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Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.

Marelli M, Smith JJ, Jung S, Yi E, Nesvizhskii AI, Christmas RH, Saleem RA, Tam YY, Fagarasanu A, Goodlett DR, Aebersold R, Rachubinski RA, Aitchison JD - J. Cell Biol. (2004)

Bottom Line: Among these proteins, eight novel peroxisome-associated proteins were identified.Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p.Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

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Rho1p binds Pex25p and Pex30p. GST-Rho1p and GST were immobilized on glutathione Sepharose and incubated with whole cell lysates derived from strains expressing TAP-tagged peroxins. Whole cell lysates (bottom) and bound fractions (top and middle) were resolved by SDS-PAGE, and TAP chimeras were detected by Western blotting. (top) Proteins bound to GST-Rho1p. Note that Rho1p interacts strongly with Pex25p and Pex30p. (middle) No interactions were detected with GST alone. (bottom) Yeast lysates showing the migration of each chimera. Cross-reacting bands are indicated (asterisks). (B) The distribution of GFP-Rho1p was observed in oleic acid-induced pex6Δ, pex15Δ, pex22Δ, pex25Δ, and pex30Δ cells. Note that GFP-Rho1p is not localized to peroxisomes in pex25Δ or pex6Δ cells. Bar, 10 μm.
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fig8: Rho1p binds Pex25p and Pex30p. GST-Rho1p and GST were immobilized on glutathione Sepharose and incubated with whole cell lysates derived from strains expressing TAP-tagged peroxins. Whole cell lysates (bottom) and bound fractions (top and middle) were resolved by SDS-PAGE, and TAP chimeras were detected by Western blotting. (top) Proteins bound to GST-Rho1p. Note that Rho1p interacts strongly with Pex25p and Pex30p. (middle) No interactions were detected with GST alone. (bottom) Yeast lysates showing the migration of each chimera. Cross-reacting bands are indicated (asterisks). (B) The distribution of GFP-Rho1p was observed in oleic acid-induced pex6Δ, pex15Δ, pex22Δ, pex25Δ, and pex30Δ cells. Note that GFP-Rho1p is not localized to peroxisomes in pex25Δ or pex6Δ cells. Bar, 10 μm.

Mentions: To investigate the role of Rho1p on the peroxisome membrane, we sought physical interaction data between Rho1p and known peroxins. Escherichia coli–produced GST-Rho1p or GST alone was immobilized on glutathione resin, and yeast extracts containing TAP-tagged peroxins (Pex2p, 3p, 4p, 5p, 6p, 7p, 8p, 11p, 12p, 13p, 15p, 17p, 19p, 22p, 25p, 27p, 29p, 30p, 31p, and 32p) were incubated with the resin. Bound fractions were analyzed by Western blotting to detect the protein A chimeras. Strong interactions were observed between GST-Rho1p and Pex25p and Pex30p (Fig. 8 A). Pex25p is a cytosolically exposed peripheral peroxisomal membrane protein that appears to play a role with Pex11p and Pex27p in the regulation of peroxisome number and size, perhaps by controlling peroxisome membrane fission (or fusion; Rottensteiner et al., 2003; Tam et al., 2003). Pex30p forms a complex with Pex31p and Pex32p, and this family of integral membrane proteins also plays a role in regulating peroxisome size and number (Vizeacoumar et al., 2003).


Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.

Marelli M, Smith JJ, Jung S, Yi E, Nesvizhskii AI, Christmas RH, Saleem RA, Tam YY, Fagarasanu A, Goodlett DR, Aebersold R, Rachubinski RA, Aitchison JD - J. Cell Biol. (2004)

Rho1p binds Pex25p and Pex30p. GST-Rho1p and GST were immobilized on glutathione Sepharose and incubated with whole cell lysates derived from strains expressing TAP-tagged peroxins. Whole cell lysates (bottom) and bound fractions (top and middle) were resolved by SDS-PAGE, and TAP chimeras were detected by Western blotting. (top) Proteins bound to GST-Rho1p. Note that Rho1p interacts strongly with Pex25p and Pex30p. (middle) No interactions were detected with GST alone. (bottom) Yeast lysates showing the migration of each chimera. Cross-reacting bands are indicated (asterisks). (B) The distribution of GFP-Rho1p was observed in oleic acid-induced pex6Δ, pex15Δ, pex22Δ, pex25Δ, and pex30Δ cells. Note that GFP-Rho1p is not localized to peroxisomes in pex25Δ or pex6Δ cells. Bar, 10 μm.
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Related In: Results  -  Collection

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fig8: Rho1p binds Pex25p and Pex30p. GST-Rho1p and GST were immobilized on glutathione Sepharose and incubated with whole cell lysates derived from strains expressing TAP-tagged peroxins. Whole cell lysates (bottom) and bound fractions (top and middle) were resolved by SDS-PAGE, and TAP chimeras were detected by Western blotting. (top) Proteins bound to GST-Rho1p. Note that Rho1p interacts strongly with Pex25p and Pex30p. (middle) No interactions were detected with GST alone. (bottom) Yeast lysates showing the migration of each chimera. Cross-reacting bands are indicated (asterisks). (B) The distribution of GFP-Rho1p was observed in oleic acid-induced pex6Δ, pex15Δ, pex22Δ, pex25Δ, and pex30Δ cells. Note that GFP-Rho1p is not localized to peroxisomes in pex25Δ or pex6Δ cells. Bar, 10 μm.
Mentions: To investigate the role of Rho1p on the peroxisome membrane, we sought physical interaction data between Rho1p and known peroxins. Escherichia coli–produced GST-Rho1p or GST alone was immobilized on glutathione resin, and yeast extracts containing TAP-tagged peroxins (Pex2p, 3p, 4p, 5p, 6p, 7p, 8p, 11p, 12p, 13p, 15p, 17p, 19p, 22p, 25p, 27p, 29p, 30p, 31p, and 32p) were incubated with the resin. Bound fractions were analyzed by Western blotting to detect the protein A chimeras. Strong interactions were observed between GST-Rho1p and Pex25p and Pex30p (Fig. 8 A). Pex25p is a cytosolically exposed peripheral peroxisomal membrane protein that appears to play a role with Pex11p and Pex27p in the regulation of peroxisome number and size, perhaps by controlling peroxisome membrane fission (or fusion; Rottensteiner et al., 2003; Tam et al., 2003). Pex30p forms a complex with Pex31p and Pex32p, and this family of integral membrane proteins also plays a role in regulating peroxisome size and number (Vizeacoumar et al., 2003).

Bottom Line: Among these proteins, eight novel peroxisome-associated proteins were identified.Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p.Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Systems Biology, Seattle, WA 98103, USA.

ABSTRACT
We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

Show MeSH
Related in: MedlinePlus