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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 contain heterotypic peroxisomes. (A) The distribution of peroxisomal enzymes in wild-type and rho1 mutant cells was analyzed by subcellular fractionation. Whole cell lysates (L), postnuclear supernatants (PNS), and 20KgS fractions enriched for cytosol (loaded at one cell equivalent) and 20KgP fractions enriched for peroxisomes and mitochondria (loaded at five cell equivalents) were analyzed by Western blotting using anti-SKL antibodies, which recognizes PTS-1 containing proteins Fox2p, Mls1p, Cta1p, and Mdh3p, and anti-Pot1p antibodies. In rho1 cells, the PTS1-containing proteins Fox2p and Mls1p were not detected in the 20KgP fraction, whereas PTS2-containing Pot1p was partially mislocalized to the 20KgS. (B) rho1-2A and RHO1-2A cells synthesizing the peroxisomal reporters DsRed-PTS1 and Pot1p-GFP were incubated in oleic acid medium at 27°C. A series of optical sections were obtained by confocal microscopy, and the positions of peroxisomes were determined from the signals of the Pot1p-GFP and DsRed-PTS1 reporters. Heterotypic peroxisomes containing Pot1p-GFP or DsRed-PTS1 were numerous in rho-2A cells (arrowheads) but were rarely observed in cells of the complemented strain, RHO1-2A. Bar, 10 μm.
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fig7: rho1 cells contain heterotypic peroxisomes. (A) The distribution of peroxisomal enzymes in wild-type and rho1 mutant cells was analyzed by subcellular fractionation. Whole cell lysates (L), postnuclear supernatants (PNS), and 20KgS fractions enriched for cytosol (loaded at one cell equivalent) and 20KgP fractions enriched for peroxisomes and mitochondria (loaded at five cell equivalents) were analyzed by Western blotting using anti-SKL antibodies, which recognizes PTS-1 containing proteins Fox2p, Mls1p, Cta1p, and Mdh3p, and anti-Pot1p antibodies. In rho1 cells, the PTS1-containing proteins Fox2p and Mls1p were not detected in the 20KgP fraction, whereas PTS2-containing Pot1p was partially mislocalized to the 20KgS. (B) rho1-2A and RHO1-2A cells synthesizing the peroxisomal reporters DsRed-PTS1 and Pot1p-GFP were incubated in oleic acid medium at 27°C. A series of optical sections were obtained by confocal microscopy, and the positions of peroxisomes were determined from the signals of the Pot1p-GFP and DsRed-PTS1 reporters. Heterotypic peroxisomes containing Pot1p-GFP or DsRed-PTS1 were numerous in rho-2A cells (arrowheads) but were rarely observed in cells of the complemented strain, RHO1-2A. Bar, 10 μm.

Mentions: Remarkably, when we examined the distribution of marker proteins in the rho1-2A strain, it appeared that peroxisomes contained a different complement of proteins than peroxisomes of wild-type cells. Wild-type and rho1 cells were incubated at 27°C in fatty acid–containing medium, and subcellular fractions were prepared by differential centrifugation (Smith et al., 2002). As expected, Western blot analysis showed that the peroxisomal proteins Fox2p, Mls1p, Cta1p, Mdh3p, and Pot1p localized primarily to the peroxisome-enriched 20KgP fraction of wild-type cells; however, only Cta1p and Mdh3p localized to the 20KgP fraction of rho1 cells, whereas Fox2p, Mls1p, and Pot1p were not efficiently pelleted to the 20KgP containing “normal” high-density peroxisomes (Fig. 7 A). These data suggest that rho1 mutants were unable to incorporate all peroxisomal proteins with normal efficiency into high-density peroxisomes. Although it appeared that some proteins, such as Fox2p, were degraded due to their mislocalization, the peroxisomal proteins that remained in the 20KgS could be pelleted at higher g-force (200,000 g; unpublished data), suggesting that at least some of the mislocalized proteins were present in smaller, lighter membrane-bound compartments.


