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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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Prioritization of candidates. (A) 306 candidate proteins identified by ICAT MS/MS are listed alphabetically, and their peroxisome enrichment scores (PE) for ICAT I or ICAT II are represented by shaded squares. See Tables S1 and S2 for details. (B) 52 candidates with PE values > 0.65 in ICAT II, and which were also quantified in ICAT I, were clustered with a Spearman similarity metric into two groups (Groups 1 and 2). Also listed are 46 candidates with high PE values quantified in ICAT II alone (Group 3). Known peroxisomal proteins are indicated with an asterisk. (C) Yeast mutants of selected candidates from Groups 1 and 3 were assayed for their ability to grow on rich medium (YPB) containing glucose (Dx) or an oleic acid/lauric acid mixture (OL), and, as controls, the nonfermentable carbon sources glycerol (Gl) and acetate (Ac) at 25°C. Growth was assayed 2 d (Dx), 4 d (Gl and Ac), and 7 d (OL) after spotting. Slowly growing strains (bottom panel) were also examined after 3 d (Dx), 8 d (Gl and Ac), or 20 d (OL) of growth at 25°C.
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fig2: Prioritization of candidates. (A) 306 candidate proteins identified by ICAT MS/MS are listed alphabetically, and their peroxisome enrichment scores (PE) for ICAT I or ICAT II are represented by shaded squares. See Tables S1 and S2 for details. (B) 52 candidates with PE values > 0.65 in ICAT II, and which were also quantified in ICAT I, were clustered with a Spearman similarity metric into two groups (Groups 1 and 2). Also listed are 46 candidates with high PE values quantified in ICAT II alone (Group 3). Known peroxisomal proteins are indicated with an asterisk. (C) Yeast mutants of selected candidates from Groups 1 and 3 were assayed for their ability to grow on rich medium (YPB) containing glucose (Dx) or an oleic acid/lauric acid mixture (OL), and, as controls, the nonfermentable carbon sources glycerol (Gl) and acetate (Ac) at 25°C. Growth was assayed 2 d (Dx), 4 d (Gl and Ac), and 7 d (OL) after spotting. Slowly growing strains (bottom panel) were also examined after 3 d (Dx), 8 d (Gl and Ac), or 20 d (OL) of growth at 25°C.

Mentions: A plot of ICAT ratios for all proteins quantified by either ICAT I (Fig. 1 C) or ICAT II (Fig. 1 D) showed a normal distribution with a pronounced shoulder extending in the direction of higher ICAT ratios. The position of each protein on the abscissa represents its ICAT ratio and approximates its relative enrichment (Ti8PP versus Ti8PM for ICAT I and AP versus Ti8PP for ICAT II). These ratios are dependent on the limitations of MS, subcellular fractionation, and biochemical fractionation. Therefore, the probability of being enriched in the enriched peroxisomal membrane fraction (PE) as a function of its ICAT ratio was determined for each protein (see online supplemental material). Essentially, the distribution of ICAT ratios was modeled using Gaussian distributions, and the mixture model was fitted to the data using an expectation-maximization algorithm. The model was adjusted to take advantage of the fact that some of the identified proteins were previously shown to be peroxisomal, but was not adjusted to account for proteins thought to be “contaminants” (with the exception of ribosomal proteins, which were ignored and not included in the analysis). Importantly, as proteins might be localized to multiple organelles, this approach permitted the inclusion of proteins previously localized to other cellular compartments. However, it should be noted that the method presented here is general and can be applied in a completely unsupervised manner when no relevant prior information is available as to the protein constituents of a particular subcellular compartment (unpublished data). The data from two independent ICAT I and ICAT II experiments were combined, and where two PE scores were obtained for a protein, the scores were averaged (Fig. 2).


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)

Prioritization of candidates. (A) 306 candidate proteins identified by ICAT MS/MS are listed alphabetically, and their peroxisome enrichment scores (PE) for ICAT I or ICAT II are represented by shaded squares. See Tables S1 and S2 for details. (B) 52 candidates with PE values > 0.65 in ICAT II, and which were also quantified in ICAT I, were clustered with a Spearman similarity metric into two groups (Groups 1 and 2). Also listed are 46 candidates with high PE values quantified in ICAT II alone (Group 3). Known peroxisomal proteins are indicated with an asterisk. (C) Yeast mutants of selected candidates from Groups 1 and 3 were assayed for their ability to grow on rich medium (YPB) containing glucose (Dx) or an oleic acid/lauric acid mixture (OL), and, as controls, the nonfermentable carbon sources glycerol (Gl) and acetate (Ac) at 25°C. Growth was assayed 2 d (Dx), 4 d (Gl and Ac), and 7 d (OL) after spotting. Slowly growing strains (bottom panel) were also examined after 3 d (Dx), 8 d (Gl and Ac), or 20 d (OL) of growth at 25°C.
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Related In: Results  -  Collection

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fig2: Prioritization of candidates. (A) 306 candidate proteins identified by ICAT MS/MS are listed alphabetically, and their peroxisome enrichment scores (PE) for ICAT I or ICAT II are represented by shaded squares. See Tables S1 and S2 for details. (B) 52 candidates with PE values > 0.65 in ICAT II, and which were also quantified in ICAT I, were clustered with a Spearman similarity metric into two groups (Groups 1 and 2). Also listed are 46 candidates with high PE values quantified in ICAT II alone (Group 3). Known peroxisomal proteins are indicated with an asterisk. (C) Yeast mutants of selected candidates from Groups 1 and 3 were assayed for their ability to grow on rich medium (YPB) containing glucose (Dx) or an oleic acid/lauric acid mixture (OL), and, as controls, the nonfermentable carbon sources glycerol (Gl) and acetate (Ac) at 25°C. Growth was assayed 2 d (Dx), 4 d (Gl and Ac), and 7 d (OL) after spotting. Slowly growing strains (bottom panel) were also examined after 3 d (Dx), 8 d (Gl and Ac), or 20 d (OL) of growth at 25°C.
Mentions: A plot of ICAT ratios for all proteins quantified by either ICAT I (Fig. 1 C) or ICAT II (Fig. 1 D) showed a normal distribution with a pronounced shoulder extending in the direction of higher ICAT ratios. The position of each protein on the abscissa represents its ICAT ratio and approximates its relative enrichment (Ti8PP versus Ti8PM for ICAT I and AP versus Ti8PP for ICAT II). These ratios are dependent on the limitations of MS, subcellular fractionation, and biochemical fractionation. Therefore, the probability of being enriched in the enriched peroxisomal membrane fraction (PE) as a function of its ICAT ratio was determined for each protein (see online supplemental material). Essentially, the distribution of ICAT ratios was modeled using Gaussian distributions, and the mixture model was fitted to the data using an expectation-maximization algorithm. The model was adjusted to take advantage of the fact that some of the identified proteins were previously shown to be peroxisomal, but was not adjusted to account for proteins thought to be “contaminants” (with the exception of ribosomal proteins, which were ignored and not included in the analysis). Importantly, as proteins might be localized to multiple organelles, this approach permitted the inclusion of proteins previously localized to other cellular compartments. However, it should be noted that the method presented here is general and can be applied in a completely unsupervised manner when no relevant prior information is available as to the protein constituents of a particular subcellular compartment (unpublished data). The data from two independent ICAT I and ICAT II experiments were combined, and where two PE scores were obtained for a protein, the scores were averaged (Fig. 2).

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