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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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Actin assembly on peroxisomes is controlled by Rho1p and Pex25p. The subcellular distribution of actin relative to that of peroxisomes was analyzed by double fluorescence confocal microscopy. Yeast deletion mutants expressing the peroxisomal reporter Pot1p-GFP were induced in oleic acid at 30 or 23°C (for rho1 and vps1 rho1) for 16 h, and actin was labeled with phalloidin-RITC. Peroxisomes colocalized with actin patches in rho1 (rho1-2A) and vps1 rho1 (vps1Δ rho1 POT1-G), pex25 (pex25Δ POT1-G), and pex11 pex25 (pex11Δ pex25Δ POT1-G) cells but not in wild-type (POT1-G), vps1 (vps1Δ POT1-G), and pex11 (pex11Δ POT1-G) cells. Bar, 10 μm.
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fig9: Actin assembly on peroxisomes is controlled by Rho1p and Pex25p. The subcellular distribution of actin relative to that of peroxisomes was analyzed by double fluorescence confocal microscopy. Yeast deletion mutants expressing the peroxisomal reporter Pot1p-GFP were induced in oleic acid at 30 or 23°C (for rho1 and vps1 rho1) for 16 h, and actin was labeled with phalloidin-RITC. Peroxisomes colocalized with actin patches in rho1 (rho1-2A) and vps1 rho1 (vps1Δ rho1 POT1-G), pex25 (pex25Δ POT1-G), and pex11 pex25 (pex11Δ pex25Δ POT1-G) cells but not in wild-type (POT1-G), vps1 (vps1Δ POT1-G), and pex11 (pex11Δ POT1-G) cells. Bar, 10 μm.

Mentions: The positions of peroxisomes and actin patches were analyzed in wild-type and mutant cells, including rho1 cells, containing a genomically encoded Pot1p-GFP chimera to mark peroxisomes. Cells were induced to proliferate peroxisomes, and their actin was labeled with phalloidin-RITC. The relative positions of peroxisomes and actin were determined by double label confocal microscopy (Fig. 9). In wild-type cells, peroxisomes and actin patches showed different localizations, although coincident staining was occasionally observed. However, in rho1-2A cells, peroxisomes and actin patches exhibited virtually exclusive colocalization. Although actin has been proposed to be involved in peroxisome localization within S. cerevisiae (Hoepfner et al., 2001), these data provide evidence for the existence of actin patches on peroxisomes and specifically a role for Rho1p in the organization of actin on this organelle. Remarkably, actin patches were also present on peroxisomes in cells lacking Pex25p, which is required for the proper localization of Rho1p to peroxisomes (Fig. 9). As a comparison, we investigated the dependence of actin localization on Pex11p and Vps1p, which are also implicated in peroxisome division and segregation. In pex11Δ and vps1Δ cells, actin was distributed as in wild-type cells. These data, together with the physical interaction data, suggest that the docking of Rho1p to Pex25p is important for dynamic assembly and disassembly of actin on peroxisomes. Interestingly, vps1Δ rho1 and pex11Δ pex25Δ double mutants also showed accumulation of actin on peroxisomes, which suggests that the majority of actin is reorganized/disassembled before organelle fission and that PEX25 is epistatic to PEX11.


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)

Actin assembly on peroxisomes is controlled by Rho1p and Pex25p. The subcellular distribution of actin relative to that of peroxisomes was analyzed by double fluorescence confocal microscopy. Yeast deletion mutants expressing the peroxisomal reporter Pot1p-GFP were induced in oleic acid at 30 or 23°C (for rho1 and vps1 rho1) for 16 h, and actin was labeled with phalloidin-RITC. Peroxisomes colocalized with actin patches in rho1 (rho1-2A) and vps1 rho1 (vps1Δ rho1 POT1-G), pex25 (pex25Δ POT1-G), and pex11 pex25 (pex11Δ pex25Δ POT1-G) cells but not in wild-type (POT1-G), vps1 (vps1Δ POT1-G), and pex11 (pex11Δ POT1-G) cells. Bar, 10 μm.
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Related In: Results  -  Collection

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fig9: Actin assembly on peroxisomes is controlled by Rho1p and Pex25p. The subcellular distribution of actin relative to that of peroxisomes was analyzed by double fluorescence confocal microscopy. Yeast deletion mutants expressing the peroxisomal reporter Pot1p-GFP were induced in oleic acid at 30 or 23°C (for rho1 and vps1 rho1) for 16 h, and actin was labeled with phalloidin-RITC. Peroxisomes colocalized with actin patches in rho1 (rho1-2A) and vps1 rho1 (vps1Δ rho1 POT1-G), pex25 (pex25Δ POT1-G), and pex11 pex25 (pex11Δ pex25Δ POT1-G) cells but not in wild-type (POT1-G), vps1 (vps1Δ POT1-G), and pex11 (pex11Δ POT1-G) cells. Bar, 10 μm.
Mentions: The positions of peroxisomes and actin patches were analyzed in wild-type and mutant cells, including rho1 cells, containing a genomically encoded Pot1p-GFP chimera to mark peroxisomes. Cells were induced to proliferate peroxisomes, and their actin was labeled with phalloidin-RITC. The relative positions of peroxisomes and actin were determined by double label confocal microscopy (Fig. 9). In wild-type cells, peroxisomes and actin patches showed different localizations, although coincident staining was occasionally observed. However, in rho1-2A cells, peroxisomes and actin patches exhibited virtually exclusive colocalization. Although actin has been proposed to be involved in peroxisome localization within S. cerevisiae (Hoepfner et al., 2001), these data provide evidence for the existence of actin patches on peroxisomes and specifically a role for Rho1p in the organization of actin on this organelle. Remarkably, actin patches were also present on peroxisomes in cells lacking Pex25p, which is required for the proper localization of Rho1p to peroxisomes (Fig. 9). As a comparison, we investigated the dependence of actin localization on Pex11p and Vps1p, which are also implicated in peroxisome division and segregation. In pex11Δ and vps1Δ cells, actin was distributed as in wild-type cells. These data, together with the physical interaction data, suggest that the docking of Rho1p to Pex25p is important for dynamic assembly and disassembly of actin on peroxisomes. Interestingly, vps1Δ rho1 and pex11Δ pex25Δ double mutants also showed accumulation of actin on peroxisomes, which suggests that the majority of actin is reorganized/disassembled before organelle fission and that PEX25 is epistatic to PEX11.

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