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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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Rho1p associates dynamically with peroxisomes. The distribution of GFP-Rho1p was observed in glucose-, glycerol-, and oleic acid–incubated cells. GFP-Rho1p localized to intracellular membrane structures in glucose- and glycerol-incubated cells. In conditions that induce peroxisomes (oleic acid), GFP-Rho1p localized to distinct punctate structures. (B) In oleic acid–induced vps1Δ cells, which contain few peroxisomes, GFP-Rho1p localized to one or two punctate structures per cell. However, in pex3Δ cells, which are defective in peroxisome biogenesis, GFP-Rho1p failed to accumulate in punctate structures. Bar, 10 μm.
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fig5: Rho1p associates dynamically with peroxisomes. The distribution of GFP-Rho1p was observed in glucose-, glycerol-, and oleic acid–incubated cells. GFP-Rho1p localized to intracellular membrane structures in glucose- and glycerol-incubated cells. In conditions that induce peroxisomes (oleic acid), GFP-Rho1p localized to distinct punctate structures. (B) In oleic acid–induced vps1Δ cells, which contain few peroxisomes, GFP-Rho1p localized to one or two punctate structures per cell. However, in pex3Δ cells, which are defective in peroxisome biogenesis, GFP-Rho1p failed to accumulate in punctate structures. Bar, 10 μm.

Mentions: We considered the possibility that the reason Rho1p had previously not been found to be associated with peroxisomes was that yeast cells are generally grown in glucose medium, which is a condition that represses peroxisome biogenesis (Veenhuis et al., 1987). Therefore, we visualized GFP-Rho1p under conditions that repress (glucose), derepress (glycerol), or proliferate (oleic acid) peroxisomes (Fig. 5 A). Although the GFP-Rho1p signal was diffuse under all growth conditions, in glycerol and glucose the signal appeared most intense at the cell periphery and on internal membranes mainly surrounding the vacuole. This localization is consistent with current knowledge, as Rho1p has been previously localized to the plasma and endomembranes, particularly at sites of growth (for reviews see van Aelst and D'Souza-Schorey, 1997; Hall, 1998; Madden and Snyder, 1998) and has recently been shown to be required for vacuole membrane fusion (Eitzen et al., 2001). Strikingly, when cells were incubated in oleic acid medium, distinct punctate structures dominated the fluorescence signal, suggesting that Rho1p was recruited to peroxisomes upon their induction. Several mutants that affect the abundance of peroxisomes supported this interpretation, as fewer of these punctate structures were observed in vps1Δ cells (Fig. 5 B), which contain fewer and larger peroxisomes than wild-type cells (Hoepfner et al., 2001), and no punctate structures were observed in pex3Δ cells, which lack detectable peroxisomes.


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)

Rho1p associates dynamically with peroxisomes. The distribution of GFP-Rho1p was observed in glucose-, glycerol-, and oleic acid–incubated cells. GFP-Rho1p localized to intracellular membrane structures in glucose- and glycerol-incubated cells. In conditions that induce peroxisomes (oleic acid), GFP-Rho1p localized to distinct punctate structures. (B) In oleic acid–induced vps1Δ cells, which contain few peroxisomes, GFP-Rho1p localized to one or two punctate structures per cell. However, in pex3Δ cells, which are defective in peroxisome biogenesis, GFP-Rho1p failed to accumulate in punctate structures. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172632&req=5

fig5: Rho1p associates dynamically with peroxisomes. The distribution of GFP-Rho1p was observed in glucose-, glycerol-, and oleic acid–incubated cells. GFP-Rho1p localized to intracellular membrane structures in glucose- and glycerol-incubated cells. In conditions that induce peroxisomes (oleic acid), GFP-Rho1p localized to distinct punctate structures. (B) In oleic acid–induced vps1Δ cells, which contain few peroxisomes, GFP-Rho1p localized to one or two punctate structures per cell. However, in pex3Δ cells, which are defective in peroxisome biogenesis, GFP-Rho1p failed to accumulate in punctate structures. Bar, 10 μm.
Mentions: We considered the possibility that the reason Rho1p had previously not been found to be associated with peroxisomes was that yeast cells are generally grown in glucose medium, which is a condition that represses peroxisome biogenesis (Veenhuis et al., 1987). Therefore, we visualized GFP-Rho1p under conditions that repress (glucose), derepress (glycerol), or proliferate (oleic acid) peroxisomes (Fig. 5 A). Although the GFP-Rho1p signal was diffuse under all growth conditions, in glycerol and glucose the signal appeared most intense at the cell periphery and on internal membranes mainly surrounding the vacuole. This localization is consistent with current knowledge, as Rho1p has been previously localized to the plasma and endomembranes, particularly at sites of growth (for reviews see van Aelst and D'Souza-Schorey, 1997; Hall, 1998; Madden and Snyder, 1998) and has recently been shown to be required for vacuole membrane fusion (Eitzen et al., 2001). Strikingly, when cells were incubated in oleic acid medium, distinct punctate structures dominated the fluorescence signal, suggesting that Rho1p was recruited to peroxisomes upon their induction. Several mutants that affect the abundance of peroxisomes supported this interpretation, as fewer of these punctate structures were observed in vps1Δ cells (Fig. 5 B), which contain fewer and larger peroxisomes than wild-type cells (Hoepfner et al., 2001), and no punctate structures were observed in pex3Δ cells, which lack detectable peroxisomes.

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