr150p-prA and Ydr479p-prA are primarily integral peroxisomal membrane proteins. (A) A PNS fraction was divided by centrifugation into a supernatant (20KgS) fraction enriched for cytosol and a pellet (20KgP) fraction enriched for peroxisomes and mitochondria. Equivalent portions of each fraction were analyzed. Immunoblotting detected the protein A chimeras shown, including that of the peroxisomal protein Pex17p. (B) Yhr150p-prA and Ydr479p-prA cofractionate with peroxisomes. Organelles in the 20KgP fraction were separated by isopycnic centrifugation on a discontinuous Nycodenz gradient. Fractions were collected from the bottom of the gradient, and equal portions of each fraction were analyzed by immunoblotting. Fractions enriched for peroxisomes and mitochondria were identified by immunodetection of the protein A chimera of Pex17p and Sdh2p, respectively. (C) Peroxisomes purified by isopycnic density gradient centrifugation were lysed by treatment with 10 mM Tris-HCl, pH 8.0, releasing matrix proteins to a supernatant fraction (Ti8S) after centrifugation. The membrane-containing pellet fraction (Ti8P) was treated with 0.1 M Na2CO3, pH 11.3, and then subjected to centrifugation to yield a supernatant fraction (CO3S) enriched for peripherally associated membrane proteins and a pellet fraction (CO3P) enriched for integral membrane proteins. Equal portions of the respective supernatant and pellet fractions were analyzed by immunoblotting. Immunodetection of thiolase, Pex17p-prA, and Pex3p-prA marked the fractionation profiles of a matrix, peripheral membrane, and integral membrane protein, respectively.
Mentions: Subcellular fractionation and organelle extraction were used to establish if Yhr150p and Ydr479p are associated with peroxisomes and to determine their suborganellar locations. Cells expressing Yhr150p-prA, Ydr479p-prA, and Pex17p-prA were incubated in oleic acid–containing medium and subjected to subcellular fractionation to yield postnuclear supernatant (PNS) fractions. The PNS fractions were subjected to further centrifugation to yield a supernatant fraction (20KgS) enriched for cytosol and a crude organellar pellet fraction (20KgP). Equal portions of the PNS, 20KgS, and the 20KgP were analyzed by immunoblotting. Yhr150p-prA, Ydr479p-prA, and Pex17p-prA all preferentially localized to the 20KgP fraction (Fig. 4 A). Peroxisomes were isolated from the 20KgP fractions of each of the strains expressing Yhr150p-prA, Ydr479p-prA, and Pex17p-prA. The gradients were fractionated, and equal portions of each fraction were analyzed by immunoblotting (Fig. 4 B). Yhr150p-prA and Ydr479p-prA coenriched with the peroxisomal peroxin Pex17p-prA and not with the mitochondrial protein, Sdh2p. Therefore, both microscopic analysis and subcellular fractionation showed Yhr150p and Ydr479p to be peroxisomal proteins. Some amount of Ydr479p-prA was always present in the 20KgS fraction and in the lighter fractions during the gradient isolation of peroxisomes. Whether this represents a selective liberation of a soluble form of Ydr479p-prA during the isolation of peroxisomes remains undetermined. It is unlikely that Ydr479p-prA is found in a cellular compartment in addition to peroxisomes, as immunostaining for Ydr479p-prA yielded exclusively a punctate pattern corresponding to the punctate pattern of peroxisomes defined by DsRed–PTS1 (Fig. 3). In addition, this punctate compartment does not correspond to mitochondria, as the fluorescence pattern generated with the mitochondria-specific dye MitoTracker did not overlap with the punctate pattern generated by detection of Ydr479p-prA by immunofluorescence (unpublished data).

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