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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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Sample preparation and analysis. (A) An organellar 20KgP fraction was subjected to isopycnic density gradient centrifugation and analyzed by SDS-PAGE and Coomassie blue staining (top panel). Fractions enriched for peroxisomes (EP) or mitochondria (M) were identified by Western blotting as shown. Equal amounts of protein derived from each of the hypotonically lysed M and EP fractions were combined and analyzed by ICAT MS/MS. (B) Peroxisomal membranes isolated from a yeast strain synthesizing Pex11p-pA were affinity purified (AP) from a fraction enriched for peroxisomal membranes (Ti8PP). Equal cellular equivalents of each were analyzed by SDS-PAGE and silver staining. Equal amounts of protein from the 20KgP, Ti8PP, and AP fractions were analyzed by Western blotting. Ti8PP and AP fractions were analyzed by ICAT MS/MS. (C and D) Histograms of ICAT ratios (heavy:light) for 192 proteins quantified in ICAT I (C) and 193 proteins quantified in ICAT II (D). The distributions were modeled by two overlapping Gaussian curves using a partially supervised mixture model Expectation-Maximization algorithm. Note that because of the nature of the data in ICAT I (dominance by mitochondrial proteins with low ICAT ratios and relatively few peroxisomal proteins with high ratios), the ICAT ratios in this experiment were transformed to their square root for modeling. For any quantified protein, the probability of being enriched (p(E); dashed line) or not being enriched (p(U); solid line) with peroxisomes was calculated as a function of its ICAT ratio.
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fig1: Sample preparation and analysis. (A) An organellar 20KgP fraction was subjected to isopycnic density gradient centrifugation and analyzed by SDS-PAGE and Coomassie blue staining (top panel). Fractions enriched for peroxisomes (EP) or mitochondria (M) were identified by Western blotting as shown. Equal amounts of protein derived from each of the hypotonically lysed M and EP fractions were combined and analyzed by ICAT MS/MS. (B) Peroxisomal membranes isolated from a yeast strain synthesizing Pex11p-pA were affinity purified (AP) from a fraction enriched for peroxisomal membranes (Ti8PP). Equal cellular equivalents of each were analyzed by SDS-PAGE and silver staining. Equal amounts of protein from the 20KgP, Ti8PP, and AP fractions were analyzed by Western blotting. Ti8PP and AP fractions were analyzed by ICAT MS/MS. (C and D) Histograms of ICAT ratios (heavy:light) for 192 proteins quantified in ICAT I (C) and 193 proteins quantified in ICAT II (D). The distributions were modeled by two overlapping Gaussian curves using a partially supervised mixture model Expectation-Maximization algorithm. Note that because of the nature of the data in ICAT I (dominance by mitochondrial proteins with low ICAT ratios and relatively few peroxisomal proteins with high ratios), the ICAT ratios in this experiment were transformed to their square root for modeling. For any quantified protein, the probability of being enriched (p(E); dashed line) or not being enriched (p(U); solid line) with peroxisomes was calculated as a function of its ICAT ratio.

Mentions: In the first approach, ICAT I, organelles from oleic acid-grown cells were collected and separated by isopycnic density gradient centrifugation. Intact organelles were collected from both peak peroxisome (Fig. 1 A, fractions 8–10) and peak mitochondrial (Fig. 1 A, fractions 2 and 3) fractions and hypotonically lysed, and the membrane-enriched fractions were collected by centrifugation. The resulting membrane-enriched fractions derived from peroxisome (Ti8PP) and mitochondrial (Ti8PM) peak fractions were labeled separately with heavy and light ICAT reagent, respectively, and analyzed by MS (see online supplemental material).


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)

Sample preparation and analysis. (A) An organellar 20KgP fraction was subjected to isopycnic density gradient centrifugation and analyzed by SDS-PAGE and Coomassie blue staining (top panel). Fractions enriched for peroxisomes (EP) or mitochondria (M) were identified by Western blotting as shown. Equal amounts of protein derived from each of the hypotonically lysed M and EP fractions were combined and analyzed by ICAT MS/MS. (B) Peroxisomal membranes isolated from a yeast strain synthesizing Pex11p-pA were affinity purified (AP) from a fraction enriched for peroxisomal membranes (Ti8PP). Equal cellular equivalents of each were analyzed by SDS-PAGE and silver staining. Equal amounts of protein from the 20KgP, Ti8PP, and AP fractions were analyzed by Western blotting. Ti8PP and AP fractions were analyzed by ICAT MS/MS. (C and D) Histograms of ICAT ratios (heavy:light) for 192 proteins quantified in ICAT I (C) and 193 proteins quantified in ICAT II (D). The distributions were modeled by two overlapping Gaussian curves using a partially supervised mixture model Expectation-Maximization algorithm. Note that because of the nature of the data in ICAT I (dominance by mitochondrial proteins with low ICAT ratios and relatively few peroxisomal proteins with high ratios), the ICAT ratios in this experiment were transformed to their square root for modeling. For any quantified protein, the probability of being enriched (p(E); dashed line) or not being enriched (p(U); solid line) with peroxisomes was calculated as a function of its ICAT ratio.
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Related In: Results  -  Collection

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fig1: Sample preparation and analysis. (A) An organellar 20KgP fraction was subjected to isopycnic density gradient centrifugation and analyzed by SDS-PAGE and Coomassie blue staining (top panel). Fractions enriched for peroxisomes (EP) or mitochondria (M) were identified by Western blotting as shown. Equal amounts of protein derived from each of the hypotonically lysed M and EP fractions were combined and analyzed by ICAT MS/MS. (B) Peroxisomal membranes isolated from a yeast strain synthesizing Pex11p-pA were affinity purified (AP) from a fraction enriched for peroxisomal membranes (Ti8PP). Equal cellular equivalents of each were analyzed by SDS-PAGE and silver staining. Equal amounts of protein from the 20KgP, Ti8PP, and AP fractions were analyzed by Western blotting. Ti8PP and AP fractions were analyzed by ICAT MS/MS. (C and D) Histograms of ICAT ratios (heavy:light) for 192 proteins quantified in ICAT I (C) and 193 proteins quantified in ICAT II (D). The distributions were modeled by two overlapping Gaussian curves using a partially supervised mixture model Expectation-Maximization algorithm. Note that because of the nature of the data in ICAT I (dominance by mitochondrial proteins with low ICAT ratios and relatively few peroxisomal proteins with high ratios), the ICAT ratios in this experiment were transformed to their square root for modeling. For any quantified protein, the probability of being enriched (p(E); dashed line) or not being enriched (p(U); solid line) with peroxisomes was calculated as a function of its ICAT ratio.
Mentions: In the first approach, ICAT I, organelles from oleic acid-grown cells were collected and separated by isopycnic density gradient centrifugation. Intact organelles were collected from both peak peroxisome (Fig. 1 A, fractions 8–10) and peak mitochondrial (Fig. 1 A, fractions 2 and 3) fractions and hypotonically lysed, and the membrane-enriched fractions were collected by centrifugation. The resulting membrane-enriched fractions derived from peroxisome (Ti8PP) and mitochondrial (Ti8PM) peak fractions were labeled separately with heavy and light ICAT reagent, respectively, and analyzed by MS (see online supplemental material).

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