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 contain heterotypic peroxisomes. (A) The distribution of peroxisomal enzymes in wild-type and rho1 mutant cells was analyzed by subcellular fractionation. Whole cell lysates (L), postnuclear supernatants (PNS), and 20KgS fractions enriched for cytosol (loaded at one cell equivalent) and 20KgP fractions enriched for peroxisomes and mitochondria (loaded at five cell equivalents) were analyzed by Western blotting using anti-SKL antibodies, which recognizes PTS-1 containing proteins Fox2p, Mls1p, Cta1p, and Mdh3p, and anti-Pot1p antibodies. In rho1 cells, the PTS1-containing proteins Fox2p and Mls1p were not detected in the 20KgP fraction, whereas PTS2-containing Pot1p was partially mislocalized to the 20KgS. (B) rho1-2A and RHO1-2A cells synthesizing the peroxisomal reporters DsRed-PTS1 and Pot1p-GFP were incubated in oleic acid medium at 27°C. A series of optical sections were obtained by confocal microscopy, and the positions of peroxisomes were determined from the signals of the Pot1p-GFP and DsRed-PTS1 reporters. Heterotypic peroxisomes containing Pot1p-GFP or DsRed-PTS1 were numerous in rho-2A cells (arrowheads) but were rarely observed in cells of the complemented strain, RHO1-2A. Bar, 10 μm.
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Related In: Results  -  Collection

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fig7: rho1 cells contain heterotypic peroxisomes. (A) The distribution of peroxisomal enzymes in wild-type and rho1 mutant cells was analyzed by subcellular fractionation. Whole cell lysates (L), postnuclear supernatants (PNS), and 20KgS fractions enriched for cytosol (loaded at one cell equivalent) and 20KgP fractions enriched for peroxisomes and mitochondria (loaded at five cell equivalents) were analyzed by Western blotting using anti-SKL antibodies, which recognizes PTS-1 containing proteins Fox2p, Mls1p, Cta1p, and Mdh3p, and anti-Pot1p antibodies. In rho1 cells, the PTS1-containing proteins Fox2p and Mls1p were not detected in the 20KgP fraction, whereas PTS2-containing Pot1p was partially mislocalized to the 20KgS. (B) rho1-2A and RHO1-2A cells synthesizing the peroxisomal reporters DsRed-PTS1 and Pot1p-GFP were incubated in oleic acid medium at 27°C. A series of optical sections were obtained by confocal microscopy, and the positions of peroxisomes were determined from the signals of the Pot1p-GFP and DsRed-PTS1 reporters. Heterotypic peroxisomes containing Pot1p-GFP or DsRed-PTS1 were numerous in rho-2A cells (arrowheads) but were rarely observed in cells of the complemented strain, RHO1-2A. Bar, 10 μm.
Mentions: Remarkably, when we examined the distribution of marker proteins in the rho1-2A strain, it appeared that peroxisomes contained a different complement of proteins than peroxisomes of wild-type cells. Wild-type and rho1 cells were incubated at 27°C in fatty acid–containing medium, and subcellular fractions were prepared by differential centrifugation (Smith et al., 2002). As expected, Western blot analysis showed that the peroxisomal proteins Fox2p, Mls1p, Cta1p, Mdh3p, and Pot1p localized primarily to the peroxisome-enriched 20KgP fraction of wild-type cells; however, only Cta1p and Mdh3p localized to the 20KgP fraction of rho1 cells, whereas Fox2p, Mls1p, and Pot1p were not efficiently pelleted to the 20KgP containing “normal” high-density peroxisomes (Fig. 7 A). These data suggest that rho1 mutants were unable to incorporate all peroxisomal proteins with normal efficiency into high-density peroxisomes. Although it appeared that some proteins, such as Fox2p, were degraded due to their mislocalization, the peroxisomal proteins that remained in the 20KgS could be pelleted at higher g-force (200,000 g; unpublished data), suggesting that at least some of the mislocalized proteins were present in smaller, lighter membrane-bound compartments.

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