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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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rho1 cells have fewer and smaller peroxisomes. (A) RHO1-2A and rho1-2A cells synthesizing Pot1p-GFP were incubated in oleic acid medium for 16 h at the semi-permissive temperature of 27°C and analyzed by confocal microscopy. Bar, 10 μm. (B) rho1-2A and RHO1-2A cells were incubated in oleic acid medium at the permissive temperature of 23°C and processed for EM. N, nucleus; L, lipid body; P, peroxisome; V, vacuole; M, mitochondrion. Bar, 0.5 μm. (C) A histogram of the areas of peroxisomes calculated for 105 randomly chosen cell images of each strain is shown.
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fig6: rho1 cells have fewer and smaller peroxisomes. (A) RHO1-2A and rho1-2A cells synthesizing Pot1p-GFP were incubated in oleic acid medium for 16 h at the semi-permissive temperature of 27°C and analyzed by confocal microscopy. Bar, 10 μm. (B) rho1-2A and RHO1-2A cells were incubated in oleic acid medium at the permissive temperature of 23°C and processed for EM. N, nucleus; L, lipid body; P, peroxisome; V, vacuole; M, mitochondrion. Bar, 0.5 μm. (C) A histogram of the areas of peroxisomes calculated for 105 randomly chosen cell images of each strain is shown.

Mentions: To investigate a role for Rho1p in peroxisome function, we first examined the peroxisome phenotypes and morphological characteristics of mutants of Rho1p. Thus, fluorescently labeled peroxisomes, detected by Pot1p-GFP, were monitored in mutant cells. Cells were incubated at permissive and semi-permissive temperatures on fatty acid medium, and peroxisome size and abundance were analyzed by confocal microscopy. At the permissive temperature of 23°C, peroxisome morphology in rho1-2A cells was similar to that observed in RHO1-2A cells (unpublished data). However, at the semi-permissive temperature of 27°C, peroxisomes in rho1-2A cells were smaller than peroxisomes in RHO1-2A cells (Fig. 6 A). In addition, rho1-2A cells appeared to have fewer peroxisomes than RHO1-2A cells, but these counts were restricted by the ability to detect peroxisomes by fluorescence microscopy. Therefore, peroxisomes that fell below the limit of resolution or contained small amounts of Pot1p-GFP were not quantified.


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)

rho1 cells have fewer and smaller peroxisomes. (A) RHO1-2A and rho1-2A cells synthesizing Pot1p-GFP were incubated in oleic acid medium for 16 h at the semi-permissive temperature of 27°C and analyzed by confocal microscopy. Bar, 10 μm. (B) rho1-2A and RHO1-2A cells were incubated in oleic acid medium at the permissive temperature of 23°C and processed for EM. N, nucleus; L, lipid body; P, peroxisome; V, vacuole; M, mitochondrion. Bar, 0.5 μm. (C) A histogram of the areas of peroxisomes calculated for 105 randomly chosen cell images of each strain is shown.
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Related In: Results  -  Collection

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fig6: rho1 cells have fewer and smaller peroxisomes. (A) RHO1-2A and rho1-2A cells synthesizing Pot1p-GFP were incubated in oleic acid medium for 16 h at the semi-permissive temperature of 27°C and analyzed by confocal microscopy. Bar, 10 μm. (B) rho1-2A and RHO1-2A cells were incubated in oleic acid medium at the permissive temperature of 23°C and processed for EM. N, nucleus; L, lipid body; P, peroxisome; V, vacuole; M, mitochondrion. Bar, 0.5 μm. (C) A histogram of the areas of peroxisomes calculated for 105 randomly chosen cell images of each strain is shown.
Mentions: To investigate a role for Rho1p in peroxisome function, we first examined the peroxisome phenotypes and morphological characteristics of mutants of Rho1p. Thus, fluorescently labeled peroxisomes, detected by Pot1p-GFP, were monitored in mutant cells. Cells were incubated at permissive and semi-permissive temperatures on fatty acid medium, and peroxisome size and abundance were analyzed by confocal microscopy. At the permissive temperature of 23°C, peroxisome morphology in rho1-2A cells was similar to that observed in RHO1-2A cells (unpublished data). However, at the semi-permissive temperature of 27°C, peroxisomes in rho1-2A cells were smaller than peroxisomes in RHO1-2A cells (Fig. 6 A). In addition, rho1-2A cells appeared to have fewer peroxisomes than RHO1-2A cells, but these counts were restricted by the ability to detect peroxisomes by fluorescence microscopy. Therefore, peroxisomes that fell below the limit of resolution or contained small amounts of Pot1p-GFP were not quantified.

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